Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip


The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: NAAT platform architecture.
Figure 2: Example structure of DNA probes and melting signals for inverse fluorescence transduction versus conventional fluorescence methods for identifying the L511P mutation in the MTB gene rpoB.
Figure 3: Structure of the CMOS biochip and its individual components.
Figure 4: On-chip detection and quantification of respiratory viruses.
Figure 5: MTB drug-resistance mutation (DRM) panel.
Figure 6: Example MCA probe designs for SNP identification in view of DNA secondary structures.


  1. 1

    World Health Organization Department of Communicable Disease Surveillance and Response. WHO Global Strategy for Containment of Antimicrobial Resistance (Health Organization, Geneva, 2001).

  2. 2

    World Health Organization. Antimicrobial Resistance: 2014 Global Report on Surveillance (World Health Organization, Geneva, 2014).

  3. 3

    Baker, S. A return to the pre-antimicrobial era? Science 347, 1064–1066 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Lushniak, B.D. Antibiotic resistance: a public health crisis. Public Health Rep. 129, 314–316 (2014).

    Article  Google Scholar 

  5. 5

    Michael, C.A., Dominey-Howes, D. & Labbate, M. The antimicrobial resistance crisis: causes, consequences, and management. Front. Public Health 2, 145 (2014).

    Article  Google Scholar 

  6. 6

    Bartlett, J.G., Gilbert, D.N. & Spellberg, B. Seven ways to preserve the miracle of antibiotics. Clin. Infect. Dis. 56, 1445–1450 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Gelband, H. & Laxminarayan, R. Tackling antimicrobial resistance at global and local scales. Trends Microbiol. 23, 524–526 (2015).

    CAS  Article  Google Scholar 

  8. 8

    Caliendo, A.M. et al. Better tests, better care: improved diagnostics for infectious diseases. Clin. Infect. Dis. 57 (Suppl. 3), S139–S170 (2013).

    Article  Google Scholar 

  9. 9

    Fakruddin, M. et al. Nucleic acid amplification: alternative methods of polymerase chain reaction. J. Pharm. Bioallied Sci. 5, 245–252 (2013).

    Article  Google Scholar 

  10. 10

    Yang, S. & Rothman, R.E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis. 4, 337–348 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Espy, M.J. et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev. 19, 165–256 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Rao, A., Fader, B. & Hocker, K. in Molecular Diagnostics Techniques and Applications for the Clinical Laboratory 327–340 (Academic Press, 2010).

  13. 13

    Neuzil, P., Zhang, C., Pipper, J., Oh, S. & Zhuo, L. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Res. 34, e77 (2006).

    Article  Google Scholar 

  14. 14

    Maltezos, G. et al. Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: a proof of principle. Appl. Phys. Lett. 97, 264101 (2010).

    Article  Google Scholar 

  15. 15

    Blair, J.M.A., Webber, M.A., Baylay, A.J., Ogbolu, D.O. & Piddock, L.J.V. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13, 42–51 (2015).

    CAS  Article  Google Scholar 

  16. 16

    Jia, B. et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 45, D566–D573 (2017).

    CAS  Article  Google Scholar 

  17. 17

    Liu, B. & Pop, M. ARDB—Antibiotic Resistance Genes Database. Nucleic Acids Res. 37, D443–D447 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Hillemann, D., Rüsch-Gerdes, S. & Richter, E. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacteriumtuberculosis strains and clinical specimens. J. Clin. Microbiol. 45, 2635–2640 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Gaudet, M., Fara, A.G., Beritognolo, I. & Sabatti, M. in Single Nucleotide Polymorphisms: Methods and Protocols 415–424 (Humana Press, 2009).

  20. 20

    Mhlanga, M.M. & Malmberg, L. Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25, 463–471 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Poritz, M.A. et al. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 6, e26047 (2011).

    CAS  Article  Google Scholar 

  22. 22

    De Wilde, B. et al. Target enrichment using parallel nanoliter quantitative PCR amplification. BMC Genomics 15, 184 (2014).

    Article  Google Scholar 

  23. 23

    Spurgeon, S.L., Jones, R.C. & Ramakrishnan, R. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One 3, e1662 (2008).

    Article  Google Scholar 

  24. 24

    Toumazou, C. et al. Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nat. Methods 10, 641–646 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Chandler, D.P. et al. Integrated amplification microarrays for infectious disease diagnostics. Microarrays 1, 107–124 (2012).

    Article  Google Scholar 

  26. 26

    Popowitch, E.B., O'Neill, S.S. & Miller, M.B. Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP fast multiplex assays for detection of respiratory viruses. J. Clin. Microbiol. 51, 1528–1533 (2013).

    Article  Google Scholar 

  27. 27

    Goodwin, S., McPherson, J.D. & McCombie, W.R. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 17, 333–351 (2016).

    CAS  Article  Google Scholar 

  28. 28

    Plummer, J.D., Deal, M.D. & Griffin, P.B. Silicon VLSI Technology: Fundamentals, Practice and Modeling (Prentice Hall, New York, 2000).

  29. 29

    Hassibi, A., Vikalo, H., Riechmann, J.L. & Hassibi, B. Real-time DNA microarray analysis. Nucleic Acids Res. 37, e132 (2009).

    Article  Google Scholar 

  30. 30

    Manickam, A., Chevalier, A., McDermott, M., Ellington, A.D. & Hassibi, A. A CMOS electrochemical impedance spectroscopy (EIS) biosensor array. IEEE Trans. Biomed. Circuits Syst. 4, 379–390 (2010).

    Article  Google Scholar 

  31. 31

    Jang, B., Cao, P., Chevalier, A., Ellington, A. & Hassibi, A. in IEEE International Solid-State Circuits Conference—Digest of Technical Papers 436–437 (IEEE, 2009).

  32. 32

    Wang, H., Chen, Y., Hassibi, A., Scherer, A. & Hajimiri, A. in IEEE International Solid-State Circuits Conference—Digest of Technical Papers 438–439 (IEEE, 2009).

  33. 33

    Wittwer, C.T. High-resolution DNA melting analysis: advancements and limitations. Hum. Mutat. 30, 857–859 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Ririe, K.M., Rasmussen, R.P. & Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Sanchez, J.A., Pierce, K.E., Rice, J.E. & Wangh, L.J. Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc. Natl. Acad. Sci. USA 101, 1933–1938 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Peirson, S.N., Butler, J.N. & Foster, R.G. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31, e73 (2003).

    Article  Google Scholar 

  38. 38

    Thiel, A.J., Frutos, A.G., Jordan, C.E., Corn, R.M. & Smith, L.M. In situ surface plasmon resonance imaging detection of DNA hybridization to oligonucleotide arrays on gold surfaces. Anal. Chem. 69, 4948–4956 (1997).

    CAS  Article  Google Scholar 

  39. 39

    Guo, X. Surface plasmon resonance based biosensor technique: a review. J. Biophotonics 5, 483–501 (2012).

    CAS  Article  Google Scholar 

  40. 40

    Fritz, J., Cooper, E.B., Gaudet, S., Sorger, P.K. & Manalis, S.R. Electronic detection of DNA by its intrinsic molecular charge. Proc. Natl. Acad. Sci. USA 99, 14142–14146 (2002).

    CAS  Article  Google Scholar 

  41. 41

    Xiao, Y., Lai, R.Y. & Plaxco, K.W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat. Protoc. 2, 2875–2880 (2007).

    CAS  Article  Google Scholar 

  42. 42

    Burg, T.P. et al. Weighing of biomolecules, single cells and single nanoparticles in fluid. Nature 446, 1066–1069 (2007).

    CAS  Article  Google Scholar 

  43. 43

    Arlett, J.L., Myers, E.B. & Roukes, M.L. Comparative advantages of mechanical biosensors. Nat. Nanotechnol. 6, 203–215 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Schena, M. DNA Microarrays: A Practical Approach (Oxford University Press, 1999).

  45. 45

    Marras, S.A. in Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols 3–16 (Humana Press, 2006).

  46. 46

    Ramsay, N. et al. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase. J. Am. Chem. Soc. 132, 5096–5104 (2010).

    CAS  Article  Google Scholar 

  47. 47

    Turner, D.H., Sugimoto, N. & Freier, S.M. Thermodynamics and kinetics of base-pairing and of DNA and RNA self-assembly and helix coil transition. Nucleic Acids 1, 201–227 (1990).

    Google Scholar 

  48. 48

    Lee, J.S., Hornsey, R.I. & Renshaw, D. Analysis of CMOS photodiodes. II. Lateral photoresponse. IEEE Trans. Electron Dev. 50, 1239–1245 (2003).

    CAS  Article  Google Scholar 

  49. 49

    Manickam, A. et al. A fully integrated CMOS fluorescence biochip for DNA and RNA testing. IEEE J. Solid-State Circuits 52, 2857–2870 (2017).

    Article  Google Scholar 

  50. 50

    Macleod, H.A. Thin-film Optical Filters (CRC Press, 2001).

  51. 51

    Zhang, F. et al. Chemical vapor deposition of three aminosilanes on silicon dioxide: surface characterization, stability, effects of silane concentration and cyanine dye adsorption. Langmuir 26, 14648–14654 (2010).

    CAS  Article  Google Scholar 

Download references


The authors thank J. SantaLucia and the staff of DNA Software for their technical support and suggestions related to thermodynamic simulations for primer and probe design. Research reported in this publication was supported by the National Human Genome Research Institute of the National Institutes of Health under award R44HG007626. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information




A.H. conceived the technology. A.H., R.G.K. and G.S. co-supervised the project and wrote the manuscript with input from the other authors. Integrated biochip and optoelectronic components were designed and built by A.M., R. Singh, M.W.M., M.M., M.H., N.W. and E.K. In silico assay design and signal processing algorithms were developed by S.B., J.E., R. Sinha, P.K., B.H. and H.V. Assay implementation and performance validation on clinical samples were executed by P.N., G.D., K.A.J., T.V., G.M., K.B.J., L.P., M.P.S., P.M., B.A.P. and Y.L. Key technical contributions for chemistry, mechanical design and fluidics were provided by P.G., L.B., P.S., N.G., M.T.T., R.B.M. and S.C.

Corresponding author

Correspondence to Arjang Hassibi.

Ethics declarations

Competing interests

All of the authors listed in this paper were employees or contractors of InSilixa, Inc., with the exception of B.H., H.V. and B.A.P., who were academic collaborators on the project.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hassibi, A., Manickam, A., Singh, R. et al. Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip. Nat Biotechnol 36, 738–745 (2018).

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing