Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish
Source abstract
Abstract
The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage-a bacteria-specific virus nanoparticle-as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ∼3 and ∼5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.
Source: europepmc · Quality: candidate unverified · Source record ↗
Research context
OpenAlex topics: Advanced biosensing and bioanalysis techniques · Biosensors and Analytical Detection · Bacteriophages and microbial interactions
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
References (37)
Show source-provided references
1. Hamburg, M. A. & Collins, F. S. The path to personalized medicine. New Engl. J. Med. 363, 301–304 (2010).10.1056/NEJMp1006304 ↗
2. Taton, T. A., Mirkin, C. A. & Letsinger, R. L. Scanometric DNA array detection with nanoparticle probes. Science 289, 1757–1760 (2000).10.1126/science.289.5485.1757 ↗
3. Valentini, P. et al. Gold-nanoparticle-based colorimetric discrimination of cancer-related point mutations with picomolar sensitivity. ACS Nano 7, 5530–5538 (2013).10.1021/nn401757w ↗
4. Cao, Y. C., Jin, R. & Mirkin, C. A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).10.1126/science.297.5586.1536 ↗
5. Qiu, F., Jiang, D., Ding, Y., Zhu, J. & Huang, L. L. Monolayer-barcoded nanoparticles for on-chip DNA hybridization assay. Angew. Chem. Int. Ed. 47, 5009–5012 (2008).10.1002/anie.200800435 ↗
6. Zhou, X., Cao, P., Tian, Y. & Zhu, J. Expressed peptide assay for DNA detection. J. Am. Chem. Soc. 132, 4161–4168 (2010).10.1021/ja9081827 ↗
7. Nam, J.-M., Stoeva, S. I. & Mirkin, C. A. Bio-bar-code-based DNA detection with PCR-like sensitivity. J. Am. Chem. Soc. 126, 5932–5933 (2004).10.1021/ja049384+ ↗
8. Stoeva, S. I., Lee, J.-S., Smith, J. E., Rosen, S. T. & Mirkin, C. A. Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes. J. Am. Chem. Soc. 128, 8378–8379 (2006).10.1021/ja0613106 ↗
9. Mao, C. et al. Viral assembly of oriented quantum dot nanowires. Proc. Natl Acad. Sci. USA 100, 6946–6951 (2003).10.1073/pnas.0832310100 ↗
10. Mao, C. et al. Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires. Science 303, 213–217 (2004).10.1126/science.1092740 ↗
11. Liu, A., Abbineni, G. & Mao, C. Nanocomposite films assembled from genetically engineered filamentous viruses and gold nanoparticles: Nanoarchitecture- and humidity-tunable surface plasmon resonance spectra. Adv. Mater. 21, 1001–1005 (2009).10.1002/adma.200800777 ↗
12. Mao, C., Wang, F. & Cao, B. Controlling nanostructures of mesoporous silica fibers by supramolecular assembly of genetically modifiable bacteriophages. Angew. Chem. Int. Ed. 51, 6411–6415 (2012).10.1002/anie.201107824 ↗
13. Wang, F., Nimmo, S., Cao, B. & Mao, C. B. Oxide formation on biological nanostructures via a structure-directing agent: Towards an understanding of precise transcription. Chem. Sci. 3, 2639–2645 (2012).10.1039/c2sc00583b ↗
14. Eber, F. J., Eiben, S., Jeske, H. & Wege, C. Bottom-up-assembled nanostar colloids of gold cores and tubes derived from tobacco mosaic virus. Angew. Chem. Int. Ed. 52, 7203–7207 (2013).10.1002/anie.201300834 ↗
15. Cao, B., Zhu, Y., Wang, L. & Mao, C. B. Controlled alignment of filamentous supramolecular assemblies of biomolecules into centimeter-scale highly ordered patterns by using nature-inspired magnetic guidance. Angew. Chem. Int. Ed. 52, 11750–11754 (2013).10.1002/anie.201303854 ↗
16. Mao, C., Liu, A. & Cao, B. Virus-based chemical and biological sensing. Angew. Chem. Int. Ed. 48, 6790–6810 (2009).10.1002/anie.200900231 ↗
17. Mohan, K., Donavan, K. C., Arter, J. A., Penner, R. M. & Weiss, G. A. Sub-nanomolar detection of prostate-specific membrane antigen in synthetic urine by synergistic, dual-ligand phage. J. Am. Chem. Soc. 135, 7761–7767 (2013).10.1021/ja4028082 ↗
18. Cuervo, A. et al. Structural characterization of the bacteriophage T7 tail machinery. J. Biol. Chem. 288, 26290–26299 (2013).10.1074/jbc.M113.491209 ↗
19. Chan, L. Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005).10.1038/msb4100025 ↗
20. Slootweg, E. J. et al. Fluorescent T7 display phages obtained by translational frameshift. Nucleic Acids Res. 34, e137 (2006).10.1093/nar/gkl600 ↗
21. Tsuboyama, M. & Maeda, I. Combinatorial parallel display of polypeptides using bacteriophage T7 for development of fluorescent nano-bioprobes. J. Biosci. Bioeng. 116, 28–33 (2013).10.1016/j.jbiosc.2013.01.015 ↗
22. Huang, Y. et al. Programmable assembly of nanoarchitectures using genetically engineered viruses. Nano Lett. 5, 1429–1434 (2005).10.1021/nl050795d ↗
23. Garcia-Doval, C. & Raaij, M. J. V. Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers. Proc. Natl Acad. Sci. USA 109, 9390–9395 (2012).10.1073/pnas.1119719109 ↗
24. Dunn, J. J. & Studier, F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477–535 (1983).10.1016/S0022-2836(83)80282-4 ↗
25. Johnson, S. M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005).10.1016/j.cell.2005.01.014 ↗
26. Esquela-Kerscher, A. & Slack, F. J. Oncomirs—microRNAs with a role in cancer. Nature Rev. Cancer 6, 259–269 (2006).10.1038/nrc1840 ↗
27. Lin, P. Y., Yu, S. L. & Yang, P. C. MicroRNA in lung cancer. Br. J Cancer 103, 1144–1148 (2010).10.1038/sj.bjc.6605901 ↗
28. Bousquet, M., Harris, M. H., Zhou, B. & Lodish, H. F. MicroRNA miR-125b causes leukemia. Proc. Natl Acad. Sci. USA 107, 21558–21563 (2010).10.1073/pnas.1016611107 ↗
29. Monya, B. qPCR: Quicker and easier but don’t be sloppy. Nature Methods 8, 207–212 (2011).10.1038/nmeth0311-207 ↗
30. Kumar, M. S. et al. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc. Natl Acad. Sci. USA 105, 3903–3908 (2008).10.1073/pnas.0712321105 ↗
31. Serwer, P. & Pichler, M. E. Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels. J. Virol. 28, 917–928 (1978).10.1128/jvi.28.3.917-928.1978 ↗
32. Xu, T. et al. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113–121 (2009).10.1002/hep.22919 ↗
33. Jusufović, E. et al. let-7b and miR-126 are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non-small-cell lung cancer. PLoS ONE 7, e45577 (2012).10.1371/journal.pone.0045577 ↗
34. Liu, Z. L., Wang, H., Liu, J. & Wang, Z. X. MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN. Mol. Cell Biochem. 372, 35–45 (2013).10.1007/s11010-012-1443-3 ↗
35. Thellin, O. et al. Housekeeping genes as internal standards: Use and limits. J. Biotechnol. 75, 291–295 (1999).10.1016/S0168-1656(99)00163-7 ↗
36. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034 (2002).10.1186/gb-2002-3-7-research0034 ↗
37. Dheda, K. et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 37, 112–119 (2004).10.2144/04371RR03 ↗