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Simultaneous DNA amplification and detection using a pH-sensing semiconductor system

Nature Methods volume 10, pages 641–646 (2013)Cite this article

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Abstract

We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis.

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Figure 1: Correlation between pH signal and amplification.
Figure 2: Schematic representation of pH-sensing integrated circuit platform.
Figure 3: On-chip amplification and detection using pH-PCR and pH-LAMP.
Figure 4: Evaluation of integrated circuit sensitivity.
Figure 5: SNP typing of CYP2C19*2 and CYP2C19*17.

References

  1. Holland, P.M., Abramson, R.D., Watson, R. & Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280 (1991).

    Article  CAS  Google Scholar 

  2. Higuchi, R., Fockler, C., Dollinger, G. & Watson, R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Nat. Biotechnol. 11, 1026–1030 (1993).

    Article  CAS  Google Scholar 

  3. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real-time quantitative PCR. Genome Res. 6, 986–994 (1996).

    Article  CAS  Google Scholar 

  4. Wittwer, C.T., Herrmann, M.G., Moss, A.A. & Rasmussen, R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130 (1997).

    Article  CAS  Google Scholar 

  5. Toumazou, C. & Purushothaman, S. Sensing apparatus and method. US patent 7,686,929 (2002).

  6. Toumazou, C., Purushothaman, S. & Ou, C.-P. qPCR using solid-state sensing. US patent 7,888,015 (2007).

  7. Bousse, L., De Rooij, N.F. & Bergveld, P. Operation of chemically sensitive field-effect sensors as a function of the insulator-electrolyte interface. IEEE Trans. Electron. Dev. 30, 1263–1270 (1983).

    Article  Google Scholar 

  8. Bausells, J., Carrabina, J., Errachid, A. & Merlos, A. Ion-sensitive field-effect transistors fabricated in a commercial CMOS technology. Sens. Actuators B Chem. 57, 56–62 (1999).

    Article  CAS  Google Scholar 

  9. Jakobson, C.G., Dinnar, U., Feinsod, M. & Nemirovsky, Y. Ion-sensitive field-effect transistors in standard CMOS fabricated by post processing. IEEE Sens. J. 2, 279–287 (2002).

    Article  CAS  Google Scholar 

  10. Bergveld, P., DeRooij, N.F. & Zeme, J.N. Physical mechanisms for chemically sensitive semiconductor devices. Nature 273, 438–443 (1978).

    Article  CAS  Google Scholar 

  11. Bergveld, P. ISFET, theory and practice. IEEE Sens. Conf. Toronto 1–26 (IEEE, 2003).

  12. Purushothaman, S., Toumazou, C. & Georgiou, J. Towards fast solid state DNA sequencing. IEEE Int. Symp. on Circuits and Systems 169–172 (IEEE, 2002).

  13. Purushothaman, S., Toumazou, C. & Ou, C.-P. Protons and single nucleotide polymorphism detection: a simple use for the Ion Sensitive Field Effect Transistor. Sens. Actuators B Chem. 114, 964–968 (2006).

    Article  CAS  Google Scholar 

  14. Garner, D.M. et al. A multichannel DNA SoC for rapid point-of-care gene detection. IEEE International Solid-State Circuits Conference 57, 492–493 (2010).

    Google Scholar 

  15. Rothberg, J.M. et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–352 (2011).

    Article  CAS  Google Scholar 

  16. Thewes, R. et al. Sensor arrays for fully-electronic DNA detection on CMOS. IEEE International Solid-State Circuits Conference. Digest of Technical Papers 350–473 (2002).

  17. Pourmand, N. et al. Direct electrical detection of DNA synthesis. Proc. Natl. Acad. Sci. USA 103, 6466–6470 (2006).

    Article  CAS  Google Scholar 

  18. Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63 (2000).

    Article  CAS  Google Scholar 

  19. Matsuo, T. & Esashi, M. Methods of isfet fabrication. Sens. Actuators 1, 77–96 (1981).

    Article  CAS  Google Scholar 

  20. Errachid, A., Zine, N., Samitier, J. & Bausells, J. FET-based chemical sensor systems fabricated with standard technologies. Electroanalysis 16, 1843–1851 (2004).

    Article  CAS  Google Scholar 

  21. Hiratsuka, M. In vitro assessment of the allelic variants of cytochrome P450. Drug Metab. Pharmacokinet. 27, 68–84 (2012).

    Article  CAS  Google Scholar 

  22. Tsao, H., Chin, L., Garraway, L.A. & Fisher, D.E. Melanoma: from mutations to medicine. Genes Dev. 26, 1131–1155 (2012).

    Article  CAS  Google Scholar 

  23. Benlloch, S. et al. Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology. J. Mol. Diagn. 8, 540–543 (2006).

    Article  CAS  Google Scholar 

  24. Niemz, A., Ferguson, T.M. & Boyle, D.S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29, 240–250 (2011).

    Article  CAS  Google Scholar 

  25. Yeung, S.S.W., Lee, T.M.H. & Hsing, I.-M. Electrochemistry-based real-time PCR on a microchip. Anal. Chem. 80, 363–368 (2008).

    Article  CAS  Google Scholar 

  26. Deféver, T. et al. Real-time electrochemical monitoring of the polymerase chain reaction by mediated redox catalysis. J. Am. Chem. Soc. 131, 11433–11441 (2009).

    Article  Google Scholar 

  27. Burns, M. An integrated nanoliter DNA analysis device. Science 282, 484–487 (1998).

    Article  CAS  Google Scholar 

  28. Neuzil, P., Pipper, J. & Hsieh, T.M. Disposable real-time microPCR device: lab-on-a-chip at a low cost. Mol. Biosyst. 2, 292–298 (2006).

    Article  CAS  Google Scholar 

  29. Ottesen, E.A., Hong, J.W., Quake, S.R. & Leadbetter, J.R. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314, 1464–1467 (2006).

    Article  CAS  Google Scholar 

  30. Wong, H.S. & White, M.H. A self-contained CMOS integrated pH sensor. International. Electron Devices Meeting, Technical Digest 658–661 (1988).

  31. Heyries, K.A. et al. Megapixel digital PCR. Nat. Methods 8, 649–651 (2011).

    Article  CAS  Google Scholar 

  32. Stedtfeld, R.D. et al. Gene-Z: a device for point of care genetic testing using a smartphone. Lab Chip 12, 1454–1462 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T.S.K.T. Lim for his support and vision in semiconductor-based DNA analysis, T. Cass for his guidance and expert opinion, and the entire DNA Electronics team for their contributions to our wider work.

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Author notes

  1. Alpesh Patel, David M Garner and Chan-Ju A Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. DNA Electronics Ltd., London, UK

    Christofer Toumazou, Leila M Shepherd, Samuel C Reed, Ginny I Chen, Alpesh Patel, David M Garner, Chan-Ju A Wang, Chung-Pei Ou, Krishna Amin-Desai, Panteleimon Athanasiou, Hua Bai, Ines M Q Brizido, Benjamin Caldwell, Daniel Coomber-Alford, Karen S Jordan, John C Joyce, Maurizio La Mura, Daniel Morley, Sreekala Sathyavruthan, Sara Temelso, Risha E Thomas & Linglan Zhang

  2. Department of Electrical and Electronic Engineering, Imperial College London, South Kensington Campus, London, UK

    Christofer Toumazou & Pantelis Georgiou

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  1. Christofer Toumazou
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Contributions

C.T. invented the technology and supervised the project with S.C.R. and L.M.S.; D.M.G., H.B., B.C., P.G. and J.C.J. designed the chips and electronics; A.P., P.A. and D.C.-A. designed the integrated test card, fluidics and electrochemistry; C.-P.O., C.-J.A.W., M.L.M., L.Z., A.P. and G.I.C. designed the molecular biological methods and assays; G.I.C., K.A.-D., K.S.J., R.E.T., D.C.-A., D.M., S.S., S.T. and I.M.Q.B. performed the research; G.I.C., L.M.S., S.C.R., C.-P.O., C.-J.A.W. and P.G. wrote the manuscript.

Corresponding authors

Correspondence to Christofer Toumazou or Leila M Shepherd.

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Competing interests

C.T., L.M.S., S.C.R., G.I.C., A.P., D.M.G., C.-P.O., K.A.-D., M.L.M., H.B., L.Z., K.S.J., P.A., D.C.-A., B.C., R.E.T. and D.M. are employees of DNA Electronics Ltd. and have stock or stock options in the company.

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Toumazou, C., Shepherd, L., Reed, S. et al. Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nat Methods 10, 641–646 (2013). https://doi.org/10.1038/nmeth.2520

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