DNA detection on lateral flow test strips: enhanced signal sensitivity using LNA-conjugated gold nanoparticles Shiva K. Rastogi, CharLene M. Gibson, Josh R. Branen, D. Eric Aston, Larry Branen and Patrick J. Hrdlicka Department of Chemistry, University of Idaho, Moscow, ID, USA, Email: srastogi@uidaho.edu Experimental Data and Results Experimental Data and Results Abstract A lateral flow test strip assay, enabling sensitive detection of DNA specific to the foodborne pathogen Escherichia coli (E. coli) O157:H7, is described. The use of LNA-conjugated gold nanoparticle probes, along with signal amplification protocols, results in minimum detectable concentrations of ~0.4 nM.
Fig. 3: Images of test strips and peak areas using the DNA-AuNP signaling probes for E. coli O157:H7 target detection, before (A) and after (B) signal amplification. Target DNA ET was added at a concentration of 12.5, 6.25, 3.13, 1.56 or 0.78 nM (strips 1-5, respectively). “C” and “T” denotes control and test lines respectively.
Introduction * There is considerable interest in developing sensitive and specific DNA detection platforms for use in fundamental research and clinical applications for rapid detection of pathogens, bioterrorism agents and biomarkers of genetic diseases,1 in laboratory and point-of-care settings.
Fig. 6 : Images of test strips and peak areas using DNA-AuNP signaling probes specific for E. coli O157:H7 in the presence of noncomplementary DNA target NT (S. typimurium) before (A) or after (B) signal amplification. NT was added at a concentration of 1.56, 3.13, 6.25, 12.5 or 25.0 nM (strips 1-5, respectively).
Fig. 7: Images of test strips and peak areas using LNA-AuNP probes specific for E. coli O157:H7 in presence NT (S. typimurium) before (A) and after (B) signal amplification. NT was added at a conc. of 25 or 50 nM (strips 1 and 2, respectively).
* Lateral flow test strips (LFTS) are used in antibody-based pregnancy2 and have also been used for detection of bacteria3 in point-of-care settings.4
* Limitations of LFTS include low signal intensity, which can compromise colorimetric discrimination at low target concentrations. * Several signal amplification strategies have been developed, including optical,5 electrochemical,6 and enzymatic approaches.7 These approaches are effective but introduce additional preparation and instrumentation steps.
* Here we describe a simple LFTS assay8 that enables sensitive detection of DNA specific to E. coli O157:H7, a food-borne pathogen9 that poses considerable health risk to humans.10
Fig. 1 Schematic illustration of signal generation on LFTS: (A) sample is applied on sample pad (sp); (B) samples containing DNA target result in formation of recognition complexes at the conjugation pad (cp); (C+D) capture of recognition complexes in test zone (tz) and of excess AuNP probes in control zone (cz), results in two pink lines; (E) in absence of target, (F+G) excess AuNP probe are captured in the control zone resulting in one pink line; (H) amplification of test and control signals through gold deposition; ap = absorbent pad.
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Table 1: Naming and sequences of oligonucleotides (ON) used in this study. LNA monomers are shown in lower underline cases ON ET DP LP TP CP NT
Sequences 3’-CGGCCTTGTCAAGATAGTCCGTACCGAGAACTAC 5’-/5ThioMC6-D//iSp18/GCCGGAACAGTTC 5’-/5ThioMC6-D//iSp18/GCCGGaACaGTtC 5’-TATCAGGCATGGCTCTTGATG/iSp18//3BioTEG 3’-CGGCCTTGTCAAG/iSp18//5Biosg 3’-GGTGCAAGCCCGTTAAGCAATAACCGCTAT
Fig.4 Detection of E. coli O157:H7 DNA target ET (0–12.5 nM) before and after signal amplification. AuNP conjugated to DNA probe DP were used. ‘‘S/N’’ = signal-tonoise ratio.
Fig. 8 Comparison of signal-to-noise ratios in LFTS assay using either unmodified or LNA-modified AuNP signaling probes.
Conclusion 1. LNA monomers containing DNA signal reporting probes showed improved detection on LFTS. 2. Gold ions deposition produced amplification of test line signal. 3. A novel methodology has been develop for specific and amplified detection of target DNA.
Acknowledgement & Contact
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Financial support from the BANTech. Center at the University of Idaho, USDA (No. 2009-34479-19833 and 2010-34479- 20715) and Idaho NSF EPSCoR is gratefully acknowledged.
References 1. Lee, O. S. et al. J. Phys. Chem. Lett., 2010, 1, 1781. 2. Nagatani, N. et al. Adv. Mater., 2006, 7, 270. 3. Li, C. Z. et al. Biosens. Bioelectron., 2011, 26, 4342. 4. Gubala, V. et al. et al. Anal. Chem., 2012, 84, 487. 5. Yang, H. et al. Nanoscale Res. Lett., 2010, 5, 875. 6. Liu, G. Anal. Chem., 2007, 79, 7644. 7. Mao, X. Anal. Chem., 2009, 81, 1660. 8. Rastogi, S. K. et al. Chem. Comm., 2012, 48, 7714. 9. Aslam, M. Food Microbiol., 2003, 20, 345. 10. Jokerst, J. C. Anal. Chem., 2012, 84, 2900.
Fig. 2: Images of test strips and peak areas using LNA-AuNP signaling probes specific for E. coli O157:H7 target detection, before (A) and after (B) signal amplification. Target DNA ET was added at a concentration of 0, 3.13, 1.56, 0.78 or 0.39 nM (strips 1-5, respectively). “C” and “T” denotes control and test lines respectively.
Fig. 5 Detection of E. coli O157:H7 DNA target ET (0-3.13 nM) before and after signal amplification. AuNP conjugated to LNA modified probe LP were used.
Shiva K Rastogi & Patrick J Hrdlicka Department of Chemistry University of Idaho, Moscow, ID 83844-2343, USA Email: srastogi@uidaho.edu; hrdlicka@uidaho.edu