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Integrating programmable DNAzymes with electrical readout for rapid and culture-free bacterial detection using a handheld platform

Abstract

The detection and identification of bacteria currently rely on enrichment steps such as bacterial culture and nucleic acid amplification to increase the concentration of target analytes. These steps increase assay time, cost and complexity, making it difficult to realize a truly rapid point-of-care test. Here we report the development of an electrical assay that uses electroactive RNA-cleaving DNAzymes (e-RCDs) to identify specific bacterial targets and subsequently release a DNA barcode for transducing a signal onto an electrical chip. Integrating e-RCDs into a two-channel electrical chip with nanostructured electrodes provides the analytical sensitivity and specificity needed for clinical analysis. The e-RCD assay is capable of detecting 10 CFU (equivalent to 1,000 CFU ml–1) of Escherichia coli selectively from a panel containing multiple non-specific bacterial species. Clinical evaluation of this assay using 41 patient urine samples demonstrated a diagnostic sensitivity of 100% and specificity of 78% at an analysis time of less than one hour compared with the several hours needed for currently used culture-based methods.

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Fig. 1: Engineering the e-RCD assay.
Fig. 2: Bacterial detection using the e-RCD assay.
Fig. 3: Evaluation of the specificity of the e-RCD assay.
Fig. 4: Preclinical evaluation of the e-RCD assay for the detection of E. coli in urine.
Fig. 5: Demonstration of a point-of-care platform for E. coli detection in unprocessed urine.

Data availability

All relevant data presented in this study are provided in the article and its Supplementary Information. Source data are provided with this paper.

References

  1. 1.

    Klein, E. Y. et al. Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc. Natl Acad. Sci. USA 115, E3463–E3470 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Ecker, D. J. et al. Ibis T5000: a universal biosensor approach for microbiology. Nat. Rev. Microbiol. 6, 553–558 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations 1–16 (Review on Antimicrobial Resistance, 2014).

  4. 4.

    Lam, B. et al. Solution-based circuits enable rapid and multiplexed pathogen detection. Nat. Commun. 4, 2001 (2013).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  5. 5.

    Davenport, M. et al. New and developing diagnostic technologies for urinary tract infections. Nat. Rev. Urol. 14, 296–310 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Nemr, C. R. Nanoparticle-mediated capture and electrochemical detection of methicillin-resistant Staphylococcus aureus. Anal. Chem. 91, 2847–2853 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7.

    Lapierre, M. A., O’Keefe, M., Taft, B. J. & Kelley, S. O. Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers. Anal. Chem. 75, 6327–6333 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Snodgrass, R. et al. A portable device for nucleic acid quantification powered by sunlight, a flame or electricity. Nat. Biomed. Eng. 2, 657–665 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Hung, G.-C., Nagamine, K., Li, B. & Lo, S.-C. Identification of DNA signatures suitable for use in development of real-time PCR assays by whole-genome sequence approaches: use of streptococcus pyogenes in a pilot study. J. Clin. Microbiol. 50, 2770–2773 (2012).

    PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Young, H. et al. PCR testing of genital and urine specimens compared with culture for the diagnosis of chlamydial infection in men and women. Int. J. STD AIDS 9, 661–665 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Liu, J. et al. An integrated impedance biosensor platform for detection of pathogens in poultry products. Sci. Rep. 8, 16109 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  12. 12.

    Nidzworski, D. et al. A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond. Sci. Rep. 7, 15707 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Drummond, T. G., Hill, M. G. & Barton, J. K. Electrochemical DNA sensors. Nat. Biotechnol. 21, 1192–1199 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Heerema, S. J. & Dekker, C. Graphene nanodevices for DNA sequencing. Nat. Nanotechnol. 11, 127–136 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Li, C.-H., Chen, K.-W., Yang, C.-L., Lin, C.-H. & Hsieh, K.-C. A urine testing chip based on the complementary split-ring resonator and microfluidic channel. In 2018 IEEE Micro Electro Mechanical Systems (MEMS) 1150–1153 (IEEE, 2018); https://doi.org/10.1109/MEMSYS.2018.8346765

  16. 16.

    Point-of-Care Diagnostics Market by Testing (Glucose, Lipids, HbA1c, HCV, HIV, Influenza, Urinalysis, Hematology, Cancer, Pregnancy, PT/INR), Platform (Lateral Flow, Immunoassay), Mode (Prescription, OTC), End-UserGlobal Forecast to 2022 (Markets and Markets, 2018).

  17. 17.

    Li, Y. Toward an efficient DNAzyme. Biochemistry 36, 5589–5599 (1997).

  18. 18.

    Feldman, A. R., Leung, E. K. Y., Bennet, A. J. & Sen, D. The RNA-cleaving bipartite DNAzyme is a distinctive metalloenzyme. ChemBioChem 7, 98–105 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Oh, S. S., Plakos, K., Lou, X., Xiao, Y. & Soh, H. T. In vitro selection of structure-switching, self-reporting aptamers. Proc. Natl Acad. Sci. USA 107, 14053–14058 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Tang, S., Tong, P., Li, H., Tang, J. & Zhang, L. Ultrasensitive electrochemical detection of Pb2+ based on rolling circle amplification and quantum dots tagging. Biosens. Bioelectron. 42, 608–611 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Shen, L. et al. Electrochemical DNAzyme sensor for lead based on amplification of DNA–Au bio-bar codes. Anal. Chem. 80, 6323–6328 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Zhang, X.-B., Kong, R.-M. & Lu, Y. Metal ion sensors based on DNAzymes and related DNA molecules. Annu. Rev. Anal. Chem. 4, 105–128 (2011).

    CAS  Article  Google Scholar 

  23. 23.

    Huang, J.-Y. et al. A high-sensitivity electrochemical aptasensor of carcinoembryonic antigen based on graphene quantum dots-ionic liquid-nafion nanomatrix and DNAzyme-assisted signal amplification strategy. Biosens. Bioelectron. 99, 28–33 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Li, C., Tao, Y., Yang, Y., Xiang, Y. & Li, G. In vitro analysis of DNA–protein interactions in gene transcription using DNAzyme-based electrochemical assay. Anal. Chem. 89, 5003–5007 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Chen, J. et al. An ultrasensitive electrochemical biosensor for detection of DNA species related to oral cancer based on nuclease-assisted target recycling and amplification of DNAzyme. Chem. Commun. 47, 8004 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Xiao, Y., Rowe, A. A. & Plaxco, K. W. Electrochemical detection of parts-per-billion lead via an electrode-bound DNAzyme assembly. J. Am. Chem. Soc. 129, 262–263 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Shamah, S. M., Healy, J. M. & Cload, S. T. Complex target SELEX. Acc. Chem. Res. 41, 130–138 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Sun, Y., Chang, Y., Zhang, Q. & Liu, M. An origami paper-based device printed with DNAzyme-containing DNA superstructures for Escherichia coli detection. Micromachines 10, 531 (2019).

    PubMed Central  Article  Google Scholar 

  29. 29.

    Zhang, W., Feng, Q., Chang, D., Tram, K. & Li, Y. In vitro selection of RNA-cleaving DNAzymes for bacterial detection. Methods 106, 66–75 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Ali, M. M., Aguirre, S. D., Lazim, H. & Li, Y. Fluorogenic DNAzyme probes as bacterial indicators. Angew. Chem. Int. Ed. 50, 3751–3754 (2011).

    CAS  Article  Google Scholar 

  31. 31.

    Tram, K., Kanda, P., Salena, B. J., Huan, S. & Li, Y. Translating bacterial detection by DNAzymes into a litmus test. Angew. Chem. Int. Ed. 53, 12799–12802 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Zaouri, N., Cui, Z., Peinetti, A. S., Lu, Y. & Hong, P. Y. DNAzyme-based biosensor as a rapid and accurate verification tool to complement simultaneous enzyme-based media for: E. coli detection. Environ. Sci. Water Res. Technol. 5, 2260–2268 (2019).

    CAS  Article  Google Scholar 

  33. 33.

    Flores-Mireles, A. L., Walker, J. N., Caparon, M. & Hultgren, S. J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Zhi, L. et al. White emission magnetic nanoparticles as chemosensors for sensitive colorimetric and ratiometric detection, and degradation of ClO and SCN in aqueous solutions based on a logic gate approach. Nanoscale 7, 11712–11719 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Kalofonou, M. & Toumazou, C. An ISFET based analogue ratiometric method for DNA methylation detection. In 2014 IEEE International Symposium on Circuits and Systems (ISCAS) 1832–1835 (IEEE, 2014); https://doi.org/10.1109/ISCAS.2014.6865514

  36. 36.

    Yan, Y. et al. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification. Sci. Rep. 6, 18810 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Rahi, A., Sattarahmady, N. & Heli, H. Zepto-molar electrochemical detection of Brucella genome based on gold nanoribbons covered by gold nanoblooms. Sci. Rep. 5, 18060 (2016).

    Article  CAS  Google Scholar 

  38. 38.

    Aguirre, S., Ali, M., Salena, B. & Li, Y. A sensitive DNA enzyme-based fluorescent assay for bacterial detection. Biomolecules 3, 563–577 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  39. 39.

    Weng, D. & Landau, U. Direct electroplating on nonconductors. J. Electrochem. Soc. 142, 2598–2604 (1995).

  40. 40.

    Mandler, D. & Bard, A. J. A new approach to the high resolution electrodeposition of metals via the feedback mode of the scanning electrochemical microscope. J. Electrochem. Soc. 137, 1079 (1990).

    CAS  Article  Google Scholar 

  41. 41.

    Werner, A. Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number. Nucl. Acids Res. 39, e17 (2011).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  42. 42.

    Bard, A. J., Shea, T. V., Crayston, J. A., Kittlesen, G. P. & Wrighton, M. S. Digital simulation of the measured electrochemical response of reversible redox couples at microelectrode arrays: consequences arising from closely spaced ultramicroelectrodes. Anal. Chem. 58, 2321–2331 (1986).

    CAS  Article  Google Scholar 

  43. 43.

    Niwa, O., Morita, M. & Tabei, H. Electrochemical behavior of reversible redox species at interdigitated array electrodes with different geometries: consideration of redox cycling and collection efficiency. Anal. Chem. 62, 447–452 (1990).

    CAS  Article  Google Scholar 

  44. 44.

    Zhang, C. & Park, S. Simple technique for constructing thin-layer electrochemical cells. Anal. Chem. 60, 1639–1642 (1988).

    CAS  Article  Google Scholar 

  45. 45.

    Horny, M. C. et al. Electrochemical DNA biosensors based on long-range electron transfer: investigating the efficiency of a fluidic channel microelectrode compared to an ultramicroelectrode in a two-electrode setup. Lab Chip 16, 4373–4381 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Willner, I., Shlyahovsky, B., Zayats, M. & Willner, B. DNAzymes for sensing, nanobiotechnology and logic gate applications. Chem. Soc. Rev. 37, 1153–1165 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Barfidokht, A. & Gooding, J. J. Approaches toward allowing electroanalytical devices to be used in biological fluids. Electroanalysis 26, 1182–1196 (2014).

    CAS  Article  Google Scholar 

  48. 48.

    Sabaté del Río, J., Henry, O. Y. F., Jolly, P. & Ingber, D. E. An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids. Nat. Nanotechnol. 14, 1143–1149 (2019).

  49. 49.

    Liang, J., Chen, Z., Guo, L. & Li, L. Electrochemical sensing of l-histidine based on structure-switching DNAzymes and gold nanoparticle-graphene nanosheet composites. Chem. Commun. 47, 5476–5478 (2011).

  50. 50.

    Park, K. S. Nucleic acid aptamer-based methods for diagnosis of infections. Biosens. Bioelectron. 102, 179–188 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Liu, M., Chang, D. & Li, Y. Discovery and biosensing applications of diverse RNA-cleaving DNAzymes. Acc. Chem. Res. 9, 2273–2283 (2017).

    Article  CAS  Google Scholar 

  52. 52.

    Akobeng, A. K. Understanding diagnostic tests 3: receiver operating characteristic curves. Acta Paediatr. 96, 644–647 (2007).

    PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Allen, J. B. & Larry, R. F. Electrochemical Methods: Fundamentals and Applications 2nd edn, 1505–1506 (Wiley, 2002).

  54. 54.

    Cinti, S., Moscone, D. & Arduini, F. Preparation of paper-based devices for reagentless electrochemical (bio)sensor strips. Nat. Protoc. 14, 2437–2451 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

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Acknowledgements

We acknowledge J. Yang for her support in SEM imaging of nanostructured gold electrodes. We thank M. Gaskin for his support towards preparing and characterizing the clinical urine samples used in this study. We acknowledge the kind help of J. Gu with the ROC plot. We thank Y. Lu for helpful discussions. The work is supported by the Natural Science and Engineering Research Council (NSERC) of Canada. L.S. and T.H. are supported by the Canada Research Chair programme. The electron microscopy was carried out at the Canadian Centre for Electron Microscopy (CCEM), a national facility supported by the NSERC and McMaster University.

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Authors

Contributions

R.P. designed and performed electrochemical experiments and co-wrote the manuscript. D.C. designed and performed molecular experiments and edited the manuscript. M.S. supervised and helped in designing the preclinical study. T.H. supervised the assay’s performance in biological samples and edited the manuscript. Y.L. supervised the overall assay and experimental design and edited the manuscript. L.S. contributed to the overall project design and supervision and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Yingfu Li or Leyla Soleymani.

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The authors declare no competing interests.

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Peer review information Nature Chemistry thanks Alexis Vallée-Bélisle and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Notes 1–9, Figs. 1–11, and Tables 1 and 2.

Source data

Source Data Fig. 1

Kinetics study data.

Source Data Fig. 2

Calibration plot data.

Source Data Fig. 3

Specificity data.

Source Data Fig. 4

Clinical testing data.

Source Data Fig. 5

Clinical testing data using chip-based pretreatment.

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Pandey, R., Chang, D., Smieja, M. et al. Integrating programmable DNAzymes with electrical readout for rapid and culture-free bacterial detection using a handheld platform. Nat. Chem. (2021). https://doi.org/10.1038/s41557-021-00718-x

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