Young Scientists Journal Vol 3 issue 8 2010

Volume 3 Issue 8 Jan-May 2010

Nanotechnology special issue

YOUNG SCIENTISTS

journal

Carbon nanotubes

Silicon dioxide particles glued to AFM Tip

Spray dried juice particles

Iron silate crystals

Gold hexagon

Stober silicon dioxide particles

Did you know that the sports drink Gatorade(R) was invented at the University of Florida in the 1960s by Dr. Robert Cade and his team, using UF football players as test subjects?

Dr. Cade’s legacy as scientist, inventor, musician, and humanitarian philantropist will be continued through the Cade Museum Foundation. www.cademuseum.org Follow us Facebook and Twitter

The Junior Cade Prize for Innovation The Cade Museum Foundation (http://www.cademuseum.org) is sponsoring an annual competition - beginning in 2010 - to recognize and promote Young Innovators and Inventors all over the world. The Prize winner will be selected from the Intel/ISEF finalists and awarded to the superior project that is outstanding in innovation and creativity and has the potential for commercialization as a new product or process.

The award will consist of a cash prize. “We want to harness the potential of the brilliant ideas springing forth from the brightest young scientists in the world." said Richard Miles, the Foundation's Executive Director.

The Cade Museum Foundation | www.cademuseum.org | info@cademuseum.org 530 W University Avenue | Gainesville, FL 32601 | USA +352-381-7100

Young Scientists Journal

Volume 3 | Issue 8 | Jan - May 2010

Editorial Board Chief Editor: Pamela Barraza Flores, Mexico Lead Editor: Courtney Williams, UK

Editorial Team Members Team Leader: Courtney Williams, UK Christopher Barry, UK Kim Dunn, UK Natalie Hackman, UK Aaron Hakim, Canada Otana Jakpor, USA Kartik Madiraju, Canada Muna Oli, USA Kimi Tur, UK Alex Lee, Hong Kong Jarred Goodman, USA Jonathan Wang, USA

Publicity Team Izy Wingrad, UK Cleodie Swire, UK

Technical Team Team Leader: Malcolm Morgan, UK Jacob Hamblin-Pyke, UK Andrew Sultana, UK Jeffrey Chan, UK

Content Sub-Team: Team Leader: George Harvey, UK Sam Gearing, UK (Multimedia) Will Goldsmith, UK Tim Perkins, UK

Young Advisory Board Jonathan Rogers, UK Malcolm Morgan, UK

Internationals Advisory Board Team Leader: Christina Astin, UK Ghazwan Butrous, UK Mark Orders, UK Joanne Manaster, USA Paul Soderberg, USA Andreia Azevedo-Soares, UK Lee Riley, USA Paul Soderberg, USA Corky Valenti, USA Anna Grigoryan, USA / Armenia Vince Bennett, USA Don Eliseo Lucero-Prisno, UK Mike Bennett, USA Linda Crouch, UK Tony Grady, USA Steven Chambers, UK Ian Yorston, UK Thijs Kouwenhoven, China Charlie Barclay, UK

This magazine web-based Young Scientists Journal is online journal open access journal (www.ysjournal.com). It has been in existence since June 06 and contains articles written by young scientists for young scientists. It is where young scientists get their research and review articles published.

Published by MEDKNOW PUBLICATIONS AND MEDIA PVT. LTD. B5-12, Kanara Business Center, Off Link Road, Ghatkopar (E), Mumbai - 400075, INDIA. Phone: 91-22-6649 1818 Web: www.medknow.com

Young Scientists Journal All rights reserved. No part of this publication may be reproduced, or transmitted, in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the editor.

The Young Scientists Journal and/ or its publisher cannot be held responsible for errors or for any consequences arising from the use of the information contained in this journal.

The appearance of advertising or product information in the various sections in the journal does not constitute an endorsement or approval by the journal and/or its publisher of the quality or value of the said product or of claims made for it by its manufacturer.

The Journal is printed on acid free paper.

Web sites: www.ysjournal.com E-mails:editor@ysjournal.com

Volume 3 | Issue 8 | Jan - May 2010

Contents...

Editorial Malcolm Morgan.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Nanotechnology special issue editor's note Muna Oli.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Nanotechnology Introduction to nanotechnology Muna Oli.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Interview with Dr. Andrew Maynard Muna Oli.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Sunscreen: A catch-22 Cole Blum and Samantha Larsen .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 11 Synthetic, 'inorganic DNA' as a means to high-density molecular electronics Matthew Kapelewski .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 15 Aptamer conjugated gold nanorods for targeted nanothermal radiation of Glioblastoma cancer cells (A novel selective targeted approach to cancer treatment) Muna Oli.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Increasing the efficiency of a hybrid polymer photovoltaic cell with polymer nanofiber complexes of varied thickness Nathan Monroe .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 Antibody-coated magnetic nanoparticles: Targeting and treating cancer Philip Schlenoff .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33 Synthesis of fluorescent silica nanoparticles conjugated with rgd peptide for detection of invasive human breast cancer cells Shamik mascharak .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 37

Young Scientist Notes Building from the Ground up: Nanostructures to microstructures Steven Noyce .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42

Young Scientist Blogs The best six days ever Courtney Williams.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

Published by MEDKNOW PUBLICATIONS & MEDIA PVT. LTD. B5-12, Kanara Business Center, Off Link Rd, Ghatkopar (E), Mumbai - 400075, INDIA. Phone: 91-22-6649 1818 Web: www.medknow.com

Editorial It seems like a lifetime has passed since I first became involved with the Young Scientists Journal. In early 2006 myself and several other interested students met in one of the physics laboratories of The King’s School Canterbury with our head of science (Christina Astin) and a stranger. This stranger turned out to be Ghazwan Butrous, the proverbial ‘man with a plan’. Ghazwan’s idea was simple, young scientists across the world should be able to come together, share their work, discuss ideas, and make friends. At the time there we many magazines for young people interested in science, but these where all written by adults. Ghazwan wanted an organisation which published articles by young scientists for young scientists, a phase we still use as our strap line today.

It took about six months to put together the first issue, meeting every Thursday at 2pm we carefully edited the articles, formatted pictures and designed the first draft of the website. The publication of the first issue was an important mile stone for us; and strong sense of achievement was felt by the team for a job well done. At the time I naively felt that in a few years we would be rubbing shoulders with the likes of Nature and Science.

It quickly became apparent that the path to greatness is not a smooth one; we have had to weather all kinds of problems, with hindsight it would be easy to say that things could have been done better, blame could be assigned and frustrations vented. Yet this would be missing the bigger picture that the Young Scientists Journal still exists. Not only does it exist but it has grown from a small group at The King’s School Canterbury, to a multinational group of like-minded individuals all striving towards a common goal. We have survived because the core idea survives, even during the darkest days when everything looked like it might fall apart; I never questioned the core principle of connecting young scientists around the world.

The Young Scientists Journal has yet to become the one of the great journals of science, but it still has great potential, many great ideas take years to become a reality, and it is only though dedication and careful nurturing that we will achieve our goals. I would like to thank all the people who have stuck by the Young Scientists Journal over the years, all the authors, editors, advisors and especially Ghazwan and Christina, for we could not have come this far without you.

As I turned 21 this year, I must hand over the reins of the Young Scientists Journal to a younger generation; I have great confidence in Pamela Barraza Flores, and I wish her and the team the best of luck as they take the Young Scientists Journal to new heights. They have however, not got rid of me completely as I will now join the International Advisory Board and continue to assist the editorial team in any way I can.

If you have had the perseverance to read this far, I would like to reward you with a pearl of wisdom shared with me at the beginning of my journey with the Young Scientist Journal. “Nobody is truly old, while they are still doing research.” I have yet to amass the necessary years to vouch for this personally, but from all the fascinating people I have met on my journey so far, there is certainly a correlation between people who love their research and love life. I am now perusing a career in science, and if you are reading this you may be considering it to. I would strongly encourage you to stick with it, it may not always be easy, but the first time you discover something that nobody has ever found before, you will understand what all the effort is for.

Malcolm Morgan Editor DOI: 10.4103/0974-6102.68731

Young Scientists Journal | 2010 | Issue 8

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Nanotechnology special issue editor's note Science is constantly evolving, but few fields are as wide spread, or exponentially rapidly evolving as nanotechnology. The ability to recreate the world using atom sized particles harbors so much potential.

It’s an exciting field that branches many fields, and affects everyone. Most people have heard of the term nanotechnology, but few really know what it means. In this issue, we hope to enlighten and teach you about what it is, what it does, and where you can find it.

This collection of young researchers, world renowned experts, and many of explanations and definitions will hopefully allow you to open your eyes and see the power of the small. Nanotechnology caught my attention about five years ago, and I’ve been doing various research projects on it since, ranging from toxic effects of certain nanoparticles, to the toxicity of consumer products containing silver nanoparticles, to my current research using nanotechnology to target and kill cancer cells.

Working on this issue has been amazing. I’ve learned so much; not only about nanotechnology applications I didn’t even know existed, but also the editing process, communicating with authors, and the publication process. The authors, editors, advisors and other collaborators have been so kind,

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efficient and helpful, a real pleasure to work with.

I would especially like to thank Dr. Ghazwan Butrous for offering me this opportunity, and I’d like to give a shout out to my family, who have been so supportive of me throughout this whole process.

So, without anymore procrastination (I’ve done plenty of that while editing), I invite you to immerse yourself in nanotechnology, and find out the big news on the science of the small.

Sincerely,

Muna Oli

Special Nanotechnology issue Editor age: 17 years Eastside High School, 1201 Southeast 43rd Street Gainesville, FL 32641-7698 DOI: 10.4103/0974-6102.68732

Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Introduction to nanotechnology

Muna Oli Eastside High School, Email: munaoli92@gmail.com DOI:10.4103/0974-6102.68733

Nanotechnology- The word alone is enough to make anyone scratch their head. Science of the small? How does that work? Why use nanotechnology? Are they saying that smaller is better? These are all questions that arise when talking about nanotechnology. As this field becomes more popular and useful in science, the understanding seems to be getting more and more complex, and every time a solution is found, 20 other questions appear. But, let’s start with what nanotechnology actually is [Figure 1].

“Nanotechnology is the understanding and control of matter at dimensions between approximately 1 and 100 nanometers, where unique phenomena enable novel applications. Encompassing nanoscale science, engineering, and technology, nanotechnology involves imaging, measuring, modeling, and manipulating matter at this length scale.” (nano.gov).

Basically, this means that nanotechnology is the science of the small…..the really small. By “cutting” elements down into nanometer size pieces, they

Figure 1: The scale of things- Examples of both natural and manmade items, and how their size measures up on the scale of things

Young Scientists Journal | 2010 | Issue 8

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become nanomaterial. A nanometer is one billionth of a meter, or about 1/80,000th the thickness of a human hair (Lane).

Why do we want to make extremely small nanomaterial? At the smaller scale, some elemental qualities are extremely different. Take silver for example. As a large piece of metal we use it for jewelry or welding. However, when it’s reduced to a nanoparticle form, the properties completely change. The silver nanoparticles become extremely antibacterial. Any element that’s reduced changes both chemical and physical properties. One of the main changes is surface area. The surface area of a nanoparticle is much higher than of a large piece of the same material. This allows for more interaction and contact with anything it is around.

Its charge, zeta potential and structure can also change. By using specific reduction methods and chemicals, you can make nanomaterials into numerous shapes including rods, spheres and pyramids [Figure 2]. Different sizes and shapes change not only the chemical properties, but also the physical properties.

Gold changes color depending on the size. As a large bar of gold, it’s gold. As you decrease the size, it becomes blue, purple, and at extremely small sizes it’s red. Because of its remarkable properties,

nanomaterials become an ideal candidate for an array of applications, in many different fields. Currently you can find nanoproducts in everything from socks, to hospital supplies, computers and even medicine.

Each field has different uses for the nanomaterials, and the products and discoveries that can be made with them are endless.We will next discuss the variety of uses for nanomaterial.

Medical The medical field has a very large variety of needs for nanomaterials. Everything from keeping equipment sterile to drug delivery systems and even potential diagnostics/cancer treatments can be accomplished through nanotechnologies. Silver nanoparticles are used for sterility. A large problem at hospitals is secondary infections from dirty medical instruments or equipment. Covering the objects with a layer of silver nanorods has solved this problem. With its antibacterial properties and high surface area, it is an ideal coating on equipment to prevent bacterial contamination (http://www.azonano.com/news. asp?newsID=6068). Drug delivery systems and treatment options are still in the research stage, but some are very close to becoming commercially available. The concept of having a “capsule” to deliver drugs or other treatments has been a possibility for decades. But until now, it has not had much success.

A nanovehicle has become a promising idea. Due to the fact that humans can control the structure and properties of nanovehicles much more efficiently than liposomes, it seems that a more controlled, precise delivery system could be accomplished (http://www. ncbi.nlm.nih.gov/pmc/articles/PMC1949907/pdf/ nihms26595.pdf).

The engineering field is extremely large. Even so, many of the subfields have a very high interest in nanotechnology.

Engineering-solar panelsnanosurfaces

Figure 2: A chart of the various shapes nanomaterial can be made into (sciencedaily.com)

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Conserving energy and reusable energy is a very important topic for scientists, especially with all the other environmental problems we are facing.

Solar cells have been proven to be one of the more efficient ways to harvest solar energy. However, solar cells are still extremely costly. Titanium-dioxide nanoparticles have been found to double the Young Scientists Journal | 2010 | Issue 8

efficiency to convert ultraviolet light into energy. This is accomplished by adding a single wall of carbon nanotubes to the titanium dioxide nanoparticles.

Quantum dots have also been considered to be used for this purpose. (http://www.technologyreview.com/ energy/18259/?a=f).

Environment-filters By combining nano silver and nano carbon, researchers are discovering ways to make a filter that removes not only bacteria and fungi, but even contaminants and deodorizers. Aqulic, a company near London has already commercialized their version. Aqulic has designed a shower head that filters water to remove any contaminants using silver, carbon, copper and zinc. Copper and zinc result in oxidation reduction through a chemical reaction. This occurs by molecules changing into different elements using the transfer of electrons.

The nanosilver disables enzymes that allow microorganisms to take in oxygen, thus killing the microorganisms.. The nanocarbon is used as a filter and deodorizer (http://www.aqulic.com/ technology/aquilc_nanotechnology/). Aqulic and numerous other companies are paving the way for cleaner water. Once methods are established and cost of manufacturing can be reduced, this will be extremely useful in places in Africa and Asia, where clean water is scarce.

Consumer products With over $1billion to fund nanotechnology annually, nanotechnology has become the largest publicly funded science initiative since the space era (http://nanotechwire.com/news. asp?nid=1580&ntid=116&pg=74). This has led to the application of over 600 products containing nanoproducts that have been put on the market, and about three new products are being released each week (http://www.sciencedaily.com/ releases/2008/04/080424102505.htm). Everything from sunscreen, makeup, refrigerators have nanomaterials hidden in them. Consumers know nothing about the product, but put it straight in their basket to purchase. These nano products in the industry have multiple abilities that make them attractive to buyers. Self cleaning windows, anti-odor socks, antibacterial clothes and even clear sunscreen alleviates common problems we have in everyday life. The only problem that remains is the question of toxicity.

Problems

Nanofood

With the endless possibilities of nanotechnologies, it seems like the small will soon rule the world. However, are we sure that’s what we really want? Although there has been much hype with all the benefits of nanotechnology, it seems that no one is aware of the harmful effects of nanotechnology. Is it dangerous to work with things this small? Could it backfire? How do elements this small affect the environment? These are very important questions that need to be addressed before it’s too late.

Not only are scientists using nanotechnology to improve peoples’ lifestyle, they’re also trying to improve people’s health. “Nanofood” has become a field of interest to many scientists. These scientists hope to make guilty pleasures, such as doughnuts, into something much healthier. One of their plans is to “re-engineer” ingredients so the healthy ingredients release nutrients and the unhealthy ones pass straight through (http://www. sciencedaily.com/releases/2009/02/090214162746. htm). They are also looking into using nanoparticles as “nutraceuticals”, where vitamins or nutrients are transported in nano capsules into the body (http:// www.environmentalleader.com/2009/02/24/top-10uses-of-nanotechnology-in-food/).

As mentioned before, nanomaterials are very reactive due to their large surface area. While these are good for some applications, it can be extremely detrimental to the environment. Silver nanoparticles are a perfect example of this. The antimicrobial properties are extremely useful in the hospital settings, but if the nanoparticles were to leak out and end up in the environment, a decrease in bacteria could be detrimental to the ecosystem. Some environmental scientists have studied effects of various nanomaterials at different trophic levels. It has been shown that some nanoparticles can travel through a primate’s nose and settle in its brain (http://www.sciencedaily.com/ releases/2004/04/040407081930.htm). Because nanoparticles are so small, they can travel into the

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body through almost any route: skin, nose, orally, and no one knows quite what happens when they are inside the body.

These are just a few results that have been found.

Many other studies have shown no toxicity from nanomaterials at all. Either way, this is not meant to halt the nanotechnology industry. Most scientists agree that nanotechnology is a good thing to have.

They are just saying that regulations need to be stricter. As of right now, the Environmental Protection Agency (EPA), Food and Drug Administration (FDA), etc. don’t know what nanotechnology is, and therefore can’t regulate it. This can potentially be detrimental if products are released and problems occur due to a lack of understanding. The scienitifc community needs to understand the full spectrum of nanomaterials, both the pros and cons. Only then can they determine standards that need to be set, and rules that should be applied for safety.

Conclusion Nanotechnology-the small taking over the world. Is this good or bad? The real answer is that it hasn’t been decided yet, but most likely the good will win. The infinite number of uses that have been discovered, and the new uses that are found everyday point to a very promising future for them. Though much more research needs to be done, it seems that pros outweigh the cons. With this being said, a lot more research needs to be done to establish this fact before the consumer world is overwhelmed with nanomaterials.

Lane, Earl. “Experts Explore the Dilemma of Regulating Nanotech and Other New Technologies”. 18 May 2009. Ed. AAAS. AAAS. <http://www.aaas.org/news/ releases/2009/0519stpf_emerging_tech.shtml.

About the Author Muna Oli is a senior at Eastside High School in Florida. Aside from research, she enjoys photography, traveling, running and reading. She hopes to pursue a combined MD-PhD degree in the future.

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Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Interview with Dr. Andrew Maynard

Muna Oli Eastside High School, Email: munaoli92@gmail.com DOI:10.4103/0974-6102.68735

Dr. Andrew Maynard, a world expert in nanotechnology served as the chief scientific advisor for the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for scholars. He recently moved to Michigan and is currently the director of the University of Michigan Risk Science Center. He has served on various international committees which help oversee nanotechnology and is internationally recognized as a research leader and lecturer in the fields of aerosol characterization and the implications of nanotechnology to health and the environment. He received his Ph.D. at the University of Cambridge.

Follow him on his blog http://2020science.org/.

MO (Muna Oli): Hello, we’re here with Dr. Andrew Maynard to discuss nanotechnology for the YSJ. AM (Dr. Andrew Maynard): Thank you, Muna.

MO: Let’s start at the basics. How would you define nanotechnology (NT) in simple terms that everyone understands?

AM: This is an impossible task. I have to say that up front. Defining NT, one of the problems we have is there is no one definition, so I always joke that if you get 10 nanotechnologists (NTists) in the same room you will get at least 20 different definitions of NT. The one that I find most helpful though is probably one from the chemist Rick Smalley, famous for his work on carbon nanotubes and fullerenes, and he used to describe NT as the art and science of making stuff that does stuff at the nanometer scale. I like it because it’s vague. And one thing you begin to realize with NT is that there’s very little preciseness here; it’s somewhat avague and grey area. But it also captures this idea of art as well as science. So you’ve Young Scientists Journal | 2010 | Issue 8

got the science there; things behaving in an unusual way at the nanometer scale. But then you have the art of actually using it in very creative ways. So that’s the definition I usually use. But it still doesn’t really help people understand what all the fuss is about, so in addition to that I try and explain what is unusual and exciting about NT using thee terms: smallness, strangeness and sophistication.

Really briefly: smallness - we all know you can do things with small objects you can’t do with larger ones. Think of a car trying to get into a small space.

A small car can get in; a large car cannot. You can extend that idea to the nanoscale – a scale just a little larger than atoms. You can do stuff with it you can’t do at a larger scale. You can get to places you can’t get to with larger materials, which might be important for treating diseases for instance.

Then think about strangeness - this is where things get weird. With some materials their properties change very radically if you start creating them with structures at the nanometer scale. The best example is perhaps gold. So you take gold. We know that gold is gold in color and doesn’t corrode, which is why we use it in jewelry. But when you take that same gold and make it into particles at about 5nm in diameter, all of a sudden it changes color - it becomes red. And it becomes chemically highly reactive - very strange.

That’s an aspect about working at the nanoscale level that excites a lot of people. Because a lot of times they discover that they can do things they never thought they could do before. So that’s a real driver and important aspect of NT: working at this level, objects behave very differently.

The third term is sophistitication. And the analogy I 7

usually use here is that people are programmed to build things. Little kids pick up blocks from an early age. The older they get, the more sophisticated they get, and the smaller the building blocks they use. What we have now are the ultimate building blocks of nanotechnology - we can now work at the scale of atoms and molecules. So we have a high level of sophistication and that enables us to take existing technologies and make them better. Most things around us work pretty well, but don’t work as well as they could because of defects at the atom level. So this new sophistication helps us to fix those imperfections. But then we can also start to create brand new technologies with this NT. MO: How small exactly is a nanometer (nm)?

AM: The obvious answer is that a nm is just a little bit larger than a typical atom. It’s hard to get a clear intuitive understanding about how small we’re talking about. Even scientists find it hard. One analogy is comparing the size of an atom to something on the human scale such as a tennis ball. And it gives you an idea of what we’re talking about. So you look at the difference between one nm and a tennis ball. The difference in size between those is equivalent to the difference in size between the tennis ball compared to the size of the moon. So that’s the scale we’re spanning. It’s a huge difference in size.

MO: Why do people care about nanotechnology, where can consumers find it?

AM: If you’re talking about consumers (people buying stuff), we can find it in all sorts of products. Five years ago, the organization I worked with, the Woodrow Wilson Center Project on Emerging NT, asked this question, because it was hard to find information on where people were coming in contact with NT. So we started looking at consumer products. We developed this online database of consumer products (http:// www.nanotechproject.org/consumer) which now contains well over a 1000 entries and it contains everything from things like sunscreens and cosmetics to sporting goods like tennis rackets and golf clubs and even electronics, items associated with foods, etc. In almost any class of consumer products it seems, people have found out you can improve the products by using NT. To give you a sense of how broad it is, if you go out and buy sunscreen, there’s a good chance that there are nanoparticles of titanium oxide in it to make it work more effectively.

Or if you use an MP3 player – an iPod for instance 8

it does what it does because it is engineered at the nanoscale.

MO: Since it’s becoming more and more common in our everyday lives, do you think that people need to be wary of it?

AM: I don’t think people need to be wary of it, but it would be helpful if people were aware of it for a number of reasons. One is so people can make informed decisions, and there you have two aspects.

On the one hand you have technology that very clearly can make products better, so that’s something someone might want to take advantage of when buying a product. On the other hand, you have a very powerful new technology which could potentially cause harm if not used appropriately or wisely. People need to know what they’re buying, what’s in it, what the uncertainty surrounding the technology is and how that might be the cause of harm or benefit for the user. So yes I think that people need to be aware that NT is used in a large spectrum of consumer products but not necessarily wary.

MO: With all this talk on NT, being aware of it evokes the question is NT good or bad?

AM: NT is both good and bad. You can see there’s huge potential for good applications. Think of it in terms of having a greater ability to manipulate matter and materials. Think about the big challenges faced by society at the moment. Things like access to renewable energy for instance or clean technology where you’re not polluting the world. In each of these areas we could do a lot, but in every single case we run into problems where we cannot solve the challenges we face with conventional technologies.

We need new ideas and innovation. We can now see where we can innovate and extend our current abilities by engineering matters at the nanometer scale. That’s where NT is very exciting - helping solve important challenges. On the down side, we have to learn how to use it wisely and responsibly. Just like any other technology, it can cause harm - you have to learn how to use it so it doesn’t cause harm. So back to the original question - is NT good or bad?

NT has tremendous potential to be good as long as we get it right.

MO: You’ve been on a lot of international committees looking at regulating NT and how NT is used. Could you explain how the government and international organizations are regulating NT? Young Scientists Journal | 2010 | Issue 8

AM: One of the things we are trying to do is to work out what the challenges are ahead of time. Industry, regulators and other organizations are trying to work out how to get nanotechnology right from the get-go.

This has led to interesting conversations, initiatives, etc. but there are a lot of questions that are trying to be answered. There are a lot of international initiatives going on to work out some of the problems.

Representatives from many different organizations are trying to grapple with these issues. Big international standards organizations for instance, that develop consensus standards, have been looking at how they can develop standards for NT for responsible use.

Similarly, the Organization for Economic Cooperation and Development (OECD) which is an organization of first world governments, is enabling governments to work together to try to understand potential barriers to developing NT responsibly, how to overcome these barriers, how nanomaterials might cause harm, and how to test and ensure their safe use. There is close collaboration with both scientists developing NT and regulators to see how you can organize and develop it safely.

MO: There’s a lot of science fiction about NT taking over the world. A great example is Michael Crichton’s book ‘Prey’. How logical is the story? Is it possible?

Will NT take over the world?

AM: NT will not take over the world in terms of science fiction like ‘Prey’ where nanobots self-organize and take over. It makes for a great story but it is science fiction - not science fact.

MO: What is your opinion on NT, the future of it, and where do you think it is going to take us?

AM: This is a difficult question to answer. It is going in a hundred and one directions at once. The ability to manipulate matter at that fine, fine scale will lead us to some great advances. That is without question. The more effectively we can manipulate materials at the scale of molecules and atoms, the more effectively we craft and manipulate the world around us to do things the way we would like them to be done. We’re going to be seeing tremendous advances in that respect in terms of stronger and lighter materials, in terms of electronics and photonics. Then we are also getting into the world of biology - this is where it gets really interesting. People describe biology as nanotechnology that works - think of DNA and how the whole biological process works. But what we are finding now as people are getting more adept Young Scientists Journal | 2010 | Issue 8

at working with materials at the nanoscale, they are beginning to interface that with biology. And that’s where you have an explosive combination; people are beginning to integrate nanoscale materials with biological systems and are coming up with completely new and innovative technologies. That’s definitely something to watch in the future.

MO: How can NT make people’s life better?

AM: There are a number of things you can do with NT that we haven’t been able to do before. One is being able to use energy more wisely and to find better ways of utilizing energy. Think about the energy crisis. We would love to be able to harness the sun’s energy more effectively. We haven’t been able to do it well in the past. But what we’re seeing with NT is a whole suite of new technologies that allow us to do that better. One of the particularly exciting ones is printable electronics, where you can effectively create ink that contains NP and you can use something that looks similar to an inkjet printer and print electronics – including solar cells - on a variety of different surfaces.

So you potentially go from a fabrication plant that costs millions of dollars to a printer that costs only 100,000’s of dollars to make solar cells. And you can effectively use this technology to print solar cells onto a variety of surfaces, leading to cheap and versatile solar cells.

Another area is water purification. There are immense challenges in getting clean water to people who need it when they need it. There aren’t too many solutions for providing people with clean water. So we are trying to work at the molecular scale to get rid of the stuff we don’t want in the water such as dissolved salts or contaminants, and to do that with minimal energy input. This is an area where nanotechnology can have a tremendous impact.

A third area is disease treatment, especially treatment of cancer. Cancer has always been high on the radar, but we really haven’t made that much progress toward treating many cancers. What we have now, is a crude set of tools. When you look at something like chemotherapy, you may treat a tumor, which is a localized cluster of cancerous cells in the body, by flooding the whole organism with toxins, and just hope that the cancerous cells die before the rest of the body does. That’s very crude medicine. With NT, we can begin to target the cancer cells specifically.

We can create very small “machines” that can be programmed to do multiple things. So you can take 9

these particles and program them to seek out and attach to cancerous cells once they’re in the body, let physicians know when they are in place and use them to say something about local environment. And then you can program them to respond to an external signal - in most cases to destroy cancer cells on demand. Targeting cancer cells is a significant step for getting rid of the cancer cells without harming the rest of the body.

MO: What is the role of young people in NT? AM: One of the obvious roles is having the imagination to use this NT in novel ways. One thing we are seeing is where NT works best is where it breaks down boundaries between conventional ways of thinking.

We have a problem in science where people are locked in their disciplines. But in reality the exciting innovation happens at the interface between multiple fields. Not only between scientists, but when scientists start talking to social scientists, who start talking to economists, and others in different disciplines. In NT, we’re seeing the sparks where people cross those boundaries. The next generations of young people aren’t necessarily constrained by those specific discipline boundaries. So as long as they are not bound, and develop or maintain an interdisciplinary outlook, I think that will be very exciting. There is only so much you can do with older generations, but if the younger generation can do stuff differently, there we can establish a foothold for combining science and NT in a totally different way.

MO: To wrap up our conversation, do you have any last thoughts and opinions to share with our readers?

AM: NT is an area which such potential yet such complexity. It’s a rich area to get involved in and get excited about and can have a significant impact. It’s an area I would certainly encourage everyone to be aware of and to understand what the possibilities are, but also to approach NT with an open mind.

To see where the flaws are in people’s thinking and more importantly to be able to transcend the idea of NT, and to start thinking about how the world and things can be different if we engineer and manipulate things at the nanoscale level, working with atoms and molecules; that’s the number one thing I would say. I’d like to inspire people to think about how they can make the world a different and better place by working with this new finesse and technology.

But the other aspect of this is, don’t just get wrapped up in the science and technology, but also think about what the long term consequences are to society; to people; to themselves and to their families.

Consider the good consequences as well as the bad consequences. The reason I say this is that I think we’re seeing a change in society where we just can’t assume that, if we get science right, everything else will be right. We have to think about things in more of a holistic way. And this is just such a wonderful opportunity and a wonderful time for young people to begin to think in this alternative way, to think about how science can interface with society and how everything meshes together.

MO: Thank you Dr. Maynard for your insightful discussion and recommendations about the broad subject of NT.

About the Author Muna Oli is a senior at Eastside High School in Florida. Aside from research, she enjoys photography, traveling, running and reading. She hopes to pursue a combined MD-PhD degree in the future.

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Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Sunscreen: A catch-22

Cole Blum and Samantha Larsen University of Pennsylvania, Class of 2014, (Attended Long Beach High School in Long Beach), E-mail: coleblum@msn.com University of Pittsburgh, Class of 2013, (Attended Long Beach High School in Long Beach, N.Y.), Email: sbl12@pitt.edu DOI:10.4103/0974-6102.68738

In classical times, wealthy people fortunate enough to stay indoors were pale and proud of it, while poor farmers and workers who toiled outdoors developed dark complexions from the sun’s rays. People thus aspired to stay out of the sun and lived in a world relatively free of skin cancer. Yet today, we are constantly looking to become darker and acquire that “healthy glow” by increasing our exposure to the sun. This new trend has unfortunately arisen just as an already expanding hole threatens the ozone layer, and the prevalence of skin cancer is on the rise. However, we are confident that sunscreens can protect us from both the harmful short-term and long-term effects of the sun. Due to this sense of confidence we believe that as long as we repeatedly apply oils and lotions to our skin, the amount of time we spend in the sun is irrelevant. The question is if sunscreens in their present form are commonly accepted as the ultimate solution to the hazards of the sun, then why are more than one million new cases of skin cancer being reported each year?

Ultraviolet (UV) radiation is the type of solar radiation that is most threatening to our health.

The most energetic UV radiation, UVC (100-280 nm wavelength), is almost entirely absorbed by the ozone layer and does not pose a threat to our skin.

However, UVB and UVA radiation can penetrate the ozone layer and reach Earth’s surface. UVB radiation (280-320 nm) is dangerous and carcinogenic due to its ability to directly mutate DNA. The least energetic UV radiation, UVA (320-400 nm), has also recently been recognized as dangerous due to its ability to indirectly damage our DNA via free radical-induced oxidation. [1] The high-energy UV radiation that reaches us can ultimately cause visible sunburn of Young Scientists Journal | 2010 | Issue 8

the skin, erythema, photoaging, immunosupression, and photocarcinogenesis, among other adverse effects.[2] To prevent these adverse effects, sunscreens contain light-absorbing organic filters as well as lightreflecting and light-scattering inorganic nanoparticle filters. A combination of these two types of UV filters is necessary for broad-spectrum protection over the UVB and UVA ranges. However, these same protective compounds have been found to be either photoactive or photocatalytic when exposed to UV light with the potential to cause damage to our skin comparable to UV radiation alone. Each sunscreen product is thus a mixture of filters with known benefits and corresponding negative side effects.

The majority of organic UVB and UVA filters have their limitations. Many are photolabile: they can break down into a variety of harmful metabolites when they absorb UV light. PABA (para-aminobenzoic acid) was patented in 1943 and is known as a highly effective UVB absorber. However, it has been found to be photocarcinogenic.[3] Padimate A has been eliminated for its phototoxicity; oxybenzone is a broad-spectrum filter but is photolabile; butyl methoxydibenzoylmethane loses its photoprotection in a short period of time; octinoxate can improve the photostability of certain other filters but is a relatively weak absorber itself.[3] Recent studies have found that oxybenzones, cinnamates, salicylates, and their metabolites can significantly penetrate the skin. High levels of them were also detected in the blood and urine of human volunteer subjects.[4] The ability of these particles to enter our bloodstreams is particularly worrisome since they may have the potential to disrupt many bodily functions. The long-term effects of the penetration and breakdown 11

The main inorganic or physical filters, titanium dioxide (TiO2) and zinc oxide (ZnO), are photostable substances and depending on their size offer protection over a wide UV range. However, they are known photocatalysts: This means that UV light excites TiO2 surface electrons to jump to higher energy levels leaving behind unstable positive holes in the TiO2 lattice. Both the excited electrons and positive holes will react with nearby oxygen and hydrogen compounds (O2, OH , etc.) to produce highly reactive free radical compounds including the superoxide anion radical (O2ď&#x201A;§-) and the hydroxyl radical (ď&#x201A;§ OH) 1. When in contact with our skin, these radicals can oxidize and reduce compounds including DNA resulting in significant mutagenesis.

In sunscreens, they can also interact with organic filters such as the benzoates to produce toxic acidic products.[1] The potential solutions to this free radical damage include incorporating antioxidants such as carotenoids and vitamin C into sunscreens and coating inorganic filter particles with dimethicone, silica, or relatively photostable organic polymers.[4,5] Polymer coatings are translucent to allow UV radiation to reach the oxide particles so they can act as sunscreens.

The coatings are also able to trap electrons in their structure and suppress the production and escape of free radicals from the particle surface.[5] Photocatalytic polymerization is a novel method for synthesizing polymer coatings on TiO2 nanoparticles.

This method takes advantage of the photocatalytic property of TiO 2 to induce polymerization and bond the polymer to the particle surface to form a surrounding protective lattice.

Another aspect of the sunscreen dilemma is our desire for an aesthetically pleasing product that can be rubbed onto the skin until it becomes translucent.

In the past, products with TiO2 and ZnO mainly contained these compounds at the microparticle size (~1 Âľm) and did not blend in with our skin.

Recently, we have begun to incorporate much smaller TiO2 and ZnO nanoparticles with diameters as small as 5-100 nm into sunscreens, because they become translucent when rubbed onto the skin. However, we have done this with no regard to the potential health risks. This societal trend unfortunately exacerbates the photocatalytic hazards of these particles since they have a significantly larger 12

surface area to volume ratio for more photocatalysis [Figure 1]. In combination with these higher levels of photocatalysis, nanoparticles have a host of other characteristics that are intensified from their microparticle cousins. For one, their size allows them to penetrate the skin layer and individual skin cells more easily [Figure 2]. Nanotechnology research is new and ongoing, which means that there are little to no regulations on human exposure to these miniscule particles. Their long-term risks are not yet known and it is troubling that they are regularly incorporated into current commercial sunscreen products.

The effects of TiO 2 nanoparticle photocatalysis on individual molecules have been studied in several settings. Guanosine triphosphate (GTP), a common biomolecule, showed significant oxidative degradation over two hours when exposed to a suspension of TiO2 nanoparticles and UVA/UVB radiation [Figure 1]. Research suggests that a polymer coating in this situation has the ability to inhibit photocatalytic GTP degradation by trapping free radicals [Figure 3].

The adverse effects of both the small size and photocatalytic activity of TiO2 nanoparticles on the skin have also been explored in laboratory research.

Human dermal fibroblasts (a type of skin cell) were studied and incubated with TiO2 nanoparticles at concentrations well below the legal limit allowed

Fraction of initial GTP concentration (%)

of these photolabile compounds found within sunscreens have yet to be determined with certainty.

100

80

60 GTP 40

GTP + 0.16 mg/mL TiO2 microparticles GTP + 0.16 mg/mL TiO2 nanoparticles

20 0

0.5

1

1.5

2

300-nm UV irradiation time (hours)

Figure 1: Degradation of guanosine triphosphate (GTP) exposed to titanium dioxide particles and UV radiation. GTP is a common biomolecule used as a test subject to model the effects of TiO 2 photocatalysis. Microparticles cause 30% GTP degradation over two hours; <25 nm nanoparticles cause 50% GTP degradation over two hours due to greater surface area for photocatalysis. TiO2 concentration of 0.16 mg/ml is well below the levels found in most sunscreens (research by authors)

Young Scientists Journal | 2010 | Issue 8

Figure 2: Titanium dioxide nanoparticles penetrate the cell membrane of a human dermal fibroblast. <25 nm TiO2 nanoparticles are able to infiltrate the membranes of human dermal fibroblasts, a type of skin cell, after 48 hours of cell incubation (research by authors)

Figure 4: Effect of titanium dioxide nanoparticle exposure on human dermal fibroblasts. Cell number after 3 days of incubation with TiO2 nanoparticles at 0.1, 0.3 and 0.5 mgď&#x201A;&#x17E;mL-1. Increasing concentration of TiO2 nanoparticles results in greater cell death (From Pan, Z. et al., 2008)

coated nanoparticles were as healthy as control cells that were not exposed to nanoparticles [Figure 5]. These studies illustrate several harmful effects of TiO2 nanoparticles commonly found in sunscreens and have given us some insight into polymer coating as a potential solution.

Figure 3: Generalized model of the protective function of coating titanium dioxide nanoparticles with a biocompatible polymer. UV radiation stimulates photocatalysis on the surface of TiO2 particles, leading to the production of highly oxidative free radicals that can damage our DNA. On left, uncoated TiO2 photocatalysis leads to the destruction of GTP. On right, the polymer coating prevents free radicals from escaping the TiO2 surface and damaging GTP

in sunscreen products. In research by Zhi Pan and others, fibroblasts exposed to the nanoparticles exhibited decreased cell counts, decreased viability, and high levels of nanoparticles within the cell vesicles [Figure 4]. [6] A sonochemical polymerization method, which has similar results to photocatalytic polymerization, was then used to graft a polymer coating onto the TiO 2 nanoparticles.

Consequently, fibroblasts exposed to the polymerYoung Scientists Journal | 2010 | Issue 8

Perhaps the most disturbing fact in our search for the ultimate sunscreen is that we may never be able to test a product for long enough to rule out every potential side effect. Solar UV radiation alone and the photoactive and photolabile components of sunscreens are capable of destroying our genetic material and killing our skin cells, resulting in sunburn and carcinogenesis. However, carcinogenesis is not a result of the visible sunburn and does not result from the complete destruction of our genetic material.

Carcinogenesis does occur when UV radiation damages our cells and DNA partially but not entirely, allowing the mutations to proliferate into future generations of deep skin cells. We can measure and predict these mutations on the immediate scale, but it is logistically and ethically impossible to accurately track the real long-term effects of UV radiation on skin tissue. Furthermore, we cannot create a study to account for every human variable (age, race, gender, complexion, exposure time, geographic location, etc.) while successfully tracking the proliferation of mutations over time.

After years of sunscreen research, we have many answers, but we are left with even more questions.

We have learned that UVA radiation, which was 13

Figure 5: Effect of polymer coated titanium dioxide nanoparticle exposure on human dermal fibroblasts. Confocal images of human dermal fibroblast cells incubated with polymer coated TiO2 nanoparticles at 0.4 and 0.8 mg/mL. Cells incubated with coated nanoparticles are just as healthy as control cells, confirming that polymer coating can prevent nanoparticle penetration of the cell membrane and cell death

once thought to be less damaging than UVB, has its own indirect mechanisms of damaging our DNA. “Broad-spectrum” protection now requires both organic and inorganic filters to block out all forms of significant UV radiation. We have also discovered that UV radiation can stimulate the breakdown of the organic filters and photocatalytic reactions in the inorganic filters in sunscreens. We have attempted to solve these problems with additives and alternatives such as polymer coating. Yet it is impossible to determine if these solutions will protect us from long-term UV exposure and will prevent the gradual proliferation of mutations that lead to deadly skin cancers. We do not know every precise mechanism in photocarcinogenesis and cannot confidently determine what truly causes skin cancer in an individual. So what then is the scientist’s advice for the best protection from UV radiation? Wear a hat.

Acknowledgments The authors would like to thank Dr. Vladimir Zaitsev and Lourdes Collazo, their mentors at Stony Brook University, and Linda Trusz, their science research director at Long

Beach High School, for their time and dedication to the research. They would also like to recognize their parents who provided support and enthusiasm during the entire process.

References 1. Brezova V, Gabcova S, Dana D, Stasko A. Reactive oxygen species produced upon photoexcitation of sunscreens containing titanium dioxide (an EPR study). J Photochem Photobiol 2005;79:121-34.

2. Gil EM, Kim TH. UV-induced immune suppression and sunscreen.

Photodermal Photoimmunal Photomed 2000;16:101-10.

3. Kullavanijaya P, Lim HW. Photoprotection. J Am Acad Dermatol 2005;52:937-958.

4. Sarveiya V, Risk S, Benson HA. Liquid chromatographic assay for common sunscreen agents: Application to in vivo assessment of skin penetration and systematic absorption in human volunteers. J Chromatogr 2003;803:225-31.

5. Lee WA, Pernodet N, Li B, Lin CH, Hatchwell E, Rafailovich MH. Multicomponent polymer coating to block photocatalytic activity of TiO2 nanoparticles. Royal Soc Chem 2007;7:4815-7.

6. Pan Z, Lee W, Slutsky L, Clark RA, Pernodet N, Rafailovich MH. Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells. Small 2008;5:511-20.

About the Authors Cole Blum and Samantha Larsen: University of Pennsylvania, Class of 2014, (Attended Long Beach High School in Long Beach, N.Y), Email: coleblum@msn.com. University of Pittsburgh, Class of 2013, (Attended Long Beach High School in Long Beach, N.Y), Email: sbl12@pitt.edu

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Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Synthetic, 'inorganic DNA' as a means to high-density molecular electronics Matthew Kapelewski Penn State University, Email: mtk180@psu.edu DOI:10.4103/0974-6102.68739

Introduction Molecular electronics is one of the fastest growing fields in modern science, and with good reason.

Computers have become a part of the everyday lives of a majority of people around the globe, largely in part to the integrated circuit, seen in Figure 1. They have completely changed the way in which we communicate, do business, and learn. Constant advancement in such a field is highly desirable, as the benefits to such advancement abound. Unfortunately, the current silicon-based economy in the computer industry can only go so far, as there is a lower limit to the size of silicon based parts. Quantum computing and molecular assemblies provide an opportunity to surpass this silicon-imposed limit. Moore’s Law is a description of a trend exhibited in computers since approximately 1965; this “law” states that the number of transistors that can be placed on an integrated circuit doubles every two years. Computer chip makers wishing to push Moore’s Law to and beyond the current limits need to have the ability to shrink components within their systems to the molecular and atomic level.

A novel approach to doing exactly this is by using individual molecules as current-carrying structures.

While in its infancy relative to other fields of science, molecular computing and single-molecule computer components are becoming a more realistic way to accomplish the miniaturization necessary for advancement. Molecules such as carbon nanotubes, seen in Figure 2, and Mo6S3I6 [1] nanotubes promise to reduce the cost of these computer chips extensively if they can effectively be implemented as molecular computational tools. Such low-cost, Young Scientists Journal | 2010 | Issue 8

molecular computer chips would have far-reaching applications, from increasing computing power to better serve the ever-expanding infrastructure of the internet, to providing ultra-affordable technology to impoverished people around the globe.

Proposed “idna” architecture and formation of such structures through self-assembly Paramount to this reduction in size is control of the formation of such structures. Self-assembly is an attractive option, as mass quantities of molecular wires can easily and quickly be synthesized by virtue of the fact that the molecules will assemble themselves in the desired arrangement. The prototypical molecule to look at as a basis for self-assembly is DNA, which has obviously been succeeding as a self-assembling structure since the dawn of life. Using DNA as a model, we have begun synthesizing novel compounds that are based, in theory, on the structure of DNA. Stoichiometric coordination of metal atoms to the pyridine-based ligands in Figure 3 can be used in place of hydrogen bonding in DNA to facilitate the self-assembly of this “inorganic DNA,” or “iDNA.” The structure of some basic types of these self-assembling molecules can be seen in Figure 4. In essence, we have a chain of “base pairs” that each have a certain number of nitrogens in them in pyridine rings, which can use their lone pair of electrons to chelate to a metal atom placed nearby. An example of this chelation can be seen in Figure 5.

The pyridine based ligands are self-complementary; 15

a Figure 1: An example of an integrated circuit used in a computer

b

Figure 4: Alignment of ligands with (a) tetracoordinate and (b) hexacoordinate metals.2

Figure 2: Different types of carbon nanotubes Figure 5: Terpyridine and pyridine ligands bonding to a copper atom

1

2

3

Figure 3: Pyridine (1), bipyridine (2), and terpyridine (3) ligands

this creates the self-assembly that is a critical part of this process. The structure of the ligands is that the pyridine and pyridine derivatives bind to a polyamide backbone. These monomers are then linked through deprotection and coupling to form di- and tripeptides, and longer oligomers.

As an example, a pyridine-bipyridine-terpyridine tripeptide could be formed. This would bind in an antiparallel, self-complementary arrangement 16

to produce the complex.[2] This occurs because each of the three metal atoms that are being used has four coordination sites where the lone pair of electrons from a nitrogen atom can bind. The single pair in the pyridine will line up with the three pairs in the terpyridine to give four total pairs, while the two bipyridine ligands will align to give another four electron pairs. This is a system in which selfcomplementary chains of pyridine-based ligands form molecular wires, which can function as redox systems for multi-electron transport. Due to the structure of the molecules, electrons could hop from metal atom to metal atom down the center of the wire, effectively transporting them in one-dimension and creating a flow of electrons that constitutes a current.

Young Scientists Journal | 2010 | Issue 8

separate most of the isomers that are created, since they will have varying amounts of charged metal atoms in them. Analysis of the separation of the products of a synthesis can possibly assist in the creation of a more controlled synthesis of the desired isomer (in most cases, the antiparallel, controlled length chain).

Figure 6: Formation of several isomers from basic iDNA building blocks[3]

Separation of isomers This setup of a molecular wire using electron hopping to carry current is a long-term goal, however. At this point, the project is still in its early stages, as the systems being studied are primarily di- and tripeptide systems. We have been synthesizing these self-complementary structures and examining their properties. One of the most prominent problems with the synthesis of these simple iDNA structures, as well as longer molecular wires based on this architecture, are possible isomers that can result from variations in the synthesis. Such isomers are important because they directly affect the physical and electronic properties of the compounds. It can be seen in Figure 6, that even the simplest building blocks, such as the two bipyridine dimers and tetracoordinate metal atom, can isomerize in multiple ways. The bottom product in Figure 6 could terminate at any length, yielding many different compounds. The difference between the other two products in Figure 6 is subtler, as the only difference is in the parallel or antiparallel arrangement of the strands. This isomerization problem manifests itself even more as the complexity of the system increases with the addition of longer strands or heterometallic complexes. Since only certain isomers of the complex are desirable, it is important to have the ability to separate these compounds in some way. Using a specific solvent system (5 ACN:2 H2O:1 KNO3) in a high performance liquid chromatography (HPLC) system, we are able to separate these compounds based on charge in a reverse-phase silica column. This process is able to

Conclusion The need for novel ways to increase computing speed is crucial to the advancement of the computing industry beyond silicon. Self-assembling molecular wires are one possible solution to achieve this.

Although time is needed, this research in molecular, self-assembling wires is making strides toward achieving this goal and thus opening up new possibilities for computing power, which would benefit people around the globe.

Acknowledgments I would like to thank Dr. Mary Beth Williams (mbw@chem. psu.edu) for her guidance and oversight of this entire project. (Group website: http://research.chem.psu.edu/ mbwgroup/). Also thanks to Matt Coppock, Joy Gallagher, and Carl Myers for their helpful insight. Thanks to my parents for being there for me for the past 19 years! I am grateful for generous financial support from the National Science Foundation (CHE - 0718373) and U.S. Department of Energy (DE-FG02-08ER15986).

References 1. VrbaniÄ&#x2021; D, RemĹĄkar M, Jesih A, Mrzel A, Umek P, Ponikvar M, et al. Air-stable monodispersed Mo6S3I6 nanowires. Nanotech 2004;15:635-8.

2. Gilmartin BP, Ohr K, McLaughlin RL, Koerner R, Williams ME. Artificial oligopeptide scaffolds for stoichiometric metal binding.

J Am Chem Soc 2005;127:9546-55.

3. Myers CP, Gilmartin BP, Williams ME. AminoethylglycineFunctionalizaed Ru(bpy)32+ with pendant bipyridines selfassemble multimetallic complexes by Cu and Zn coordination.

Inorg Chem 2008;47:6738-47.

About the Author Matthew Kapelewski, Penn State University, Email: mtk180@psu.edu

Young Scientists Journal | 2010 | Issue 8

17

Nanotechnology

Aptamer conjugated gold nanorods for targeted nanothermal radiation of Glioblastoma cancer cells (A novel selective targeted approach to cancer treatment) Muna Oli Eastside High School, Email: munaoli92@gmail.com DOI:10.4103/0974-6102.68740

ABSTRACT

Selectively targeted nanothermal radiation differs from traditional cancer treatment in two main ways: by targeting specific cancer cells, and localizing treatment. This research focused on the ability to selectively target cancer cells (and cancer stem cells) by targeting the cancer with an aptamer/gold nanorod conjugate. This nano-bio molecule emits heat when excited by a harmless near infrared laser and kills cancer cells, allowing for a selective and targeted treatment. My research showed that selective targeting and killing of aptamer conjugated gold nanorods was possible to kill Glioblastoma cancer cells.

Introduction The number of cancer patients who are currently living with a diagnosed primary or metastatic brain tumors (or gliomas) in the United States is 350,000.

Mortality rate due to gliomas is almost 100%, with most patients living less than a year.[1] About 50% of gliomas are glioblastoma multiforme (GBM) tumors, one of the most dangerous and aggressive types of tumors.[2] As a prominent example, Senator Edward Kennedy died on August 27, 2009 of a Glioblastoma tumor, 15 months after the initial diagnosis.

As the fight to cure cancer continues, more treatments have become available, and mortality rates have declined for some forms of cancer.

However, brain cancer mortality has declined only by 18

5% in the last 50 years.[3] Despite substantial research efforts over the years, surgery and systemic therapy are still the most common treatments used. These treatments are painful, expensive, and have many side effects such as burns, hair loss, fatigue, nausea, and immunosuppression (http://www.cancer.gov/).

Furthermore, they donâ&#x20AC;&#x2122;t target cancer stem cells (CSC). The â&#x20AC;&#x153;cancer stem cell hypothesisâ&#x20AC;? suggests that a tumor harbors multiple types of cells, including cancer stem cells. Although they are not actual stem cells, they mimic many stem cell properties; they are resistant to traditional cancer treatments and also exhibit slow division rate.[4,5] CSC are resilient, and difficult to kill, especially because most cancer drugs only target fast dividing cells. The resistance of CSCs against traditional treatments has led scientists to believe that surviving CSCs are the cause of cancer relapses and metastasis.

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A second reason for the low efficacy and severe side effects of conventional cancer treatments is that they kill the cancer and healthy cells that exhibit rapid cell division rates (e.g., bone marrow, blood cells, hair follicles, GI cells). Treatment efficacy could be maximized and consequently, side effects minimized if treatment specifically targets cancer cells and CSC with minimal adverse effects on healthy cells using aptamers. Aptamers are oligonucleic acid molecules with a complex 3D structure that bind to a specific target molecule and can recognize virtually any class of target molecules with high affinity and specificity.[6-10] In lay language, aptamers are human synthesized strands of DNA and have the ability to target any specific cell marker.

Nanosized particles have chemical and physical properties different from their larger counterparts.

[11] Gold nanorods (GNR) in particular have the potential to kill cancer cells using hyperthermia.

Hyperthermia is the old practice of using heat to kill cancer cells though it is difficult to heat a tumor without damaging nearby tissues.[12] In addition to hyperthermia killing cancer cells directly, studies have shown it sensitizes cancer cells (which are susceptible to heat) for other systemic therapies.

[13] When GNRs are excited by a low energy Near Infrared (NIR) laser, they heat up a large area. If a specifically targeted aptamer is bound to the GNR, the aptamer conjugated gold nanorod (A-GNR) is delivered straight to the cancer cell. Upon NIR laser treatment, the GNR releases heat causing cancer cells to die, and leaving surrounding healthy cells unaffected.

The overall goal of my project was to develop a framework for treating tumors and metastasis of cancer (Specifically GBM) that addresses the shortcomings of current therapy. My specific objectives were to: (1) Synthesize and characterize gold nanorods; (2) Evaluate effects of gold nanorods (GNR) and laser on cells, and determine safe concentration of GNR; (3) To determine if nanothermal radiation is capable of killing tumor cells; (4) To investigate if aptamers can be used to target GNR to specific cells; and (5) Investigate efficacy of targeted nanothermal radiation to kill targeted tumor cells. My project is designed to target and personalize cancer treatment and to kill fast and slow growing cells by using aptamerconjugated GNRs as part of the cancer treatment scheme [Figure 1]. I hypothesized that the use of the selectively targeted nanothermal radiation using an aptamer conjugated gold nanorod would lead to a Young Scientists Journal | 2010 | Issue 8

Figure 1: An overview of the aim of the project. Conjugate aptamers and antibodies, expose to cells, heat with a NIR last, and selectively kill cancer cells

superior treatment option that targets cancer cells, and does not affect healthy cells.

Methods This experiment required numerous materials and methods, some of which are quite complicated.

I have not included them in this paper due to complexity and length.

Results Gold nanorods synthesis and characterization I synthesized 20nm x 50nm gold nanorods and coated them either with PEG or left them in CTAB, the latter is necessary for covalent modification of the nanorods like conjugation to the aptamers.

I characterized the GNR by TEM and UV VIS spectroscopy TEM pictures demonstrate uniformity and even dispersion of nanorods and confirms their size [Figure 2a]. UV VIS analysis showed that the GNR absorbance maximum was at 736nm [Figure 2b]. This analysis was performed every few months to ensure that the GNR did not aggregate or change in size or shape.

GNR emission of heat when exposed to a NIR laser Gold nanorods emission of heat when exposed to a near infrared (NIR) laser was measured using an agar phantom setup.[14] As can be seen from Figure 3, the temperature of agar was measured over 900 seconds (15min). The graph shows the steady temperature 19

a

a

b

Figure 2: (a) A transmission electron microscope (TEM) image of freshly synthesized gold nanorods was taken. The dimenstions of these nanorods were approximately 20nm x 50nm. It also ensures uniformity and even dispersion of GNR. (b) UV-VIS analysis of GNRs. Optical density of nanorods was measured from 400nm-900nm. The peak resonance absorption band was at 736, clearly in the near infrared (NIR) range

b Figure 4: Effect of gold nanorods at various concentrations on neuropshere growth. (a) Light microscope pictures of neurospheres after 7 days of exposure to cells. The only concentration that did not have any significant effect on the cells was at 1:1,000. All higher concentrations of GNR killed cells. IC50 was at the concentration 1:100, meaning that concentration killed 50% of the cells. (b) This graph shows neurosphere count after 7 days of exposure to gold nanorods. The toxic effect of higher concentrations is great enough to be considered statistically significant correlates identically with the graph. This experiment was done in replicates of three, and the data are displayed as mean ± SEM and p<0.01

Figure 3: Comparison of temperature (˚C) increase in 15min of surrounding agar (surface tissue) vs. agar with gold nanorods (Deep tissue). After about 2 minutes a temperature difference between deep tissue and surface tissue is seen. By the end of 15min, deep tissue is still increasing while surface tissue plateaued

increase over 15 minutes. It is evident that the agar with the GNR (deep tissue) heats up more rapidly than the surrounding agar (surface tissue). After 15 minutes there is an increase of over 2˚C by the deep tissue, and about 1˚C increase by surface tissue. All experiments were performed in at least triplicates.

GBM cells exposure to GNR A titration series was performed to determine a safe concentration of GNR to cells without being lethal or toxic to the cells. Figures 4 a and b show a visual and statistical representation of the effects of various gold concentrations on cell growth.

Figure 4A shows the drastic decrease in number of neurospheres at a concentration of 1:100 or higher.

Statistical significance and IC 50 was measured using the program Excel. 4B shows statistical data of the cell counts. The stars represent a statistically significant decrease in number of cells due to the concentration of GNR. It can be seen that at any concentration 1:100 and higher, the gold has obvious 20

and severe effects of the cells. It was therefore determined the concentration of 1:1,000 PEGylated GNR should be used. Figure 5 uses a nuclear stain (DAPI) and an apoptotic marker (Annexin V) to look at the health of cells when exposed to the predetermined concentration of GNR to ensure cell health was maintained.

Exposing cells to laser Figure 6A shows the long term effects from the laser by performing a neurosphere assay and counting the number of neurospheres seven days after exposure.

The cells with the laser had about twice as many neurospheres as the control cells. Figure 6b shows almost identical health in populations between control cells and cells that were exposed to the laser, indicating that the laser did not harm the cells.

Cells exposed to GNR and laser Seen from Figure 7, there was an obvious difference between all the samples except the one where cells were exposed to gold and the laser. It should be noted that the left graph (annexin V) shows an obvious decrease in cells. While the intensity of the cells with the gold and laser are not at a higher intensity, the percent of max cells, also known as the number of cells counted was about a fourth of the original number [Figure 7]. This shows that the method Young Scientists Journal | 2010 | Issue 8

kills cells extremely quickly. Dead cells decay, and are spun out during procedures preceding FACS analysis, which accounts for the low numbers.

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Figure 5: Flow Cytometry using Florescent Activated Cell Sorting was used to collect data on percent of cells that showed markers of apoptosis after 12 hours. (a) Used DAPI stain to look at nuclear staining.

Cells exposed to gold look almost identical to control cells. The same thing can be seen with (b), which looked at Annexin V (conjugated to Alexafuor 568), an apoptotic marker. Samples in both graphs look almost identical to control, further proving that the nanorods have no effect on cells

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Figure 6: (a) Neurosphere formation assay-number of neurospheres were counted seven days after exposure to the laser. Sample exposed to the laser had twice as many neurospheres as the control sample. (b) Raw Florescent Activated Cell Sorting data comparing control cells to cells exposed to laser after 24 hours. FSC on the x-axis and SSC on the y-axis shows the size and complexity of sample cells. The two samples look almost identical, indication there are no significant effects on the cells when exposed to laser

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Aptamer targeting of GBMs Specific targeting was done by attaching an aptamer to the GNR. Because an aptamer specifically synthesized for GBM cells was not available, a different aptamer was used. Sgc8 is a leukemia marker, but the same marker is commonly found on other cells, and is slightly more general than other aptamers, which is why it was chosen. This experiment was performed to measure the preference of aptamer binding to the GBM cells. Affinity was measured using flow cytometry [Figure 9A]. Since a flourescein molecule was attached to the aptamer, anything bound to the aptamer would fluoresce at 488, (excitation wavelength of fluorescein).

FACS data here shows the control (left) compared to cells exposed to the aptamer (right). Cells showed a 99.09% binding affinity to the aptamer, proving that the aptamer/gold conjugate is extremely effective in binding to cells. 9B shows a confocal image of a GBM cell. The green dots on the outer edge of the cell are aptamers fluorescing.

Aptamer specificity of GBM compared to mouse cells This experiment was performed to measure the specificity of the aptamer to the GBM cells compared to the binding of aptamers in other cells [Figure 10].

The results showed that at 1:3 aptamer/nanorods to cell ratio (compared to the original concentration used), the GBM has a much stronger signal of the aptamer than the mouse cells.

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Analysis of cancer stem cells This experiment was performed to observe cancer stem cells (CSC) within neurospheres by using carboxyfluorescein succinimidyl ester (CFSE), a proliferation indicator. Using confocal microscopy, CSC were observed after seven days of growing (Figure 8). Bright green dots inside the neurosphere are cells that have not divided as many times as the rest of the cells, a strong characteristic of CSC.

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AlexaFluor 568-A Figure 7: An analyzed series of samples from control cells to cells with gold and laser. Annexin V (AlexaFluor 568) used to analyze nuclear staining and apoptosis. The low peak show that the cells were killed when exposed to gold and laser. The higher peaks represent the control and cells exposed to one part of the method (ex: gold, laser, etc)

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Efficient killing of all GBM cells after aptamer targeted GNR treatment and laser exposure This portion of the research was to see if the A-GNR would be a reliable method for killing the cells.

Analysis was performed visually (under a microscope) and numerically. Results showed immediate death of cells [Figure 11 a and b] shows visual and numerical data of cells after incubation. The picture in Figure 11a of experimental cells shows complete 21

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Figure 8: GBM cells exposed to CFSE and examined after 7 days growing in culture. Fast growing cells (not green) and slow growing (green) cells are indicative of population variations within GBM sphere. A control, B cells exposed to GNR, C cells exposed to aptamers.

Figure 11a: Aptamer conjugated gold nanorods were exposed to cells and either exposed to laser or not (control). Cells that were incubated with A-GNR but not exposed to the laser were healthy, while the same treatment but exposure to the laser killed all cells efficiently. Image on the right shows dead cells

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Figure 9: (a) Aptamer exposed to cells and analyzed using FACS. The graphs have the florescence intensity on the x-axis (There would be high intensity if aptamers were bound to cells), and side scatter on the y-axis. The left graph is the control, with no florescence as expected.

The right graph shows cells exposed to aptamers. The high florescence intensity indi9cates a strong binding of aptamers to cells (Control). (b) A layered confocal image of two GBM cells together. The large, bright green fluorescent dots show the aptamers that have targeted and stuck onto the cell)

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Figure 11b: This bar graph shows cell counts of all the samples, from control, to cells exposed to A-GNR and laser. Cells exposed to A-GNR and laser showed an overall decrease in cell count, and a dramatic decrease in number of living cells. Control cells were hardly affected

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destruction of cells after being exposed to the laser.

The graph in Figure 11b shows the number of live and dead cells. For each sample, the left column is cell count after two hours, the right column in cell count after 16 hours. A dramatic decrease in overall 22

All parts of the original hypotheses were supported by the data and results. The results indicated that this method is credible and could potentially be used as a new treatment option for patients.

Synthesis and characterization of gold nanorods.

I have demonstrated that the GNR I made were uniformly and evenly dispersed, shown by a tight peak in the UV VIS experiment and the EM analysis.

The characterization of GNR is limited due to the fact that the particles were rod shaped and not spherical.

I was able to effectively and specifically conjugate the GNRs to specific aptamers which I used for targeted Young Scientists Journal | 2010 | Issue 8

nanothermal radiation therapy on the GBM cells.

GNR emission of heat when exposed to a NIR laser: As discussed previously, cancer cells are known to be sensitive to increased heat (hyperthermia), which is key to the success of nanothermal radiation. I have shown in an in vitro experiment [Figure 3] that GNRs cause local heat emission and that the surrounding areas plateau, after an increase of about 1-2oC, at a temperature of ~37oC, which is not harmful to healthy tissue, but causes apoptosis in most cancer cells.[15] Observe effects of GNR, laser and aptamers on cells.

Before I tested targeted nanothermal radiation therapy, the exposure of cells to A-GNR and the NIR laser, I wanted to ensure that there were no negative side effects of the gold, the aptamer or the laser by itself.

My results show that, at the ideal concentration, GNRs (1:1000), aptamers and the NIR laser gave no negative effect on the health of the cells. Too high concentration of GNR (<1:100) was shown to be toxic to the cells but apatamers at any concentration were not found to be toxic. Toxicity of GNRs could be caused by residue of CTAB, which is known to be toxic to cells.[16,17] The exposure of cells to NIR laser or to GNR and the laser has resulted in slight increase of cell numbers in 3 different experiments in the nanosphere survival assay. This can possibly be attributed to the heat from NIR laser, stimulating growth of the cells. Although this could indicate some toxic processes in the light of the hypothermic nanothermal radiation treatment, the cells should become more sensitive to heat induced killing, chemotherapy and radiation therapy after the exposure to hypothermia.[15,18] Expose cells to gold and laser - examine cell death.

Though the GNR and aptamers had no effect on the cells individually, when combined, cell death was obvious. The cells were exposed to gold for a period of time, washed, and analyzed using various apoptotic markers and DNA stains. Seen on the FACS graphs, there was no obvious difference between all the samples except the one where cells were exposed to gold and the laser, and the cells exposed to just the laser. It should be noted that the left graph (annexin V) shows an obvious decrease in cells. While the intensity of the cells with the gold and laser is not at a higher intensity, the percent of max cells, also known as the number of cells counted was about a fourth of the original number. This shows that the method kills cells extremely quickly. Dead cells decay, and are spun out during procedures preceding FACS analysis, which accounts for the low numbers.

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Analyze the presence of cancer stem cells within GBM neurospheres. Analysis of cancer stem cells: Carboxyfluorescein succinimidyl ester (CFSE) is a common dye used to look at division rate of cells. In this study, the dye was used to attempt to distinguish cancer stem cells from the â&#x20AC;&#x153;normalâ&#x20AC;? fast growing tumor cells. Using a confocal microscope with a green laser, the difference between cells was very obvious. Within neurospheres, there were a few bright green cells. This is evidence that the cell did not divide as rapidly as the majority of cells, a major characteristic of cancer stem cells. These cells were the minority of the population, only about 1% of the cells.

Selectively target A-GNR to GBM cells and measure cell death: However, because there has not been an aptamer specifically synthesized for GBM cells, a different aptamer had to be used. Sgc8 is a leukemia marker, recognizing protein tyrosine kinase 7,[8] but the same marker is commonly found on other cancer cells. The process of detecting and synthesizing an aptamer specific to GBM is currently in progress and I will repeat certain experiments once I have the GBM specific aptamer. This experiment was performed to measure the specificity of the aptamer to the GBM cells. Cells were exposed to A-GNR conjugates, incubated, and washed.

Aptamer specificity of GBM compared to mouse cells: This experiment was performed to measure the specificity of the aptamer to the GBM cells compared to the binding of aptamers in other cells.This data demonstrates the specificity of the aptamer. If an aptamer specifically used for GBM was used, the binding affinity would have been much higher. It would also show a higher binding affinity if the concentration was higher, but that would not necessarily indicate specific binding affinity.

Efficient killing of all GBM cells after aptamer targeted GNR treatment and laser exposure: This portion of the research was to see if the A-GNR would be a reliable method for killing the cells. The experimental cells completely disintegrated after being exposed to the laser. This data further proves the idea of cell selectivity, absorption of A-GNR into cell, and the efficient killing of the cancer cells.

Problems CTAB: Though there are multiple methods to synethesize nanorods, the most stable and simple method involves making seed particles and 23

allowing them to grow. This process requires cetyltrimethylammonium bromide (CTAB) for the nanorods to grow. However, CTAB is known to be cytoxic. Because gold nanorods were functionalized in various methods, the stock solution was kept in .2M CTAB. Despite the fact that nanorods were washed repeatedly before being exposed to the cells, some residue still remained. This may have accounted for some of the cell death. However, the results have shown that the death of cells due to the treatment method described are fall to significant to be completely explained by CTAB. Functionalized GNR – clearance from body; Although previous studies have been done showing that gold nanorods are not toxic, the rods can still be easily excreted through the body. A study done by Powers in 2009 showed the effect of PEGylated vs. “naked” gold nanorods in a rat. Using various techniques they concluded that naked rods were sent to the kidney within five minutes, while PEGylated rods stayed suspended through the body for about 24 hrs. All nanorods in this study were PEGylated, but this could still play a factor in specificity or absorption of GNR into cells.

Finding the right concentration for gold treatment: A large problem often encountered when working with nanosized material is consistency and the spontaneous changes in the properties of the particles. Each batch of gold nanorods made will be slightly different, no matter how precise the method is carried out. If one batch is made, and then stored, the properties could change over time. The nanorods were kept in .5M CTAB which is known to keep nanorods stable, but analysis of the nanorods batch was performed multiple times to ensure consistency in size.

Specificity of the aptamer: as previously mentioned, due to time restraints, costs, and expertise in the field, I have not yet been able to create a specific aptamer that will bind to GBM. However, this process is very feasible, and in progress. Usually this process takes about three months. However, after visiting the Burnham Institute in Orlando, Florida, they introduced me to a robot that collects data points for an experiment. This extremely complex machine can remove an ELISA plate from the incubator, analyze and replace it, without human interference. At optimal use, the robot can collect 2 million data points a day.

If access to this machine were granted, the aptamer selection method would be reduced greatly.

24

Studies ongoing and future plans This research project has been a continuation for the last year, and has kept evolving into a larger and more complex study [Figure 12]. One large aspect of this project is to be able to image and treat the tumor at the same time, thus saving time, money, and stress of the patient and health care system. This can be accomplished by attaching a fluorochrome to the aptamer which will be picked up on the imaging device.[19] By attaching the proper fluorochrome to the aptamer, a radiologist would be able to see exactly where the tumor is, and the size of it. This has an additional benefit of being able to detect or diagnose metastasis of cancer.[20] Another important aspect that is currently being researched in this field is tumor population dynamics.

Contrary to popular belief, a tumor is made up of multiple types of cells, not just one: an example being tumor stem cells. It is a growing belief in medical field that in order to treat cancer in the best possible way, an understanding of the tumor on the micro and macroscopic level is needed. A tumor is much like a mini ecosystem, with different populations, needs, etc. In order to understand how a tumor grows and reacts, and how therapy affects these populations, modeling can be used to determine and hypothesize how a tumor will react to specific disturbances.

The ability to have a specific aptamer for the specific type of cancer is essential.[21] There have been a few studies finding a specific aptamer for GBM. I am planning to find a specific aptamer for the exact cell line I’m using. I would also like to synthesize an aptamer specifically for the GBM CSC.

Figure 12: The complete nanothermal radiation treatment option

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Once optimal concentration, laser wavelength, laser time of exposure, etc. has been established, the next step would be testing this method in vivo. No matter how well an experiment works in cell culture, there is no way to stimulate the actual effect without using real animals.[22] A GBM tumor would be transplanted into the side of a lab rat or mouse, and be allowed to grow for about a month. Then, using the optimal parameters, the treatment would be used to test its specificity and efficacy in a ex-vivo live animal model. Hypothetically it should work the same, but the environments are extremely different, so itâ&#x20AC;&#x2122;s hard to determine what the overall effect would be.

Final Thoughts Whatâ&#x20AC;&#x2122;s the point of this study? The reason that I am so dedicated to this project is that the real world application of this treatment method would really enhance, possibly revolutionize cancer treatment.

More efficient, more personalized cancer treatments with fewer side effects are urgently needed, as is overcoming chemotherapy and radiation resistance of the tumors and reducing risk of metastasis by targeting cancer stem cells. This study has shown that nanothermal radiation is extremely successful in both killing the dangerous cells and causing virtually no side effects. Additionally, the combination of aptamer-targeted therapy can benefit all approaches of cancer treatment. My next goal is to prove this approach feasible with animal studies, further strengthen this approach in the near future.

References 1. Sakariassen P. Cancer stem cells as mediators of treatment resistance in brain tumors: Status and controversies. Neoplasia 2008;9:882-92.

2. Bruce JN. Glioblastona multiforme (GBM) and anaplastic astrocytoma (AA). Available from: http://www.abta.org/ siteFiles/SitePages/AB2A6786BBBE123DBA5D1353155F7813. pdf. [cited in 2007] [retrieved on 2009 Nov 9].

3. National Center for Health Statistics. Cancer Death Rates Among Women, US, 1930-2005. In U. Cancer Death Rates Among Women, 1930-2005 (Ed.), US Mortality Data 1960-2005, US Mortality Volumes 1930-1959,: National Center for Health

Statistics, CDC; 2008.

4. Ailles LE, Weissman IL. Cancer stem cells in solid tumors. Curr Opin Biotechnol 2007;18:460-6.

5. Xie Z. Brain tumor stem cells. Neurochem Res 2009;34:2055-66.

6. Cho EJ, Lee JW, Ellington AD. Applications of aptamers as sensors. Ann Rev Analyt Chem 2009;2:241-64.

7. Huang YF, Chang HT, Tan W. Cancer cell targeting using multiple aptamers conjugated on nanorods. Analyt Chem 2008;80:567-72.

8. Huang YF, Shangguan D, Liu H, Phillips JA, Zhang X, Chen Y, et al.

Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells. Chem Biol Chem 2009;10:862-8.

9. Phillips JA, Xu Y, Xia Z, Fan ZH, Tan W. Enrichment of cancer cells using aptamers immobilized on a microfluidic channel. Analyt Chem 2008;81:1033-9.

10. Shangguan D, Tang Z, Mallikaratchy P, Xiao Z, Tan W. Optimization and modifications of aptamers selected from live cancer cell lines. Chem Bio Chem 2007;8:603-6.

11. Suri SS, Fenniri H, Singh B. Nanotechnology-based drug delivery systems. J Occup Med Toxicol 2007;2:16.

12. Geoffrey von, Maltzahn, et al. "Sers-Coded Gold Nanorods as a Multifunctional Platform for Densely Multiplexed near-Infrared Imaging and Photothermal Heating." Advanced Materials 21.31 (2009): 3175-80.

13. Diagaradjane P. Modulation of in vivo tumor radiation response via gold nanoshell-mediated vascular-focused hyperthermia: Characterizing an integrated antihypoxic and localized vascular disrupting targeting strategy. Nano Lett 2008;8:1492-500.

14. Liu VG, Cowan TM, Jeong SW, Jacques SL, Lemley EC, Chen WR. Selective photothermal interaction using an 805-nm diode laser and indocyanine green in gel phantom and chicken breast tissue. Lasers Med Sci 2002;17:272-9.

15. Liu C. Energy absorption of gold nanoshells in hyperthermia therapy. IEEE Trans Nano Biosci 2008;7:206-14.

16. Smith DK, Korgel BA. The Importance of the CTAB surfactant on the colloidal seed-mediated synthesis of gold nanorods.

Langmuir 2008;24:644-9.

17. Szydlowska H, Zaporowska E, Kuszlik-Jochym K, Korohoda W, Branny J. Membranolytic activity of detergents as studied with cell viability tests. Folia Histochem Cytochem (Krakow) 1978;16:69-78.

18. Li J, Wang B, Liu P. Possibility of active targeting to tumor by local hyperthermia with temperature-sensitive nanoparticles.

Med Hypotheses 2008;71:249-51.

19. Agarwal A, Huang SW, Donnell MO, Day KC, Day M, Kotov N, et al. Targeted gold nanorod contrast agent for prostate cancer detection by photoacoustic imaging. J Appl Physics 2007;102:64-70.

20. Cai W. Applications of gold nanoparticles in cancer nanotechnology. Nanotechnol Sci Appl 2008;1:17-32.

21. Jayasena SD. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin Chem 1999;45:1628-50.

22. Marshall GP 2nd, Reynolds BA, Laywell ED. Using the neurosphere assay to quantify neural stem cells in vivo. Curr Pharm Biotechnol 2007;8:141-5.

About the Author Muna Oli is a senior at Eastside High School in Florida. Aside from research, she enjoys photography, traveling, running and reading. She hopes to pursue a combined MD-PhD degree in the future.

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25

Nanotechnology

Increasing the efficiency of a hybrid polymer photovoltaic cell with polymer nanofiber complexes of varied thickness Nathan Monroe Episcopal High School of Jacksonville, Jacksonville, FL, Massachusetts Institute of Technology, Email: Monroe@mit.edu DOI:10.4103/0974-6102.68741

Introduction The purpose of this investigation is to increase the power output of a specific polymer when utilized in a photovoltaic cell. Fossil fuels are an efficient and reliable source of energy, but they are detrimental to the environment, producing pollutants and greenhouse gases.[1] Also, the supply of fossil fuels is quickly diminishing. According to Kaufman,[2] at the rate fossil fuels are currently consumed, the supply will be exhausted within fifty years. Assuming that this statement is true, there is significant need for a new source of energy that will last a long time, be safe, clean, and easy to produce.

Photovoltaic cells convert light energy into electrical energy using the photovoltaic effect.[3] The photovoltaic effect occurs when photons of light strike the active material, the semiconductor. The electrons of semiconductor atoms are excited from the valence band (valence electrons) into the conduction band, where they can move freely between atoms. The excited electron is known as a charge carrier, or an exciton. The electron moving from its original atom into other atoms is known as exciton dissociation. Exciton dissociation results in one free, excited electron, and an electron hole, or the lack of an electron where the excited electron previously occupied. The electrons are captured by a metal electrode (the negative contact), 26

and the holes are captured by the positive contact, and used as electricity.[4] Silicon is the most common semiconductor used. The photovoltaic effect will be applied in this investigation, but with a polymer in place of silicon.

This polymer, dissolved in a liquid, can be sprayed onto materials, painted onto materials, or even printed in rolls. Its only downside is its efficiency.

Unlike conventional silicon solar panels, which have an average efficiency of 15-20%, the polymer solar panels have a typical efficiency of 2-3%.[5] Twenty percent efficiency for conventional solar panels would be perfectly acceptable if the solar panels were more practical. However, they are heavy, fragile and difficult to make. Conventional solar panels require extreme temperatures and pressures to produce, consuming large amounts of energy.[4] Polymer solar cells require no extreme temperatures or pressures, and are thus easier and cheaper to produce. Polymer solar cells have an extreme advantage over silicon solar cells because of their special application properties.

Polymer solar cells, if efficient enough, could be painted onto vehicles, buildings, or even clothing as a valuable energy source. Polymer solar cells have the added advantage of being flexible, allowing them to be painted onto curved surfaces such as cars or other vehicles. Finally, polymer solar cells have a significant weight advantage over silicon cells, weighing Young Scientists Journal | 2010 | Issue 8

significantly less. This is especially advantageous for space applications. Current rocket technology relies on liquid fuel, which is very expensive. With silicon solar panel systems weighing hundreds of pounds,[6] the fuel costs add up very quickly. Lighter, cheaper solar power systems and reduced fuel costs could be as simple as using a different paint on space shuttles or unraveling rolled lightweight polymer cells into space.

If the efficiency of the polymer solar panels could be increased, they could provide more than enough energy for sustainable development.

Currently, these polymer photovoltaic cells are very inefficient. The main reasons for this are suboptimal band alignment between electron donor and acceptor semiconductors, and low charge carrier mobilities stemming from polymer based semiconductorsâ&#x20AC;&#x2122; disorganized nature.[7] Zinc oxide has been proven to increase efficiency by promoting exciton dissociation.

This is due to the physics of the photovoltaic effect.

The photovoltaic effect and exciton dissociation occur when there is a difference in potential (Fermi energy) between two areas of the solar cell, where a high potential area is in junction with a low potential area.[4] When the polymer is illuminated, its electrons are excited, making it high potential. Zinc oxide has non-excited electrons, making it low potential. Thus, Zinc oxide provides the potential difference required for the photovoltaic effect at the junction between it and the polymer [Figure 1]. This explains the result of previous studies (year three of this investigation), where zinc oxide was mixed homogenously with the polymer. Zinc oxide had an increased efficiency when mixed with the polymer because it increased the surface area of the high potential/low potential junction, and thus the efficiency on the one molecule thick layer

between the polymer and the electrode. Because the zinc oxide only increases efficiency when it is at the interface between the polymer and the electrode, the goal of year 4 of this investigation was to increase the surface area of the zinc oxide/polymer junction by creating zinc oxide nanofibers in replacement of the homogenous mixture, and optimize nanofiber length for maximum efficiency. Year four of this investigation proved that increased nanofiber length resulted in increased surface area and increased efficiency.

However, if nanofiber length (thickness of the nanofiber layer) surpassed polymer thickness, the nanofibers penetrated completely through the polymer and caused an electrical short, ruining the devices.

The polymer is applied through a process known as spin-coating. During this process, a substrate is spinning rapidly on a platform, and the liquid polymer is dropped onto the spinning substrate. The polymer spreads out to form a film. The thickness of the polymer is a function of polymer solvent (varying surface tension and density), spin rate (centripetal force), and polymer concentration (viscosity). Previous research has shown that polymer thickness has a significant effect on efficiency.[8] Increased polymer thickness results in higher resistance and thus lower efficiency.

Essentially, there are two conflicting variables when producing hybrid organic/inorganic devices with P3HT and ZnO nanofibers. As nanofiber length increases, efficiency increases. As polymer thickness increases, efficiency decreases. The two variables are linked in that the nanofibers must be as long as possible without surpassing polymer thickness. The goal of this research was to find the optimal thickness of these two layers, the â&#x20AC;&#x153;polymer nanofiber complexâ&#x20AC;? that resulted in maximum efficiency.

Method

Figure 1: The ZnO provides the Fermi energy potential difference required for the photovoltaic effect (adapted from Sun et al., 2005)

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Before the production of solar devices it was necessary to develop procedures for reproducibly controlling the thickness of the polymer and nanofiber layers. The spin-coating process was the determining factor for polymer thickness, assuming constant polymer solvent and concentration. During spin coating, polymer solvent and concentration were kept constant at chloroform and 20g/L. Polymer films were produced with spin rates of 8210, 2620, 1110, 557, 313, and 190rpm. These rates were determined by previous research from Chang et al,[8] to predict polymer thicknesses of 130, 180, 230, 280, and 330nm based on a regression equation matching known data. Profilometry was used to determine 27

actual polymer thickness. These data were plotted, and a new regression function was calculated to relate spin rate and polymer thickness for given polymer solvents and concentrations [Figure 2]. To confirm the reproducibility of this regression, new slides were produced using the regression function. These thicknesses were measured, and a new regression function created. The difference between the first and second regression functions was less than the margin of error of the surface profiler equipment.

The zinc oxide nanofiber production procedures were also analyzed for nanofiber length. The nanofibers are produced using the sol gel process from zinc nitrate and zinc acetate precursors. Due to the surface properties of glass, zinc oxide nanofibers can not be grown directly onto glass. There must first be a flat layer of zinc oxide from which to grow the nanofibers.

This is known as the nucleation layer. The nucleation layer is produced by spin coating at 2000rpm the following solution: 0.823g Zinc Acetate, 0.223mL Ethanolamine, 5mL 2-methoxyethanol. The slides were annealed at 300 degrees for 10 minutes, initiating the reaction seen in Figure 3, and resulting in a flat layer of zinc oxide. The unwanted ions were washed away by subsequently washing the slides in acetone and isopropanol. The nanofibers were produced in a solution of 1mM zinc nitrate at 70 degrees, according to the reaction seen in Figure 4. The pH was raised to 13 using sodium hydroxide. Previous research has shown that nanofiber length is a function of the reaction time in the zinc nitrate solution. Nanofiber slides were produced with reaction times of 5, 10, 15, 20, 25, and 30 minutes. Nanofiber length was measured using scanning electron microscopy [Figure 5]. Average

fiber length was taken from three locations on each slide. The SEM images were taken from a 45 degree angle. Using the Pythagorean Theorem, the measured nanofiber length was multiplied by the square root of 2 to find actual nanofiber length. Reaction time was plotted against nanofiber length [Figure 6] and a regression function was produced to relate the two variables. This process was repeated to confirm the accuracy of the regression function.

The solar devices were produced using slides of patterned Indium Tin Oxide (ITO). Ten 1”x1” glass slides pre-coated with (ITO) underwent intense cleaning procedure using a micro weave cloth to remove dust and impurities [Figure 7]. It is important to note that the entire solar cell, when completed, is only ~1000nm thick, meaning that any dust or particle on the slide during production could be thicker than the entire solar cell, causing an electrical short and a ruined device, so the cleaning procedure is critical to the production process. The slides were placed into an ultrasonic vibrator with 100ml isopropyl alcohol for ten minutes. This was repeated with deionized water and acetone. The slides were placed into an ozone and ultraviolet light generator for thirty minutes. This was to ensure the absence of any impurity which could interfere with electrical conduction of the slide, and to enhance hydrophilic properties of the slides’ surface to facilitate spin-coating. The nanofiber layer was produced according to the procedure described above, in thicknesses (nanofiber lengths) of 100, 150, 200 and 250nm [Figure 8]. The ITO contact area was wiped with a Q-Tip soaked in ethanol prior to annealing to prevent fiber growth in this area.[9] Two slides were produced with each nanofiber length, leaving two

350

300

Polymer thickness (nm )

250 y=1670.x-0.28 R2=0.989

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150 y=1090.x-0.23 R2=0.957

100 y=2898.x 2 R =1

50

0

0

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Figure 3: The reaction mechanism for the production of a zinc oxide nucleation layer from a Zinc Acetate precursor (adapted from Bahadur et al., 2007)

-0.39

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Spin rate (RPM) Predicted Film Thickness (nm)

Actual Film Thickness (nm) Trial 1

Actual Film Thickness (nm) Trial 2

Figure 2: Polymer film thickness vs. spin rate for a given polymer solvent and concentration, using power regressions. The R-squared values for the first and second trials were 0.989 and 0.957, respectively

28

Figure 4: The reaction mechanism for the production of the zinc oxide nanofibers in solution from a zinc nitrate precursor (adapted from Bahadur et al., 2007)

Young Scientists Journal | 2010 | Issue 8

a

b

c

d

e

f

Figure 5: Scanning electron microscope images of the ZnO nanofibers. After 5 minutes reaction time (a), there was no fiber growth. After 10 minutes (b) the average fiber length was 103nm. After 15 minutes (c), the average fiber length was 151nm. After 20 minutes (d), the average fiber length was 173nm. After 25 minutes (e), the average fiber length was 212nm. After 30 minutes (f), the average fiber length was 264nm

300 250 y = 70.73e x R2=0.973

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0.044

200 150 100 50 0 10

15

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Figure 6: Reaction time vs. nanofiber length. After an exponential regression, the R-squared value was 0.973

slides with no fibers as the control. The polymer layer was applied on top of the nanofiber layer using the process described above [Figure 9]. The polymer was applied such that it was always 30nm thicker than the nanofiber layer, to account for small variances in fiber length and minor residuals in the regression function. For example, to the 100nm nanofiber device, a polymer layer was added of 130nm. This process was completed in nitrogen to prevent the oxidation of the polymer. The slides were annealed at 200 Young Scientists Journal | 2010 | Issue 8

Figure 7: The solar cell begins as a pre-patterned ITO (indium tin oxide) substrate. The ITO (grey) is transparent enough to allow light to pass through to the solar cell, yet conductive, allowing it to act as the top electrode

degrees for 1 minute, to melt the polymer between the nanofibers, further increasing the surface area of the junction. A thermal evaporator was used to achieve zero humidity and a complete vacuum environment.

Four slides at a time were placed into the thermal evaporator and each underwent application of both an 8nm BCP (bathocuproine) layer, and an aluminum electrode directly to the polymer surface of the slide [Figures 10, 11]. The BCP effectively increased the 29

Figure 8: Zinc Oxide nanofibers are applied, starting with the Zinc Oxide nucleation layer. Ethanol wipes on the ITO contact areas are used to dissolve the nucleation layer, preventing fiber growth in these areas.

The fibers (yellow) are then grown from solution

Figure 9: The active layer, P3HT (red) is applied via spin-casting. This is to ensure even thickness on all slides

Figure 10: The BCP layer (green) is applied via vacuum thermal evaporation. This effectively increases the thickness of the solar cell, reducing the chance of an electrical short due to dust or impurities

30

Figure 11: The aluminum electrodes are applied via vacuum thermal evaporation at an even thickness. Aluminum contact points are applied above the ITO for optimal contact. The circuit is completed through the polymer. Because that area is not illuminated, it does not add to the overall device area

Figure 12: The completed solar device. The device area (yellow) is the intersection of the ITO and aluminum electrodes, with 4 devices per slide. The two contact points are ITO (blue) and aluminum (pink)

thickness of the slide, reducing the chances of an electrical short due to dust or other impurities. It is also a hole-blocking layer. The use of the thermal evaporator assured intimate contact of the entire electrode with the entire surface of the polymer. The procedure of BCP layer and electrode application was repeated for all test slides. Each side of each test slide had the BCP and aluminum electrode layers applied, allowing each slide to serve as four different test trials. Each slide was inserted into a solar simulator, and attached to a semiconductor parameter analyzer, which was connected to a computer to graph and record power output [Figure 12]. Power output from the device was recorded in amperes using a sweep mode from minus Young Scientists Journal | 2010 | Issue 8

to plus one volt. This was repeated for each of four test sites on each slide, and repeated for all ten test slides. The entire process was repeated, resulting in two slides, or 8 solar cells for each test group, and two slides for the control. The process of testing each solar cell for efficiency was repeated after 72 hours of air exposure to test the effects of air exposure on the devices. All slides, polymers, and used chemicals were disposed of as per safety protocol. All results were analyzed using Microsoft Excel.

Figure 13: The calculation of power conversion efficiency from open circuit voltage (Voc), short circuit current density (Jsc), fill factor (FF), and power input (Po). All units cancel to result in power output/power input, which is mechanical efficiency

Initial power conversion efficiency

0.00045 0.0004

Young Scientists Journal | 2010 | Issue 8

0.00035 0.0003 0.00025 0.0002 0.00015 0.0001 0.00005 0 100nm

150nm

200nm

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Control (Pure P3HT)

Test group (number denotes nanofiber length)

Figure 14: Immediately after device production, there is a strong parabolic trend with increasing complex thickness. However, all nanofiber devices have an efficiency lower than the control

Power conversion efficiency after three days 1.2 1

Arbitrary unit s

Efficiency was calculated based on number taken from the J-V curve from the semiconductor parameter analyzer [Figure 13]. According to the data, nanofiber length, polymer thickness, and air exposure had an effect on solar cell efficiency. The addition of nanofibers initially decreased the efficiency compared to the control [Figure 14]. However, after 72 hours exposure to air, the nanofiber devices showed an increased efficiency, while the control decreased in efficiency [Figure 15]. This increase in efficiency may be due to the reduction of oxygen vacancies in the ZnO nanofiber crystals, and thus an increase in carrier concentrations. Some nanofiber devices had efficiencies greater than the control after 72 hours.

The device with nanofiber length/polymer thickness of 100/130nm had an efficiency 29% greater than the control. The trend of air exposure over time indicates that further exposure would continue to increase efficiency in nanofiber devices, and would likely show asymptotic behavior after the polymer and nanofibers are completely oxidized. Maximum efficiency within nanofiber devices was seen in 100/130 and 250/280 devices. This indicates that both polymer thickness and nanofiber length have an effect on efficiency, with low polymer thickness increasing efficiency in the 100/130 devices, and long nanofiber length increasing efficiency in the 250/280 devices. After 72 hours, efficiency was measured using arbitrary units. This was due to the fact that the BCP layer degrades over time, decreasing efficiency, while maintaining the trend of efficiencies over all devices, so the efficiencies themselves are irrelevant. The solar devices initially have a low efficiency due to device conditions. Due to limited polymer availability, reduced polymer concentrations were used from the optimal 32g/L. As a result, the trend of efficiencies is more important than the efficiencies themselves. This trend indicates that after 72 hours, the solar device with nanofiber length/polymer thickness of 100/130nm increases

Power conversion efficiency (%)

Results and Discussion

0.8 0.6 0.4 0.2 0

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150nm

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Control (Pure P3HT)

Test group (number denotes nanofiber length)

Figure 15: After three days, the parabolic trend is preserved with increased complex thickness, yet the nanofiber devices have an efficiency frame shifted above the control. This may be due to the oxidation of the polymer and the reduction of oxygen vacancies in the ZnO nanofiber crystals

efficiency 29% over the control, and continues to increase efficiency over time. Applying this trend to solar cells with optimal polymer concentrations would result in a final theoretical efficiency of 5.2%.

After a 2 sample T-test, the p-value when comparing the 100/130 device and the control was calculated 31

to be less than 0.0001, indicating that the results were statistically significant.

There were some minor errors in the procedures.

First, the slides were intended to be removed from the annealing oven simultaneously. However, this was not possible because it took time to remove each device from the annealing oven, so there was a 10-15 second variation. Next, there was a slight technical problem with fiber growth. The fibers were grown in a 70 degree solution. However, the solution was heated from the bottom, resulting in a temperature gradient across the slides. This would have resulted in varying fiber diameters across the slide. However, this would have had a minimal effect as the gradient was the same on all slides. Finally, the polymer is laterally conductive. This means that energy is collected on the slide in an area slightly larger than the intended device area. However, since light was only being shined on the intended device area, and the effect was the same for all devices, the efficiency ratios are preserved and the effects are minimal. These problems had miniscule effects on the final efficiencies, and any effect would have been the same across all test groups, having minimal, if any effect on the data trend. Despite these small effects, this research generated new knowledge on polymer solar cells, in hopes of one day replacing fossil fuels with solar energy, reducing humanityâ&#x20AC;&#x2122;s effects on the environment and achieving true sustainable development.

Acknowledgments I would firstly like to thank my parents Drs. Mark Monroe and Julie McKay for their never ending moral and financial support. I would also like to thank my teacher Mrs. Marion Zeiner for her guidance and help along the way. I would like to thank Dr. Jiangeng Xue for his selfless donation of time and materials. Finally, I would like to thank graduate students Ying Zheng, Jason Myers, and Bill Hammond for their time and help with equipment.

References 1. Solomon S, Plattner GK, Knutti R, Friedlingstein P. Irreversible climate change due to carbon dioxide emissions. Proc Natl Acad Sci U S A 2009;106:1704-9.

2. Kaufman A. Exploring solar energy. Prakken Publications; 1995.

3. Solar energy. In Microsoft Encarta Reference Library 2003 CD Rom]. Redmond, WA: Microsoft. 2003.

4. Sun SS, Sariciftci NS. Organic photovoltaics: Mechanisms, materials, and devices. 2005.

5. Winters J. Catching more rays. Mech Engg 2005;127:14A. 6. Sietsema R. Solar panel comparison. Available from: http:// claymore.engineer.gvsu.edu/~solar/archives/development/ solarpanel.html. [cited in 2009].

7. Ginley DS. Hybrid photovoltaic devices of polymer and ZnO nanofiber composites. Proceedings of the Fourth International Symposium on Transparent Oxide Thin Films for Electronics and Optics; vol. 426, 2005. p. 26-9.

8. Chang C, Pai C, Chen W, Samson A. Spin coating of conjugated polymers for electronic and optoelectronic applications. Thin Solid Films 2004;479:254-60.

9. Olson DC, Shaheen SE, Collins RT, Ginley DS. The effect of atmosphere and ZnO morphology on the performance of hybrid poly(3-hexylthiophene) / ZnO nanofiber photovoltaic devices. National Renewable Energy Labs; 2007.

About the Author Nathan Monroe is 19 years old and is an undergraduate at the Massachusetts Institute of Technology, studying Electrical Engineering and Physics. In his free time, he enjoys playing guitar and mandolin, singing, and running. He hopes one day to become a researcher for an electronics company, or start his own green energy company.

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Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Antibody-coated magnetic nanoparticles: Targeting and treating cancer Philip Schlenoff Maclay School (Tallahassee, Florida) DOI:10.4103/0974-6102.68743

Used as far back as the ninth century to make pots glisten gold, nanoparticles have a rich history filled with a wide variety of applications. In fact, sol-gel synthesis (silica based nanoparticle creation) became so popular in the 1990s that over 35,000 papers were published on the process. Modern day applications include medicinal and food-based uses, even being synthesized in beer bottle glass to make it less breakable.[1] Once magnetic nanoparticles were introduced, the opportunities for nanotechnology skyrocketed. Their ability to be guided, tracked, and affected by various kinds of radiation, including Radio Frequency (RF) and laser, gives them certain advantages compared to particles made of materials like silica or clay.

Because magnetic nanoparticles can be heated by RF radiation, they have been proposed for the treatment of cancer. It is envisioned that these nanoparticles can be injected intravenously, circulate, and if coated with the proper antibodies,[2] selectively target cancer cells and be ingested by them. In the next step, RF irradiation of approximately 350 kHz may be used externally to irradiate the tumor saturated with the particles. Once the temperature rises above 45oC, the cancer cells are eradicated. However, these nanoparticles tend to aggregate in the bloodstream because of the presence of proteins such as serum albumin, limiting their effectiveness in reaching the brain or metastasized tumors.[2] This project aims to solve the aggregation problem, eventually leading to a cheap, effective, and safe nanoparticle-based cancer treatment.

Young Scientists Journal | 2010 | Issue 8

Current literature on nanomedicine treatments for cancer typically involve using quantum dots or nanorods coated with gold and poly(ethylene) glycol and heating them in vivo using near infrared (NIR) laser treatment.[3-6] However, due to NIR’s low penetration depth, the usefulness of this method is limited. For tumors deep inside the body or metastasized tumors, NIR would require invasive surgeries to reach the targeted area. Radio frequency, on the other hand, penetrates the entire body without losing field strength, and can be applied to the entire body at once. Iron oxide is also less toxic to organic systems.[7] This research project had initially followed a different path. In 2007, my project entitled “What Size Nanoparticles Do Vertebrate Cells Preferentially Ingest During Endocytosis” was a study into the optimal ingestion size of lab-made sub-100nm silica “beads” for cancer cells [Figure 1]. Silica was used for its biocompatible properties[8] and each particle was coated with a zwitterion “mask,” SBS, [Figure 2] to facilitate cell ingestion by stabilizing the nanoparticles in the cell medium [Figure 3].[9] Particles were synthesized at five different sizes ranging from 12-87nm and then incubated with cells for two hours. Because the nanoparticles had been tagged with a fluorescent dye, confocal microscopy revealed the location of the ingested particles in the cells [Figure 4]. Results indicated that for the time frame used, all different sizes of nanoparticles were ingested equally and fully. The significance of this project was 33

Figure 1: Structure of tetraethyl orthosilicate (TEOS)

Figure 4: Confocal microscopy reveals the location of the fluorescent nanoparticles inside the cells; they aggregate to certain organelles

Figure 2: Structure of SBS. The close proximity of the + and – charges on the ends help stabilize particles in the blood

Figure 5: Structure of iron oxide and silica nanoparticles with SBS coating (all structures drawn using ChemDraw Pro). Particles used for 2007 experiments lacked the iron oxide core

Figure 3: Stability of high and low concentrations of iron oxide nanoparticles in cell medium, and low concentrations in salt solution.

This graph indicates that the zwitterion coated silica shell/iron oxide core particles are stable in cell medium, which mimics most destabilizing conditions of blood

not the rather uneventful results, but the experience it gave me working with nanoparticles. Furthermore, it gave me the idea of tagged nanoparticles, labeled with antibodies that could specifically target cancer tumors.

In 2008, this idea evolved into a project titled “Stealthy 34

Iron Oxide Nanoparticles: Towards the Identification and Eradication of Cancer Cells.” After reading some literature about doping silica nanoparticles with metals, it was decided that an iron oxide (Fe3O4) core would fit the goal [Figure 5]. The magnetic material inside the particle allows control over the temperature of the nanoparticles from a distance, and has no effect on healthy cells [Figure 6].[10] A unique zwitterion coating (a material which carries 0 net charge), originally designed to hide stents from the body’s immune system, provided these nanoparticles with an incredible advantage. The extremely close proximity of the positive and negative charges on the ends of SBS [Figure 2] mimics the phospholipid bilayer of the cell membrane, and masks the foreign particles from the body’s immune system and from cell receptors.[9] This allows these “stealthy” iron Young Scientists Journal | 2010 | Issue 8

oxide nanoparticles great versatility in vivo (in the bloodstream).[11]

The success of these trials led to my current research, entitled “Antibody-coated Magnetic Nanoparticles: Targeting and Treating Cancer" [Figure 9]. The goal of this project is to synthesize a viable cancer treatment for future in vivo animal trials. While initial steps for creating these finalized nanoparticles have remained the same, a final addition of antibodies allows them to target cancer cells [Figure 10]. These particles have been conjugated to the breast-cancer specific antibody, anti-HER2, enabling selective properties that will leave healthy cells untouched.

[14] At this time, the zwitterionic nanoparticles have been shown to fluoresce under secondary antibody staining (which binds to the primary antibody) 800% more intensely than the control, indicating that the primary antibodies are present on the surface of the particles.

Current and future trials involve testing these antibody-coated nanoparticles in a cell culture, to ensure the antibodies can fulfill their selective abilities. It may also prove useful to test the treatment in a flowing system. If these steps are successful, they could help zwitterionic nanoparticles become a more viable option for cancer treatments. However, Young Scientists Journal | 2010 | Issue 8

Figure 6: In order for the particles to react to Radio Frequency irradiation, they have to be superparamagnetic (zero net orientation at zero magnetic field). This graph shows that they are superparamagnetic before and after adding silica

Figure 7: After feeding and multiple washings, the cells with nanoparticles were attracted to a magnet placed near the test tube.

Also known as the “magnet test” Magnetic nanoparticles coated with SiO2 and SBS

1.25

1.20

Correlation function

After synthesizing nanoparticles with an iron oxide core, fluorescent dye, a silica shell, and a zwitterion coating, the nanoparticles were fed to prostate cancer cells for two hours.[12,13] They were exposed to metal-heating radio frequency (RF) irradiation for thirty and forty minutes, then the cell cultures’ viability was measured. For the control, nanoparticles without iron oxide at their core, the cell culture remained fully viable after incubation. For the cells that were fed magnetic nanoparticles, after RF treatment was applied, there was 16% cell death after thirty minutes.

After forty minutes, 30% of the cells had died. This was a proof-of-concept that the treatment could work effectively inside the body, which heats even faster than a cell culture, promising an even higher overall cell death count for the duration the RF was applied.

To verify that the nanoparticles were inside the cells and not on the surface, the cells were washed intensively, then imaged with confocal microscopy and subjected to a “magnet test,” where cells were observed to move in a magnetic field [Figure 7].

Dynamic Light Scattering was performed to measure the hydrodynamic radius of the particles, which averages around 25.1nm [Figure 8].

1.15

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1.05 R h=25.1 nm 1.00 1.0x10-6 1.0x10-6 1.0x10-6

0.001 tau (sec)

0.01

0.1

1.0

Figure 8: Dynamic light scattering is used to determine the hydrodynamic radius of the nanoparticles

35

References

Figure 9: Procedure for synthesizing particles, feeding them to cancer cells, and killing them with RF irradiation

Figure 10: The nanoparticles are composed of Iron Oxide, Silica, Antibodies, and a Zwitterion molecule

other questions also need to be answered before a nanoparticle based cancer treatment can work its way into the mainstream. Even though these nanoparticles are biocompatible, it is important to know how they are filtered out by the body after treatment. In addition, detailed in vivo thermal diffusion studies will reveal the amount of time to heat cancer tumors to the required 45 degrees Celsius, without damaging the surrounding tissue.

I hope to continue this research not only through college, but also as a career. Cancer kills over twenty thousand people a day[15] and putting even a small dent in that number could have a huge impact throughout the entire world.

1. Available from: http://en.wikipedia.org/wiki/Nanoparticle. [cited in 2009].

2. Brannon-Peppas L, Blanchette JO. Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 2004;56:1649-59.

3. von Maltzahn G, Centrone A, Ji-Ho P, Ramanathan R, Sailor MJ, Hatton TA, et al. SERS-coded gold nanorods as a multifunctional platform for densely multiplexed near-infrared imaging and photothermal heating. Adv Mater 2009;21:3175-80.

4. von Maltzahn G, Park JH, Agrawal A, Bandaru NK, Das SK, Sailor MJ, et al. Computationally guided photothermal tumor therapy using long-circulating gold nanorod antennas. Cancer Res 2009;69:3892-900.

5. Gao X, Cui Y, Levenson R, Chung L, Nie S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004;22:969-76.

6. Lo C, Xiao D, Choi M. Homocysteine-protected gold-coated magnetic nanoparticles: Synthesis and characterization. J Mat Chem 2007;:2418-27.

7. Casals E, Campos S, Bastus N, Puntes V. Distribution and potential toxicity of engineered inorganic nanoparticles and carbon nanostructures in biological systems. Trends Analyt Chem 2008;27:672-83.

8. Gerion D, Pinaud F, Williams SC, Parak WJ, Zanchet D, Weiss S, et al. Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ZnS semiconductor quantum dots. J Phys Chem B 2001;105:8861-71.

9. Schlenoff JB. Stabalized silica colloids. US Patent Appl 2009;20:2816.

10. Lee H, Lee E, Kim do K, Jang NK, Jeong YY, Jon S. Antibiofouling polymer-coated superparamagnetic iron oxide nanoparticles as potential magnetic resonance contrast agents for in vivo cancer imaging. J Am Chem Soc 2006;128:7383-9.

11. M. Racuciu1, D. E. Creanga, and A. Airinei. Citric-acid-coated magnetite nanoparticles for biological applications. J Eur Phys Jr E 2006;21:117-21.

12. Santra S, Tapec R, Theodoropoulou N, Dobson J, Hebard A, Tan W. Synthesis and characterization of silica-coated iron oxide nanoparticles in microemulsion: The effect of nonionic surfactants. Langmuir 2001;17:2900-6.

13. Lu Z, Dai J, Song X, Wang G, Yang W. Facile synthesis of Fe3O4/ SiO2 composite nanoparticles from primary silica particles.

Colloids Surfaces A Physicochem 2008;317:450-6.

14. Santra S, Zhang P, Wang K, Tapec R, Tan W. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal Chem 2001;73:4988-93.

15. Garcia M, Jemal A, Ward EM, Center MM, Hao Y, Siegel RL, et al. Global cancer facts & figures 2007. Atlanta, GA: American Cancer Society; 2007.

About the Author Philip Schlenoff has recently graduated from Maclay School in Florida, and has just started at the University of Florida where he is majoring in biomedical engineering. Aside from research he enjoys playing piano and reading.

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Young Scientists Journal | 2010 | Issue 8

Nanotechnology

Synthesis of fluorescent silica nanoparticles conjugated with rgd peptide for detection of invasive human breast cancer cells Shamik Mascharak Santa Cruz High School, Santa Cruz, California, Email: indiadrummer@gmail.com DOI:10.4103/0974-6102.68746

ABSTRACT

The objective of this research was to detect malignant human breast cancer cells using Fluorescent Silica Nanoparticles (FSNPs) conjugated with RGD peptide that exhibits high affinity toward integrin receptors on cancer cells. The fluorescent silica nanoparticles (FSNPs) were synthesized by the Stรถber method via controlled hydrolysis of tetraethylorthosilicate (TEOS) in a water/oil microemulsion in the presence of 3-aminopropyltriethoxysilane (APTS), fluorescein-5-isothiocyanate (FITC), and 3-(Trihydroxylsilyl)propylmethylphosphonate (THPMP). The FSNPs (70 nm in diameter) were checked for quality via TEM and fluorescence microscopy (515 nm), and confirmed to be consistent in shape and size. For tumor targeting, the FSNPs were conjugated to cyclo(Arg-Gly-Asp-D-Tyr-Cys) peptide (RGD) with the use of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) in DMSO. Both peptide-FSNP conjugates and FSNPs were added to MCF7 (benign breast cancer cells), MDA-MB 435 (transformed human breast cells), and MDA-MB 231 (metastasized breast cells) and after 2 hrs subjected to fluorescence microscopy. The FSNP-RGD peptide conjugates selectively got attached to the high concentration of integrins expressed on the surface of the cancer cells. In the case of the normal cells, the expression of integrin was low and hence such cells showed very few FSNPs on the cell surface. The results confirm that FSNP-RGD conjugates are excellent imaging tools for cancer detection. Since silica nanoparticles are inexpensive, readily synthesized, and relatively non-toxic, they afford a convenient method to identify malignant sights in cellular matrices.

Introduction Functionalization of silica nanoparticles (SiNPs) is a simple, cheap and effective strategy for the preparation of multichromophoric detection systems.

For example, dye-doped silica nanoparticles could be conveniently employed as cellular markers and tracked via their fluorescent properties. Compared to semiconductor nanocrystals (quantum dots such as Young Scientists Journal | 2010 | Issue 8

CdTe QDs), SiNPs are far less toxic due to the lack of heavy metals. The non-cytotoxic properties along with their inherent dispersivity make SiNPs a superior material for microscopic imaging in vivo. Another advantage of SiNPs is the fact that their fluorescent properties can be altered with the judicious choice of different dyes. In this project, fluorescein was chosen as it is also a cheap and effective reagent for imaging (green fluorescence). Fluorescent Silica 37

Nanoparticles (FSNPs), are conveniently synthesized following the Stöber method which involves controlled hydrolysis of tri- and tetra-alkoxy silanes. In such synthesis, the dye can be incorporated within the SiNP by adding another silane in which the dye is covalently attached to the silicon center. The dye gets covalently attached to the Si-O-Si network and is tightly incorporated within the SiNPs (no leakage).

While FSNPs can be used for a variety of imaging purposes, in this project they were used for the detection of invasive human breast cancer cells. In such a pursuit, three human breast cancer cell lines namely, MCF-7, MDA-MB-231 and MDA-MB-435 (provided by UCSC drug screening laboratory) were selected. In these cancer cell lines, the extent of regulation of the integrin family of proteins (surface receptors responsible for metastasis) is known to vary significantly. In particular, the αvβ3 integrin protein is known to be overexpressed in the MDA-MB-231 and MDA-MB-435 cells, although the degree of expression does vary. This integrin is not present on the surface of MCF-7 cells (and hence can be used as a control). The major goal of this project was to conjugate FSNPs to an exogenous ligand that could distinguish the different levels of expression of this integrin. If the conjugate could specifically bind to the αvβ3 integrin, then one can both identify the cancer cells and determine the progression of metastasis.

Integrins are heterodimeric cell surface receptors [Figure 1] that mediate adhesion between cells and communication with the extracellular matrix.[1] They bind strongly to ligands that contain an exposed arginine-glycine-aspartate (RGD) moiety. These

integrins are involved in cell growth, migration, and survival. The integrins, if not properly regulated can lead to thrombosis, inflammation and cancer. As stated before, integrins (such as the αvβ3 integrin) have been demonstrated to be present in high concentration on cancer cell surfaces (such as metastatic human breast cancer cells). Since integrins are overexpressed in malignant cells, various exogenous agents can be delivered to cancer cells via the strong integrin-RGD interaction. In this project, the high affinity of the overexpressed αvβ3 integrin (on cancer cell surface) for the RGD motif has been exploited for the detection of metastatic human breast cancer cells by conjugating the RGD peptide with the FSNPs. As described below, the RGD-decorated FSNPs indeed identified cancer cells via attachment to the cell surface due to strong RGD-αvβ3 integrin interaction. Since the integrin concentration is significantly lower on the surface of the benign MCF-7 cells, very few RGD-FSNPs were noted on such cells. The detection method is thus proven to be quite effective in identifying invasive human breast cancer cells.

Investigative Questions 1) Are RGD-peptide conjugated fluorescent silica nanoparticles (RGD-FSNPs) stable and fluorescent over long periods of time? Are they uniform in size?

2) Can RGD-FSNPs be used to distinguish MDAMB-231 (metastasized human breast cancer) and MDA-MB-435 (transformed human breast cancer) cells from benign MCF-7 breast cancer cells?

Materials and Methods

Figure 1: Structure of αvβ3 integrin showing the αv units in blue and the β3 units in orange

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A. Preparation of the Fluorescent Silica Nanoparticles (FSNP) The FSNPs were synthesized by the Stöber method[2] via controlled hydrolysis of tetraethylorthosilicate (TEOS) in a water/oil microemulsion in the presence of 3-aminopropyltriethoxysilane (APTS), fluorescein5-isothiocyanate (FITC), and 3-(Trihydroxylsilyl) propylmethylphosphonate (THPMP). APTS was used in excess in order to decorate the surface of the FSNPs with -NH2 groups (for further conjugation with RGD peptide). Since such -NH2 groups will be present as -NH3+ in physiological buffers (pH 7.4) and reduce the ζ-potential (thus promoting coagulation of FSNPs), THPMP was also added to the reaction mixture. The THPMP makes negativelyYoung Scientists Journal | 2010 | Issue 8

charged chemically inert methylphosphonate groups available on the FSNP surface thereby increasing the ζ-potential.

First, the FITC-APTS conjugate was synthesized by mixing 69 mg of APTS with 5.25 mg of FITC in 1 mL of absolute ethanol under dry N2 atmosphere. The mixture was stirred for 24 h. During the synthesis, the FITC-APTS conjugate was protected from light to prevent photobleaching. This conjugate solution was used as the fluorescent silane reagent. A waterin-oil emulsion was prepared by mixing 7.7 mL of cyclohexane (oil), 1.77 gm of TX-100 (surfactant), 1.6 mL of n-hexanol (cosurfactant) and 0.34 mL of DI water in a 30 mL rb flask for 30 min. With an interval of 10 min between 2 successive additions, 50µl of FITC-APTS conjugate, 100µl TEOS and 100µl ammonium hydroxide was added. After 30 min of stirring, 15µl of THPMP was added and the mixture was stirring was continued at room temperature. After 24 h, the microemulsion system was destabilized by adding denatured ethanol (roughly 12 mL), and the FSNPs were collected by centrifugation (2000 RPM, 10 min). The FSNPs were then repeatedly washed and centrifuged 4 times with ethanol and 2 times with DI water. For redispersing FSNPs, each centrifugation step was followed by vortexing and sonication. The final FSNPs in DI water were stored in the dark to prevent photobleaching and later checked for quality by Tranmission Electron Microscopy (TEM) imaging.

B. Conjugation of the RGD peptide to the FSNPs A batch of 25 mg of FSNP was dispersed in 1 mM Tris-citrate buffer (pH 7.4). Next, 25 mg of SPDP was dissolved in 0.5 mL of DMSO and the solution was added to the FSNP suspension. The mixture was then stirred at room temperature (under dark conditions) for 12 h. Next, the reaction mixture was spun down (2000 RPM, 10 min) to obtain the FSNP-SPDP conjugates as a fluorescent pellet. This pellet was resuspended in the same buffer and once again spun down (to remove any excess SPDP). The pellet was finally dispersed in 7 mL of tris-citrate (pH 7.4) buffer and to it was added a solution of 5 mg of cyclo(ArgGly-Asp-DTyr-Cys)[3] in 0.2 mL of DMSO. The mixture was stirred overnight at room temperature (under dark conditions). Next morning, the reaction mixture was centrifuged at 1700 RPM for 10 min to collect the RGD-FSNPs. They were finally resuspended in DI water and stored in the dark. A small batch of the Young Scientists Journal | 2010 | Issue 8

peptide-conjugated nanoparticles was used to obtain TEM images [Figure 2]. Also, their fluorescence spectrum (Perkin Elmer Fluorescence Spectrometer) showed no variation in fluorescence characteristics [Figure 3] following RGD conjugation.[4]

Figure 2: ITEM image of the FSNPs

Figure 3: Fluorescence spectra of the various samples

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C. Cell experiments All three cell lines were placed in 8-well plates with 0.5 mL of DMEM growth medium and incubated in 5% CO2 incubator for 72 h. After the cells grew to confluency (checked by visible-light microscopy), 0.25 mL of the medium was pipetted out from each well and 0.25 mL of the RGD-FSNP solution was added. The trays were placed back into the incubator for 2 h in order to allow the NPs to interact with the cells. Next, all the liquid was aspirated off and each well was carefully washed 2 times with 0.1x PBS buffer. The cells were then fixed with paraformalin fixer. After 15 min, the wells were again washed 2 times with the PBS buffer, 1 time with the quenching buffer (containing glycine) and 1 time with DI water.

Next, the cells were stained with DAPI and the well covers were removed. Fluoro-Mount drops were added on each cell cluster and slide covers were placed on the slides. The trays were then allowed to dry for 45 min. Finally, the four sides of the slides were sealed with transparent nail polish. These slides were stored in the refrigerator. A parallel set of experiments was carried out with three trays of the same cell lines and FSNPs without RGD by following the same procedure. These served as controls for my experiment. The cells were visualized with the aid of a Zeiss HAL 100 fluorescent microscope.[5,6]

some interactions with the cell surfaces were noted.

However, the FSNPs did not show specificity.

Conclusions The results shown in Figure 4 clearly indicate that the RGD-FSNPs are stable in water and exhibit strong fluorescence. Experiments also showed that the intensity does not change over days of storage. Figure 5 confirms that the RGD-FSNPs can identify cancer cells on the basis of αvβ3 integrin concentrations on cell surfaces. For example, while MCF-7 cells with an inherently minimal concentration of αvβ3 integrins show very few RGD-FSNPs on their surfaces, MDA-MB-231 cells exhibit strong

Results A. TEM Images of the FSNPs Dilute solutions of the FSNPs in ethanol were spread on TEM slides, dried under vacuum, and the TEM images were taken using a JEOL 1200 EX instrument (Dr. Yang of UCSC provided help in such measurements). The images [Figure 2] clearly showed that the particles were all spherical and of uniform (~ 70 nm) diameter.

B. Fluorescence spectra of FSNPs[7] C. Fluorescence spectra and TEM images of the RGD-FSNPs D. Results of cell experiments The merged fluorescent images clearly showed increased NP interactions with MDA-MB-231 and MDA-MB-435 cells while MCF-7 cells exhibited minimal interactions. NP association with the cells was specific to each cluster on the slide with few NPs noted outside the cell-covered areas (showing no adhesion to the slide floors). It was also evident that there were greater NP interactions with the highly invasive MDA-MB-231 cells which are known to exhibit higher expression levels of αvβ3 integrin on their cell surfaces. With FSNPs without RGD, 40

Figure 4: Fluorescence spectrum and TEM image of RGD-FSNPs

Figure 5: Images of the three cancer cell lines showing increased interactions with the RGD-FSNPs in the more invasive lines

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association with RGD-FSNPs. It is therefore possible that RGD-FSNPs could be employed to distinguish between cancer cells with varying concentrations of αvβ3 integrin and hence their likelihood of metastasis.

of their invasiveness (metastatic tendency). Further research on this detection system could lead to a cheap, effective, and non-toxic method for in vivo imaging of invasive breast cancers.

Broader impact of this work Breast cancer is the second leading cause of cancer deaths in women today (after lung cancer) and is the most common cancer among women.[8] According to the American Cancer Society, about 1.3 million women will be diagnosed with breast cancer annually worldwide and about 456,000 will die from the disease. Metastasis (to bone, liver and brain) is the primary cause of death in human breast cancer.[9] While the breast cancer rate has risen in the last 30 years in western countries, breast cancer deaths have been dropping steadily since 1990 thanks to better treatments and early detection.

Clearly, there is a need for tools for early and effective detection of breast cancer.

Acknowledgments

The RGD-FSNPs in this project has been synthesized by using very simple chemical steps performed under mild conditions (room temperature, simple centrifugation). The synthetic steps employ no severely toxic materials (like H2Te in CdTe quantum dot synthesis) and silica-based materials are known to have low cytotoxicity. Since the fluorescein dye is trapped within the silica NPs, no photobleaching is observed with these FSNPs. The synthetic protocol affords particles of uniform size and they are stable over months. This detection tool is therefore noteworthy for its easy preparation, sensitivity, and ability to distinguish human breast cancer cells with varying degrees of αvβ3 integrin regulation.

The integrin has been implicated in the pathology of metastatic breast tumors. The detection system described here demonstrates its ability to distinguish between breast cancer cells of different kinds in terms

Mr. Walter Bray of UCSC Drug Screening Facility provided the cells for imaging. Help from Mr. Michael Rose, a graduate student of the Chemistry Department of UCSC, in the synthetic experiments, in fixing and staining cells, and in the use of the Fluorescent microscope is gratefully acknowledged.

Dr. Y. Yang of UCSC provided help in obtaining the TEM images of the FSNPs. Thanks are also due to Prof. Ted Holman of UCSC for allowing use of his Fluorescence Spectrometer and to Prof. Lindsay Hinck of UCSC Biology Department for use of her Fluorescence Microscope. I’d also like to thank my parents Nandini and Pradip Mascharak for their constant support and encouragement.

References 1. "Integrin. Available from: http://www.wikipedia.com. [cited on 2009 Mar 3]. [updated on 2009 Jan 11].

2. Stober, W. Colloid Interface Science 1968. p. 62-9.

3. Peptides International. Louisville: Peptides International; 2008.

4. Hermanson GT. Bioconjugate techniques. 2nd ed. Academic Press; 2008.

5. Montalti, Marco. Plasmonics in Biology and Medicine. Vol. 3.

6. Williams DB, Carter CB. Transmission electron microscopy a textbook for materials science (4-Vol Set). New York: Plenum; 2004.

7. Skoog DA. Principles of instrumental analysis. Philadelphia: Saunders College Pub., Harcourt Brace College; 1998.

8. Breast Cancer Facts and Figures (2007-2008). American Cancer Society; 2008.

9. Felding-Habermann B, O'Toole TE, Smith JW, Fransvea E, Ruggeri ZM, Ginsberg MH, et al. Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci U S A 2001;98:1853-8.

About the Author Shamik Mascharak is 16 years old and lives in Santa Cruz, California. In his free time, he enjoys playing drums and guitar, as well as doing Tae Kwon Do (where he holds a second degree black belt). He also participates in a number of extracurricular activities such as Mock Trial and the local Kuumbwa Jazz Honor Band (where he is the drummer). Upon graduating, he wants to attend college and eventually become a researcher in a medical field.

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Young Scientist Notes

Building from the Ground up: Nanostructures to microstructures Steven Noyce American Fork High School, E-mail: steven.noyce@gmail.com DOI:10.4103/0974-6102.68751

Creativity is at the heart of existence. Throughout history, humankind has sought to create, whether it is a painting, a piece of architecture, or a machine.

Incredible feats have been realized, with increasing complexity and diversity, as technology has enabled us to create larger buildings, more vibrant images, and smaller electronics. Although countless enabling discoveries have been made, there is a common theme that has sparked the technological explosion of the last century: the utilization of the small. Miniaturization has led to everything from our advanced electronics to our convenient microfiber couches that easily wipe clean after a spill. We have seen amazing advances, but as a whole have not come anywhere near utilizing the possibilities that small scales provide. The research now presented deals not only with the creation of nano-scale structures such as Carbon Nanotubes (CNTs), but also on the assembly and use of these components in micro and macro scale devices, such as MicroElectroMechanical Systems (MEMS).

When I began my 2009 International Science and Engineering Fair (ISEF) research project, I knew exactly what I wanted to work with: Carbon Nanotubes. I was blown away by the countless unique features of these molecules; ballistic electron transport, variable semiconductivity, tensile strength greater than any other material except the related Graphene molecule, impermeability to gaseous hydrogen, Van Der Waals radii allowing huge macroscale forces, and countless other properties 42

made CNTs seem like a molecule of dreams.

And dream, I did, but for quite some time it seemed like that was all I would ever do. You see, I was working in a High School chemistry room, with extremely limited resources, and in these conditions I feared that I would never create even one measly nanotube. I worked tirelessly, however, and in time, success came. By doing extensively modified replications of experiments I had seen in journal articles, I first began synthesizing amorphous carbon product, then impure CNT/carbon compound, and eventually, a pure Carbon Nanotube product.

As my replications began to work, I started to realize ways that the processes could be improved upon. I used my physics and chemistry background to work out the math and build models for some of the ideas I was having, and then I started to try them out, again dealing with very limited supplies and apparatus.

One of the first improvements that I made was adding chlorine gas (generated from the controlled electrolysis of salt water and used with stringent safety precautions) to the flame synthesis setup I had running. The basic idea of the setup was to take a hydrocarbon gas, such as methane, mix it with hydrogen gas and combust it in a controlled flame with a chemically isolated, catalytically activated metal mesh slicing through the flame. The high thermal conductivity of the mesh transferred a large amount of heat from the combustion region on the Young Scientists Journal | 2010 | Issue 8

outside of the flame to the inner gas cone, where oxygen was not present (a necessary requirement for CNT growth). Some great chemistry then had a chance to occur in the localized high temperature regions on the surface of the mesh. First, the iron salt which the isolated mesh had been coated with experienced a heat decomposition, leaving only a mix of iron and iron oxide behind. It then became diffusively mobile, conglomerated into small nanoparticles, and was reduced to porous iron metal by the hydrogen gas. Next the hydrocarbon gas was cracked at the catalytic iron sites, often yielding carbon atoms which went into a solid state steal solution, eventually causing the iron particles to become saturated with carbon solute. Finally supersaturation and crystallization occurred, with the “crystal” being a carbon nanotube that emerged and “grew” from the iron clumps. The reason that the addition of chlorine was helpful to this process is twofold. Not only did the chlorine remove the undesirable contaminant of amorphous carbon more readily than the CNTs, but it also forms intermediate chlorocarbons that are more prone to donating carbon atoms to the iron catalyst. This improvement synthesized better quality nanotubes with less impurities with less reactant in less time.

Without going into great detail, some of the other processes that I improved upon were Hot Wire Generator synthesis (which I rigged up using tungsten filaments from old light bulbs in various arrangements), synthesis in fused sodium chloride using an exfoliated graphite precursor (pencil “lead” in a blender with some chemicals), high voltage arc synthesis, and carbon electrode arc synthesis in various media. These methods were all great, and I was thrilled with the success that I was having with them, but I sensed that there was something missing.

The end result of all of these procedures was a powdery black mess of CNTs, which, although having many incredible applications, did not seem, like the highly ordered nanostructures I had wanted to create.

I began to realize the source of my dissatisfaction.

Although the nanotubes themselves had low entropy with their precise arrangements of atoms, they had no order with respect to one another as they lay jumbled together. It seemed to me that although CNT composites and the like had an incredible amount of potential, the most exciting properties of nanotubes were at either the individual or highly organized Young Scientists Journal | 2010 | Issue 8

level. My goal became to create ordered arrays of nanotubes, and so I set out, shooting for the stars.

The only technique I had found in the literature that gave me a hint as to how I would go about accomplishing this goal was that of patterned, plasma-enhanced chemical vapor deposition. I had no idea how I could possibly get something similar to work in my meager High School lab, so I went into overdrive trying to reason out what simplifications I could make in order to set up a functional apparatus.

Although I didn't have the means, the first simplification I knew I needed to make was to set up a proper RF coil. Because of my limited access to scientific literature, I did not know if it would be possible to do the same process thermally, but I took a guess and hoped that it would, and worked out the thermodynamics so that I could sleep at night. I scavenged for materials, trying with all my might to find things that would be chemically inert, thermally stable, geometrically workable, and most of all, obtainable. I never did find an ideal solution to all those problems for the reaction chamber, since I didn't have access to quartz or porcelain tubing, but I was able to work things out with some carefully oxidized aluminum. For the substrate, I had similar troubles and settled on aluminum again, this time an alloy with a high enough melting point. For the iron catalyst, I needed precise control over the thickness, composition, and pattern. I used a Sharpie marker to replicate the photolithography process, drawing simple patterns on the substrate. I then used an electroplating technique to put down an extremely thin layer of iron on the areas where Sharpie was not preventing conductivity. Once again I used natural gas and hydrogen obtained from the electrolysis of water as my reactant gases, and a combination of an old ceramics kiln and the grills from a toaster for the heat source. After a lot of work and more luck, I had finally produced true carbon nanotube microstructures!

At this point my chemistry teacher decided he could use the work I had done to get me involved with a university research group. I had previously contacted a local university, but they had turned me down, telling me that no professors were interested in working with High School students. At this point, however, my teacher brought a faculty member from 43

the university over to my school and had him take a look at what I was doing. He seemed impressed with what I had done, and gave me the contact information of a professor who led a CNT research group. I started attending group meetings, and discovered that the work being done by the group was nearly identical to the setup I had recently finished throwing together. They informed me of their process of filling the CNT microstructures with silicon in order to make MEMS, or MicroElectroMechanical Systems, and I was immediately enthralled with the idea, as it provided a simple method of making MEMS from any material with aspect ratios tens of times higher than previous fabrication methods.

that neither require vacuum conditions to operate nor suffer from evaporative filament degradation.

A second item was a much less than paper thin speaker that operates by thermoacoustical methods and thus has no moving parts and can be bent or stretched into any shape. A final item that I made was that of a non sticky, extremely strong adhesive tape that is stable under fairly high temperatures and is able to support very high forces. After assembling these items, I explored CNTs further by suspending them in various solutions and running time series dynamic conductivity tests that provided information about their properties and how they interact with other molecules.

I was thrilled to finally have access to high quality equipment, and started doing research into the specifics of CNT synthesis so that I could get a better framework in anticipation of my future in MEMS fabrication. I studied how various conditions, such as ethylene flow rate, hydrogen flow rate, reactant composition, argon dilution, and other variables affected the growth rate, dynamics, and maximal height. I reasoned that a likely limitation on the growth height was the coating of the iron nanoparticles with amorphous carbon, but I knew that adding an oxidizer would etch the CNTs, so I devised a phased water vapor incorporation method that increased the maximal growth height by a factor of 3. I studied nanotube oxidation rates and increased the efficiency of synthesis throughput. I researched the process of iron diffusion that occurs during growth, found several easy methods of determining the post-synthesis location of the iron, and looked into the practicality of substrate reusability. The findings from these projects were great additions to the CVD (chemical vapor deposition) process, making it more efficient, extensible, and usable.

I was finally ready to use the information I had gained in an effort to improve upon the CNT MEMS fabrication process. My first project dealing with this was the â&#x20AC;&#x153;RIE-less Process,â&#x20AC;? in which I worked out some of the details of using a thermal oxidation step in order to avoid the expensive and cumbersome Reactive Ion Etch that had previously been used to remove the floor layer so that MEMS devices could be released. The problem then arose that the small amount of iron at the base of the CNT forest precluded our samples from entering some semiconductor processing machinery, so I worked on a gas phase iron removal process that was not harmful to the CNTs, but would steal the iron out from under them. After that, I became interested in using different materials for the sacrificial layer, instead of silicon or silicon dioxide. I tried chromium, aluminum, nickel, iron, and copper with some success, but it was difficult to get as good of growth atop these materials. After those attempts, I became interested in whether the number of walls on the multi-walled CNTs could be adjusted during a growth by changing reactant gas compositions.

I experimented with this briefly, and found first that by stopping reactant flow for a short time during growth, a line could be drawn across the forest, and if reactants were turned back on at different flow rates afterward, then the change in growth rate made a very slight alteration to the side-wall angle, so that by repetition of this process, a limited amount of three dimensional control over the forest structure was possible.

At this point, I was almost ready to start making CNT MEMS, but before I began, I wanted to learn more about CNT properties, and I did so by finding interesting ways to use them. My first investigations were making CNT/polymer composites of various composition. These materials have a wide range of applications, yet I focused on how strength and conductivity vary with CNT concentration. I then moved on to creating conductive CNT sheets and wires, and from them I was able to make several useful items. The first was that of incandescent light bulbs 44

Through all of the above methods and others not mentioned, I was able to improve upon many aspects of engineering structures on both the Young Scientists Journal | 2010 | Issue 8

nanoscale and the microscale. I may not have made one end all, incredible discovery, but I was able to effectively feed my creative spirit and love of learning by doing many separate experiments, which all blended together as contributions to

the singular goal of designing simple methods of advanced microfabrication through organization of nanoscale precursors. The experience I had through doing research was enlightening, and life changing.

About the Author Motivated. That's how I would describe myself. My desire has always been to learn more, to do more, and to be more.

I have saught improvement, both of myself and those around me. Although I have put forth great effort in this regard, it has been only through divinely given strength that I have had any capacity to acheive. When I was young my learning was disabled, yet I never allowed my future to appear dark. My life has been one filled with hope, and I yearn for my story and research to instill such in others and improve the world in which we live.

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Young Scientist Blogs

The best six days ever

Courtney Williams 19 Years, Imperial College London, South Kensington Campus, London, UK, Email: cewilliams7@gmail.com DOI:10.4103/0974-6102.68753

In September 2009, I was lucky enough to attend the 21st EU Contest for Young Scientists in Paris.

The EU Contest for Young Scientists brings together scientists aged 14-21 from around Europe and elsewhere in the world for six days of competition, cultural exchange and social activities. As you can see from the title it was, y’know, not too bad. This article was originally published along with tweets as a series of blog posts on the YSJ website.

of Sheffield and funded by the Nuffield Bursary Scheme, I studied background noise from whales and dolphins, and also simulated the sounds the neutrinos would make in a big water tank. I worked on it for six weeks in the summer holidays; some people at EUCYS had spent years on their projects!

The night before getting the Eurostar to Paris I only got about three hours sleep – on top of poster-related anxiety, there was the fact the presentation I wanted to have on loop on my stand refused to save on my netbook, and of course general “I don’t deserve this free holiday with lots of smart people” angst.

I managed to get to London with everything intact eventually though.

The contest was held at the Palais de la découverte – the UK team (Hannah Stuart, Robert Tann and me, plus Ellie Chambers, our “escort”) arrived too late for the scheduled set-up time (and most of the opening ceremony), but we arrived safely after getting better acquainted on the train and got to see a bit of Paris after dark. We also got bright red bags full of stuff, including bright yellow visibility jackets, though thankfully didn’t have to use them at all.

I won the chance to go to EUCYS through my national fair, the 2009 Big Bang Fair, as one of three UK representatives. My project dealt with work I did for the Acoustic Cosmic Ray Neutrino Experiment, or ACoRNE. It aimed to detect tiny particles called neutrinos by listening for the sounds they were meant to make when they were very energetic and entered seawater. For my project, carried out at the University 46

The next day, the competition began. We were all subject to “jury visits”, where we were asked about our project and judged – there were seventeen jury members altogether from a range of different fields.

Coincidentally, the head of the jury was a professor in the physics department of Imperial College London; I study theoretical physics at Imperial! He didn’t judge Hannah, Robert or me for obvious reasons, but he did come round to say “hi” and ask about our projects informally. Unlike the Big Bang Fair, all the EUCYS projects were organised by field – this was Young Scientists Journal | 2010 | Issue 8

advantageous for me because it gave me a fighting chance of understanding a few of the projects around me! I still have no clue how the jury picked winning projects, considering how high quality all the stands I saw were, and how many I couldn’t even begin to understand. In the morning we also had a lecture from a representative of the European Patent Office. I didn’t actually get a jury visit on the first day – whether that was a good thing or not is debatable!

In the evening I attended two demonstration sessions at the Palais: electromagnetism, in which we watched lightning being created artificially, and “Galileo’s Merry-go-round”, in which some of us sat on a carousel and felt and saw the effects of forces acting on us as we spun round. If you go to Paris I really recommend you visit the Palais, as I will if/when I return to Paris – it has a huge range of exhibits and the emphasis is on “hands-on” learning, though be warned that all the information on the exhibits is in French.

EUCYS has a good balance between competition and social elements – we got to do a lot of touristy things in addition to being judged and meeting fellow young scientists! One of these things was a cruise down the Seine! The weather was great that evening (though it got cold and wet later in the week) and it was great to see Paris from the water. If you want to watch a video of our cruise you can see it at http:// www.youtube.com/watch?v=sOSkI8MJLBM After our Seine cruise came something I’ve always wanted to do – we went up the Eiffel Tower for dinner! We had to wait a bit until we could go into the restaurant for dinner, so lots of us had a wander about the first floor of the Tower and took in the sights and sounds of Paris by evening. We even watched what was apparently Europe’s largest firework display across the city! Unfortunately one of the UK representatives, who shall remain nameless, managed to get lost on the second floor of the Tower...

The third day brought my first judging sessions, then in the evening a visit to the Musée des arts et métiers and two lectures by two scientists from EIROforum facilities. The European Molecular Biology Laboratory lecture given by Jacopo Lucci was Greek to me, but the one given by Paula Stella Teixeira of the European Southern Observatory on stellar and planetary formation was very interesting.

In addition to jury members coming round, there Young Scientists Journal | 2010 | Issue 8

were national organisers and researchers (with members of the public and schools coming round during certain sessions) – one person advised me to speak more slowly and pause at the end of sentences. I think more than anything else attending EUCYS helped me with talking to people whose first language wasn’t English! On the third day one jury member asked me how my project was original – despite saying that the studies I did hadn’t been carried out by the ACoRNE collaboration before and acoustic detection of neutrinos was a fairly new field (i.e. people HAVE studied marine mammals and detector responses before, just not in this context) she seemed unconvinced. It didn’t really matter too much since I wasn’t there to win, but it did leave me feeling a little annoyed. On a slightly more lighthearted note, I apparently became known as “the girl with the neutrinos” – Neutrino Girl always did strike me as an awesome superhero name...

The student helpers assigned to each country were really great; I wouldn’t have seen half the stuff I did (Notre Dame, Sacre Coeur, Montmartre, Champs d’Elysses, Arc du Triomphe...) without the student helpers organising jaunts out to various landmarks.

It meant we got to see Paris as if we were tourists, in addition to being in the city for a specific purpose.

Most people had at least one dodgy jury session, me being no exception. On the fourth day, this particular jury member let me explain half of my project, then exclaimed his disbelief, then spent five minutes reading my poster, then said “thank you” and left.

Hmph. On the other hand, it was always nice when jury members enquired about what university you were heading to, what your future plans were and so forth. Well, after the mean jury member I was just glad the rest let me finish talking!

I had seven jury members come round in total – the norm was five or six, but some people had even more than seven! On the fourth evening there was a round table with Wendelin Werner (Fields Medallist), Jean Dalibard (atomic physicist), Claudie Haigneré (spationaut, aka a French astronaut) and Pascale Cossart (bacteriologist), plus a representative of the EU Commission. Very interesting discussions took place, with many young scientists asking questions.

After, we had dinner at the nearby Café de la musique, with music provided by the Original Jackass Band and a few of the more co-ordinated among us taking to the dance floor!

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On the fifth day Hannah, Ellie and I went to the Chaillet area of Paris for shopping since we had a few hours between the last jury session and the award ceremony. Robert went with another group that visited Père Lachaise, a big cemetery where lots of famous people (including Oscar Wilde) are buried. During the morning session I had a walk round the previously unexplored life sciences stands and found that some of them were actually understandable! That’s not a bad reflection on them, just my puny physicist mind unable to cope with all the scary long words.

On the fifth evening came the award ceremony in the Palais. We were sat with Estonia, who were missing a flag (each table had small flags on them as markers) – using our initiative as young scientists we cobbled one together! The first prize of the evening was the EIROforum CERN prize – the winner gets the chance to spend a week at CERN, visiting the permanent exhibition and various experiments and departments.

I was kind of shocked when my country, then my name was read out. Kind of really shocked. To the extent where I was still shaking at the end of the ceremony. Without a speck of false modesty I thought my project was the weakest of the physics projects.

In the end a lot of the prizes went to physics projects; I can’t really comment on whether more life sciences ones deserved to win since I didn’t see or understand all of them, but I think all the prizewinners were very worthy. In particular, the first prize winners and some of the youngest contestants, Liam McCarthy and John D. O’Callaghan, for their project on a convenient test for somatic cells in cow’s milk – so simple and yet so brilliant! In addition to prizes, the awards ceremony also included magicians coming round to our tables and an incredible beat boxer!

Before the UK team caught the train back to London we headed back to Montmartre for a few hours for some last-minute shopping. I have to say that even if I hadn’t won a prize, just experiencing the contest was a sufficient prize! As well as exploring Paris and taking in lectures and museums, I got to meet young scientists from all over the world (who I still keep in touch with via the wonder that is Facebook). I don’t think I’ll ever forget the experience!

About the Author 19 Years, Imperial College London, South Kensington Campus, London, UK. Email:cewilliams7@gmail.com

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