ISyPeM II Giulia Cappi École polytechnique fédérale de Lausanne
Annual Plenary Meeting of the Nano-Tera program May 4th, 2015
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Therapeutic drug monitoring (TDM)
Imatinib exposure (ng/ml)
Candidates for therapeutic drug monitoring Modern anticancer agents Immunosuppressants Antiretrovirals Antibiotics
Courtesy of N. Widmer
enrico.accastelli@epfl.ch
time after dose (hrs)
EDMI Research Day 2012
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Therapeutic Drug Monitoring Today Transfer to and measurement at central clinical labs
Interpretation of the result by a specialized doctor
Drug Concentration
Blood sample taken at the doctor’s office or at the hospital
Target
? Patient Treatment duration (days)
http://e-sante.futura-sciences.com
Abbott "Architect ci8200"
Today this procedure severely restricts the applicability of TDM enrico.accastelli@epfl.ch
EDMI Research Day 2012
Drug quantification in body fluids Used in Clinical facilities
Agilent LC-MS/MS
enrico.accastelli@epfl.ch
Abbott "Architect ci8200” FPIA (fluorescence polarization immunoassay) CMIA (chemiluminescent microparticle immunoassay) MIA (magnetic immunoassay)
EDMI Research Day 2012
Point-of-need
Syva RapidTest® EMIT (Enzyme multiplied immunoassay technique)
4
Point-of-need therapeutic drug monitoring Hand-held ISyPeM approach toTDM
iSTAT, Abbott
Accu-Chek product lines. Roche Diagnostics
Top bench
Cobas product lines Roche Diagnostics enrico.accastelli@epfl.ch
Clonit StMicroelectronics
EDMI Research Day 2012
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ISyPeM. A solution to enable drug-based therapeutic drug monitoring
• Low-sample volumes • Miniaturized reader • Cartridge that can be stored with stable reagents enrico.accastelli@epfl.ch
• Monitoring and prediction interface for clinicians and practitioners • Local or remote
EDMI Research Day 2012
• Extension of eHealth medical standards • Data integrity
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ISyPeM: A solution to enable drug-based therapeutic drug monitoring
HOMOGENEOUS AND HETEROGENEOUS ASSAY
• Cartridge that can be stored with stable reagents enrico.accastelli@epfl.ch
In vitro selection of capture molecules for drug targets EDMI Research Day 2012
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In vitro selection of monoclonal antibodies for Tacrolimus Heinis Lab
Antibody phage display
Target: tacrolimus
Sangram Kale
Phage-antibodies were successfully selected for Tacrolimus and expressed enrico.accastelli@epfl.ch
EDMI Research Day 2012
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In vitro selection of DNAaptamers for Tobramycin
Guiducci Lab
DNA beacon aptamers Capture Selex
F. M. Spiga Now with Creoptix Sensors
Spiga F. M. et al. ACS Comb. Sci. 2015, featured on the cover of May Issue enrico.accastelli@epfl.ch EDMI Research Day 2012
No drug derivatization High affinity Specific Functional in serum 9
Low-volume sample preparation on chip Plasma extraction from whole blood Renaud Lab
Yield outperforms any passive on-chip method reported to date
David Forchelet
http://dbs-system.ch
FPIA of Tobramycin performed in glass microcapillaries Squared glass capillary 0.26
reference system
300 Âľm
Anisotropy
0.24
capillary
0.22 0.2 0.18 0.16 0.14 0
1
2
3
4
Concentration [Âľg/ml]
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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6
7
Diana Burghelea J-M Segura 10
Measurement system: miniaturization of an established assay technique Fluorescence Polarization Immunoassay Competitive assay Highly-sensitive Compatible with small volumes
Martial Geiser team
20 cm
25 cm
*
Requires derivatization of drug molecules enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Label-free quantification of drug from serum samples Heterogeneous assay based on binding kinetics
Guiducci Lab
No drug derivatization No additional reagents
Transmission Surface Plasmon Resonance Change of optical properties of the interface
200 nm
Giulia Cappi
Layers of functionalized gold nanoislands Cappi G. et al. Sensors and Actuators 2013 enrico.accastelli@epfl.ch EDMI Research Day 2012
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Hue readout of surface plasmonics Compact read-out system
CMOS image sensor Guiducci Lab
Hue readout of surface plasmonics
Cappi G. et al. Analyt. Chem. 2015 enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Measurement of binding kinetics of DNA aptamer-Tobramycin Guiducci Lab
Transmission SPR signal
KD = 200 nM, same as extracted from commercial SPR
Cappi G. et al. Analyt. Chem. 2015 enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Tobramycin quantification from serum ✔ Small molecules ✔ Complex Matrix
✔ Small molecules ✖ Complex Matrix
Chang, A. L. et al., Anal. Chem. 2014, 86, 3273−3278.
✖ Small molecules ✔ Complex Matrix
Jang, H. R., et al. Anal. Chem. 86, 814–819 (2013)
enrico.accastelli@epfl.ch Cappi G. et al. Analyt. Chem. 2015
EDMI Research Day 2012
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Supporting clinical interpretation EzeCHiel interface Population-based percentiles
Dosing schedule suggestion
Individual concentration vs time profile Yann Thoma
Div. Clin. Pharmac.
Aline Fuchs
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Data management
Health data Medical DB POC
Data security Dynamic Health Data Aggregation
Alevtina Dubovitskaya
Data Exchange between EzeCHiel and DB of the Medical “translate� from one representation to another
Institution
RSDB
MOLIS, CHUV
EzeCHiel
Dubovitskaya, A. Privacy Preserving Interoperability for Personalized Medicine, in: Swiss Medical Informatics, Swiss Society for Medical Informatics, Switzerland, 2014
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Population pharmacokinetics Meta-analysis. Implementation and validation
Div. Clin. Pharmac.
Aline Fuchs
Aziz Chaouch
Pharmacokinetic models
Br J Clin Pharmacol. 2014 Nov;78(5):1090-101
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Acknowledgements
Guiducci Lab
enrico.accastelli@epfl.ch
EDMI Research Day 2012
BACK up
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Filtration and FPIA on paper FPIA for tacrolimus with antibody selected by EPFL (tacrolimus fluorescent derivate needed) Aptamer for imatinib EzeCHiel start up and beta testers Interoperability with MOLIS database Population pharmacokinetic studies on tacrolimus and tobramycin enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Personalized Medicine and TDM  Therapeutic drug monitoring (TDM) is foundational to the concept of personalized medicine.  The concept has later evolved to include pharmacogenomic and other biomarker-driven strategies for patient segmentation In the clinics: TDM transformed drug therapy by affording the ability to characterize sources of variability in drug disposition and response to individualize drug dosing. Initially, TDM formed the key conceptual basis for personalized medicine Interest from Pharma: Personalized medicine takes into account the fact that 30% of drugs investigated in clinical trials fail because of lack of efficacy*, and its premise is that stratifying patients and diseases into molecular subtypes and treating with subtype-specific drugs will improve drug efficacy. JD Momper and JA Wagner, Clinical pharmacology & therapeutics, VOLUME 95 NUMBER 2 2014 Li and Jones Genome Medicine 2012, 4:27 T. Buclin, Who is in charge of assessing therapeutic drug monitoring? The case of imatinib. Lancet Oncology 2012 Kola I, Landis J: Can the pharmaceutical industry reduce attrition rates?Day Nat2012 Rev Drug Discov 2004, 3:711-715 enrico.accastelli@epfl.ch EDMI Research
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Research level drug quantification approaches MEDIC UCSB
LUCID EPFL
We conjugated its 3′ end to a methylene blue (MB) reporter and its 5′ end to an alkane thiol for attachment to gold working electrodes within the MEDIC chip micro- channel . Target binding induces a conformational change in the aptamer that modulates electron transfer between MB and the electrode . This modulation is expected to produce a readily measurable change in current at the MB reduction peak when the sensor is interrogated using square-wave voltammetry (SWV). Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals, Brian Scott Ferguson et al. enrico.accastelli@epfl.ch EDMI Research Sci Transl Med 5, 213ra165 (2013)
The sensor molecule works by binding the drug circulating in the patient’s bloodstream and changing color accordingly. The molecule itself is made up of four components. One component is a receptor protein, which can bind the molecules of the target drug. The second component is a small molecule similar to the target drug, which can bind the drug receptor. The third component is a light-producing enzyme called luciferase, and the fourth is a fluorophore molecule that can modify the color of the luciferase’s light when it comes close to it. Griss R, Schena A, Reymond L, Patiny L, Werner D, Tinberg CE, Baker D, Johnsson K. | . Nature Chemical Biology
Day 2012
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Where we stand. Why we are unique. Well-defined application objective (in the shortmedium term) candidate diseases clearly identified point-of-care use for therapeutic drug monitoring in clinical or private practice settings support to MDs. Development of comprehensive, flexible, but user friendly code
Unique analytical approach, combining development of novel in vitro selection protocols of drug capture molecules (small drugs, even with no receptor or antibody available) assay development (new molecules employed in traditional or alternative detection techniques)
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Where we stand. Why we are unique. (cont.) Committed to the feasibility of our solutions Data management: compliancy and interoperability Assay development: integration, sample size, sensor characteristics responding to realistic requirements for development
Leveraging nearby companies’ potential interest Pharma: Novartis, Roche, Debiopharm Assays/Systems: STMicroelectronics, Abionics, Mycartis, DBS systems, …
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Project structure and timeline enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Research areas – Internal structure Novel capture probes and quantification in complex matrices C. Guiducci, CHUV, STMicroelectronics and C. Heinis
Miniaturization of sample preparation and FPIA J.M Segura, C. Heinis, Ph. Renaud, CHUV
Interpretation and dose adjustment Database exchange and interoperability Y. Thoma, CHUV, M. Schumacher
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Internal organization in teams and team leaders System integration M. Pfeifer
Data interpretation and data management Y. Thoma
Assays for drug quantification/sem i-quantification J-M Segura
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Milestones expected by May 2015 WORKPACKAGE 1 - Sample preparation LMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUV M1.1: demonstration of volumetric sampling of blood plasma, concept validation of FP measurements in paper. Concept of the whole system architecture. WORKPACKAGE 2 - Highly selective capturing molecules for the target drugs LPTT-EPFL, CLSE-EPFL and CHUV M2.1: Phage selection and characterization of antibody specific for imatinib (will be provided to Segura for FP assay development); expression of tacrolimus-specific Ab and characterization (will be provided to Segura). M2.4: report on SELEX-driven convergence of the DNA library and choice of the 10 (aptamer) candidates WORKPACKAGE 3 - Drug detection by miniaturized systems CLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUV M3.1: fluorescent and biotin derivates M3.2: validated FPIA protocols for tacrolimus and tobramycin M3.5: choice of an opto-electronic device (including light source and detection) for the FP method and functioning table-top system. enrico.accastelli@epfl.ch
EDMI Research Day 2012
Milestones expected by May 2015 (cont.d) WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent Databases HEIG-VD, AISLab HES-SO Valais, CHUV M4.1: Local connected version of the database integrated with EzeCHiel WORKPACKAGE 5 - Demonstrators LDI-HES-SO Valais, STMicroelectronics, CHUV WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of care CHUV, ASILab HES-SO Valais, HEIG-VD M6.1: theoretical elaboration of a PKPD meta-analysis concept M6.4: validation report about the interpretation algorithms as implemented in the EzeCHieL tool
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Forthcoming milestones
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Milestones expected by Oct 2015 WORKPACKAGE 1 - Sample preparation LMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUV M1.2: concept validation of fluid transfer between two paper layers by contact and sample concentration by evaporation WORKPACKAGE 2 - Highly selective capturing molecules for the target drugs LPTT-EPFL, CLSE-EPFL and CHUV M2.5: report on the chosen aptamer binding characteristics towards the target drug M2.2: Affinity and/or stability maturation of imatinib-specific antibody according to the needs of Segura; CDR grafting of tacrolimus-specific Ab into stable IgG framework (will be provided to Segura). WORKPACKAGE 3 - Drug detection by miniaturized systems CLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUV M3.3: comparison of assays in Microsystems M3.7: full description of the requirement specification for the demonstrators to be deployed.
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Milestones expected by Oct 2015 (cont.d) WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent Databases HEIG-VD, AISLab HES-SO Valais, CHUV M4.2: Interoperable server prototype with eHealth standards WORKPACKAGE 5 - Demonstrators LDI-HES-SO Valais, STMicroelectronics, CHUV M5.1:Advanced Breadboard (MAY 2016) WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of care CHUV, ASILab HES-SO Valais, HEIG-VD M6.2: publication of a computer too implementing PKPD meta-analysis M6.5: validation report about the measurement results produced by the analytical tool
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Timeline for demonstrators 1ST YEAR Nov 2013 – Oct 2014
FUNCTIONAL MODULES
2ND YEAR Nov 2014 – Oct 2015
3RD YEAR Nov 2015 – Oct 2016
SYSTEM PROTOTYPE PRE-PROTOTYPES
MODELING SOFTWARE
SECON D TESTIN G PHASE FIRST ASSEMBLED PROTOTYPE FIRST TESTING PHASE
INTELLIGENT DATABASES
enrico.accastelli@epfl.ch
4th YEAR Nov 2016 – Oct 2017
EDMI Research Day 2012
ADVANCED PROTOTYPE
Project context and aims
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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DEADLINES and DATES
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Deadlines 28th of March send out my presentation for the REVIEW MEETING (6th of May in Bern)
13th of April updated scientific report which will cover (Nov. 1st, 2013 – March 2015). Please, send me your updated contribution, according to the usual template:https ://www.dropbox.com/sh/5asho7dmnqmcowv/AADj enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Mark your calendars
REVIEW MEETING 6th of May (afternoon) in Bern. Co-PIs ANNUAL N-T MEETING 4-5th of May in Bern. All ISyPeM participants. We have to choose a PhD student to present existing achievements of our work!
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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CLSE and ISyPeM • • •
CHUV Identification of drug candidates Comparison with standard measurement systems Samples
DNA capture molecules Analysis of matrix effects
•
J-M/HES-SO Use of DNA aptamers for FPIA assays
enrico.accastelli@epfl.ch
Label-free measurement techniques Nano-patterned surfaces
STMicroelectronics • Optical readout
EDMI Research Day 2012
•
Philippe Renaud Integration of sample preparation
39
aptamers
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Challenges in small molecules assays
Lack of suitable capture molecules
APTAMERS
ANTIBODIES
10 kDa - 30 kDa
150 kDa
Sensing in matrices: • Non specific adsorption • Interference
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Streptavidin-coated magnetic beads
Docking region (12nt) CCTTACCTAGGTGTAG-5'
Selection strategy for DNA aptamer
-CCTTACCTAGGTGTAG-5' 5'-GGAATGGATCCACATC-
(A) LoadingLonger randomized region Conversion to ssDNA conjugation beads-aptamers
Tobramycin. Selected best binders (CLSE) better or comparable to existing aptamers
Ligand-binding oligos amplified by PCR
(B) Selection exposition of the small molecule to the aptamers' pool Binding aptamers - drug
(C) Partitioning Separation of binding aptamers from the pool
Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High ThroughputEDMI Sequencing”, ACSDay Combinatorial Science, accepted for publication enrico.accastelli@epfl.ch Research 2012
42
Res
Flowcell1
Flowcell2
0 -10 -20 -30
0.2µM 2µM 20µM 200µM
Minimizing the number of cycles A T
Flowcell1
Response (RU)
30
T
T
Flowcell2
50 40 30 20 10 0 -10 -20 -30
0
100
100
C
2
200 300 Time (s)
SELEX Cycle 12
0
10
R e s p o n s e m a x (R U )
R e s p o n s e m a x (R U )
C 20
0
B
200 300 Time (s)
400
Tobramycin
0µM 0µM 0µM 0.02µM 0.2µM 2µM 20µM 200µM
SPR monitoring Specificity arises for sublibrary at cycle 8
400
30 20 10
4
0
6 2
4
8 Cycle number 6
8 Cycle number
10
10 12
12
14
14
High throughput sequencing The most enriched sequences are already visible after only two captureSELEX cycles
Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High Throughput Sequencing”, ACS Combinatorial Science, accepted for publication
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Selectivity towards Kanamycin
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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45
Surface preparation
50μm ~2 m m
Detection is label-free Does not require drug derivatization
Control aptamer
500μm
Specific aptamer
Passivation
Analysis
enrico.accastelli@epfl.ch
EDMI Research Day 2012
46 Binding kinetics of Tobramycin on SPR in serum
Tobramycin concentration range of initial serum samples: [5µM - 100µM] Derived sample characteristics: Serum 10%, Tween 20 0.01%, Tobr [0.5µM - 10µM]
Detection of sample concentration is preceded by calibration
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Determination of Tobramycin concentration in serum
47
Tobramycin concentration range of initial serum samples: [5µM - 100µM] Derived sample characteristics: Serum 10%, Tween 20 0.01%, Tobr [0.5µM - 10µM] • LoD 0.15 µM R2=0.993
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Conclusive remarks
48
Tobramycin-specific DNA aptamers successfully selected with KD <1 µM No drug derivatization Relatively simple and fast aptamer selection protocol
Tobramycin detection [0.5µM - 100µM] in serum samples Linearity up to 10 µM Label-free, direct detection Volume of serum needed: 20µl Sample preparation time: 45 min per sample (from serum) Analysis time: half an hour per sample
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Direct detection of drug molecules in patient sera Standard addition method Direct detection by SPR based on immobilized DNA aptamers specific for Tobramycin
24% error
6% error
(Tolerance in this range: 20%)
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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50
New SELEX concept
Classic
• • • •
Capture
Evolution-like iterative selection system Long(er) sequences, immobilised oligo More specificity - more possibilities for binding detection And they are selected on a surface
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Design of the assay
51
Specifications for a companion diagnostic device in TDM
Sensitivity in the physiological range Specificity in presence of concomitant drugs Simple and fast sample preparation Transferability of the analytical approach to other drugs Avoid drug derivatization Straightforward selection of new probes
Sample size efficiency enrico.accastelli@epfl.ch
EDMI Research Day 2012
52 Serum sample preparation for drug concentration analysis
Precipitation upon protein denaturation Filtering with low cut-off (eg: < 3 kDa) Anti-fouling solutions to prevent non specific adsorption on the surface
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Determination of Tobramycin concentration
53
Tobramycin concentration range of initial serum samples: [5µM - 100µM] Derived sample characteristics: Serum 10%, Tween 20 0.01%, Tobr [0.5µM - 10µM]
Some numbers: Volume of patient serum needed: 20µl Sensing region covered by aptamers: 1.05 mm2 Moles of aptamers required: 150pmol Manipulation time: ~10min per sample Run time: ~12min per cycle (2 cycles per sample)
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Variability in treatment response
2.4
Risk of adverse effects
Imatinib 400 mg qd
Drug Concentration (μg/mL)
2.0 1.6
Target
1.2 0.8
800 mg qd
0.4
Risk of inefficacy 0.0
0
1
enrico.accastelli@epfl.ch
2
3
Patient
4 5 6 7 8 9 Treatment duration (days)
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11
12
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T. Buclin, et al. “Who is in charge of assessing therapeutic drug monitoring? The case of imatinib”. Lancet Oncol. 2011;12(1):9-11.
Therapeutic drug monitoring at the point-of-care Compatibility/Suitability Acceptable and suitable setting for infield drug measurement, considering disease, frequency of monitoring, treatment toxicity, costs Need for stable and specific molecular assay for drug measurement, compatible with on field biosensors
Information and Interpretation Missing statistical population data Need for formal and accessible models of interpretation Data exchange
DA RISITEMARE
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EDMI Research Day 2012
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tSPR
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Transmission SPR. Direct detection in undiluted serum. x 10
-4
16 14 12
hue
10
KD= 0.26 ÂľM
8 6 4
Aptamer (average) Langmuir interpolation Control
2 0 0
5
10
15
20
Tobramycin concentration (ÂľM)
Undiluted serum
G. Cappi, E. Accastelli, V. Cantale, M. A. Rampi, L. Benini, C. Guiducci ., Sensors and Actuators B: Chemical, 2013 G. Cappi, Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., T. Buclin, L. Decosterd, Guiducci, C. enrico.accastelli@epfl.ch EDMI Research Day 2012 Analytical Chemistry, accepted for publications
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Portable system for array measurements Power supply
White LED Regions of interest
Diffuser Plasmo nic sensor Lens CMOS image sensor Compu ter enrico.accastelli@epfl.ch
â&#x20AC;˘ Power supply via USB â&#x20AC;˘ Real-time display of images registered EDMI Research Day 2012
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Do Hue and Peak have the same plasmonic information? • Evaluation of the response of the NIs to different RI • Test with glucose/sucrose solutions alternated with water • Elaboration from the same RGB raw data
enrico.accastelli@epfl.ch
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Validation of the hue for plasmonic evaluation Bulk refractive index change Surface binding events Small molecules in saline buffer
0.202
584
Small molecules in serum matrix
0.2015
5 8 3 .5
0.201
583 5 8 2 .5
0.2
582
0.1995
5 8 1 .5
0.199
581
0.1985 0.198 enrico.accastelli@epfl.ch
P e a k lo c a tio n (n m )
• Tobramycin 10 μM in TE 1X buffer • DNA aptamer spot
H ue
0.2005
5 8 0 .5
0
10
20
EDMI Research Day 2012
30
40
T im e (m in ) 60
50
60
70
580
Hue-peak correlation to surface events h33 c orrelation hue peak in 0TE.2 0 0 4
Surface RI change in TE buffer
0 .2 0 0 3
Hue
0.205
1E-4
H ue
0.204
0 .2 0 0 2
0.203 0.202
0 .2 0 0 1 580
0.201 0.2
580
enrico.accastelli@epfl.ch
581
582
583
Pe a k lo c aEDMI tio n Research (n m ) Day 2012
0.0715 nm 5 80 .1 5 8 0 .2 5 8 0 .3 Pe a k lo c a tio n (n m )
584 35
5 8 0 .4
Small molecules detection in TE buffer 0 .2
A p ta m e r C o n tro l
0 .1 9 9
H ue
0 .1 9 8 0 .1 9 7 0 .1 9 6 0 .1 9 5
0 .5
1
0.5 µM
1 .5
2
1 µM
Serum TE enrico.accastelli@epfl.ch
2 .5
3 3 .5 T im e (h o u rs )
2 µM
4
4 .5
5
5 µM
TE NaCl EDMI Research Day 2012
5 .5
6
10 µM +
37
TE
DNA aptamer and control spots
Array of aptamers/control HCR/blank NIs
2-channels microfluidics Tinta in FTO & NIs
N Is
0.198
H
FTO
Na Cl
H
CONTR
H
DNA
TE
0.196
H
DNA
To br 20 µM
TE
Na Cl
TE
H
Tob r 200 µM
TE
Na Cl
TE
NIS
Ref FTO
SHaptam Control HCR Blank NIs
Valore Pixel
0.194
0.192
0.19
0.188
0.186
enrico.accastelli@epfl.ch
0.184
1500 Day 20002012 2500 EDMI1000 Research
500
3000 Numero Immagine
3500
4000
4500
5000
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ISyPeM
Therapeutic drug monitoring for personalized medicine
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MULTI-SCALE APPROACH FOR POINTOF-CARE TDM
POINT-OF-CARE TESTING
Low volume blood testing Quantitative Specificity towards metabolites Miniaturized
enrico.accastelli@epfl.ch
INTERPRETATION AND DOSE ADJUSTMENT
Is the result expected? Is the drug still suitable? Prediction and dose adjustment
EDMI Research Day 2012
DATA EXCHANGE AND INTEROPERABILITY
Upload patient’s data Extension of eHealth medical standards Data integrity
65
INNOVATE IN VITRO ASSAYS
In-Check - STMicroelectronics
DNA aptamers
Monoclonal antibodies enrico.accastelli@epfl.ch
Aptamer-based analysis of tobramycin in serum samples EDMI Research Day 2012
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SUPPORT INTERPRETATION OF DRUG CONCENTRATION DATA
ezeCHiel
enrico.accastelli@epfl.ch
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EXPLOITATION PARTNERS
Division of Clinical Pharmacology Collect patient samples
Validation in field conditions
Elaborate a protocol for large-scale randomized clinical trials
Readers and prototyping
Contribution in the development of a demonstrator
enrico.accastelli@epfl.ch
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Candidate treatments for therapeutic drug monitoring
Modern anticancer agents
Immunosuppressant
IMATINIB
TACROLIMUS
1000 ng/ml 493.60 Da
Antiretrovirals EFAVIRENZ
enrico.accastelli@epfl.ch
10 ng/ml 804.02 Da
Antibiotics 2000 ng/ml 315.70 Da
TOBRAMYCIN
EDMI Research Day 2012
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1000 ng/ml 467.5 Da
From surface confined to volume confined systems
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Clinical Pharmacology, UNIL CHUV (CH) Thierry Buclin, Director Laurent Decosterd European Institute of Oncology, Milan (I) Marco Giorgio Ludwig Institute for Cancer Research (CH) Immanuel Luescher CEA LETI (F) Thomas Ernst
enrico.accastelli@epfl.ch
EPFL Microelectronic Systems Laboratory, Yusuf Leblebici CMi LMIS4, Philippe Renaud Nanophotonics and Metrology Laboratory Olivier Martin
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• ●
Dielectric properties of single cell : Discrimination ●
- Main leukocyte sub-populations (monocytes, lymphocytes, neutrophils) in human blood
(Label-Free Differential Leukocyte Counts Using a Microfabricated, Single-Cell Impedance D Holmes et al.)
Spectrometer,
- Red blood Cells ghost and RBCs fixed in glutaraldehyde
(Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation, K. Cheung et al.)
- Monocytes and dendritic cells (On-chip non-invasive and label-free cell discrimination by impedance spectroscopy, G.Schade-Kampmann et ●
viability ●
al.
- MFC-7 cell death (Label-free single cell analysis with a chip-based impedance flow cytometer, A.Pierzchalski et al)
●
- Living and dead yeast cells (Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis , G. Mernier et al)
●
Infection
- Babesia bovis infected erythrocytes (Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer, C. Küttel )
•
Dielectric properties of labels decorating the cell
Polysterene particles (Single Cell Impedance Cytometry for Identification and Counting of CD4 T-Cells in Blood Using Impedance Labels, D. Holmes et al.)
•
Change in ionic force of medium cell lysis detection
human
- CD4+ T (Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices , X. Cheng) enrico.accastelli@epfl.ch EDMI Research Day 2012 74
Back up
enrico.accastelli@epfl.ch
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Quantification of small molecules in serum EMIT (Enzyme Multiplied Immunoassay Technique ) requires derivatization of the drug molecules Drug of abuse Syva RapidTest®
FPIA: (Fluorescence Polarization Immunoassay) hard to integrate, based on antibodies requires derivatization of the drug molecules Therapeutic drug monitoring: employed only for specific diseases (difficult to interpret and measure) FPIA
enrico.accastelli@epfl.ch
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New approaches to small molecules quantification. DNA aptamer based
c
APTAMERS
ANTIBODIES
10 kDa - 30 kDa
150 kDa
Chang, A. L. et al., Anal. Chem. 2014, 86, 3273â&#x2C6;&#x2019;3278.
enrico.accastelli@epfl.ch
Y
Ferguson, B. S. et al. (2013). "Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals." Science Translational Medicine 5(213): 213ra165.
EDMI Research Day 2012
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Do our aptamers work in complex matrices? Test on SPR label-free
enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Point of care biosensor Localised Surface Plasmon Resonance
Cappi, G., Accastelli, E., Spiga, F.M., Cantale, V., Rampi, M.A., Benini, L., Guiducci, C. (2013) Mat Res Soc Symp Proc enrico.accastelli@epfl.ch
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DNA Aptamer Selection against tobramycin
Aminoglycoside antibiotic Adverse effects on kidney and ears Tested in buffer: • 0.23 µM KD • No affinity toward carbenicillin • 5 time higher KD toward kanamycin
enrico.accastelli@epfl.ch
EDMI Research Day 2012
Tobramycin
80
Normalized signal
Tobramycin on T-LSPR in undiluted serum 1.6 Aptamer 1.2 0.8 0.4 0 -0.4
10 µM 0.5
0 µM 1
20 µM 1.5
0 µM 2
40 µM 2.5
0 µM
3
3.5
60 µM 4
0 µM 4.5
80 µM 5
5.5
Time (hours)
enrico.accastelli@epfl.ch
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Point of care biosensor: can monitor small molecules? Tobramycin on T-LSPR x 10
-4
16 14 12
hue
10 8 6 4
Aptamer (average) Langmuir interpolation Control
2 0 0
5
10
15
Tobramycin concentration (µM)
• T-LSPR setup: Kd= 0.26 µM • Biacore SPR: Kd= 0.23 µM Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.) enrico.accastelli@epfl.ch
EDMI Research Day 2012
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20
Point of care biosensor: small molecules in complex matrices? Tobramycin on T-LSPR in undiluted serum
Linear trend Theoretical minimum resolvable concentration of tobramycin 3.4 µM.
Good linearity in almost all the analytical range (1-80µM) Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.) enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Making aptamers - SELEX In-vitro evolution-like process
Classic
Capture
Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission)
No need to immobilize the target: good for small molecules! enrico.accastelli@epfl.ch
EDMI Research Day 2012
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Capture-SELEX with control A B via Surface Plasmon Resonance In stream control of library affinity T T
Flowcell1
Response (RU)
T
Flowcell2
50 40 30 20 10 0 -10 -20 -30
SELEX Cycle 12
0
100
R e s p o n s e m a x (R U )
C
200 300 Time (s)
0µM 0µM 0µM 0.02µM 0.2µM 2µM 20µM 200µM 400
30 20 10 0
2
4
6
8 Cycle number
10
12
14
Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission) enrico.accastelli@epfl.ch
EDMI Research Day 2012
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enrico.accastelli@epfl.ch
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Tobramycin
enrico.accastelli@epfl.ch
Carbenicillin
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