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Signal Amplification in Field Effect-Based Sandwich Enzyme-Linked Immunosensing by Tuned Buffer Concentration with Ionic Strength Adjuster

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Abstract

Miniaturization of the sandwich enzyme-based immunosensor has several advantages but could result in lower signal strength due to lower enzyme loading. Hence, technologies for amplification of the signal are needed. Signal amplification in a field effect-based electrochemical immunosensor utilizing chip-based ELISA is presented in this work. First, the molarities of phosphate buffer saline (PBS) and concentrations of KCl as ionic strength adjuster were optimized to maximize the GOx glucose-based enzymatic reactions in a beaker for signal amplification measured by change in the voltage shift with an EIS device (using 20 μl of solution) and validated with a commercial pH meter (using 3 ml of solution). The PBS molarity of 100 μM with 25 mM KCl provided the maximum voltage shift. These optimized buffer conditions were further verified for GOx immobilized on silicon chips, and similar trends with decreased PBS molarity were obtained; however, the voltage shift values obtained on chip reaction were lower as compared to the reactions occurring in the beaker. The decreased voltage shift with immobilized enzyme on chip could be attributed to the increased Km (Michaelis–Menten constant) values in the immobilized GOx. Finally, a more than sixfold signal enhancement (from 8 to 47 mV) for the chip-based sandwich immunoassay was obtained by altering the PBS molarity from 10 to 100 μM with 25 mM KCl.

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References

  1. Selvakumar, L. S., & Thakur, M. S. (2012). Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: a novel approach. Analytica Chimica Acta, 722, 107–113.

    Article  CAS  Google Scholar 

  2. Nian, H., Wang, J., Wu, H., Lo, J.-G., Chiu, K.-H., Pounds, J. G., & Lin, Y. (2012). Electrochemical immunoassay of cotinine in serum based on nanoparticle probe and immunochromatographic strip. Analytica Chimica Acta, 713, 50–55.

    Article  CAS  Google Scholar 

  3. Perrotta, P. R., Arévalo, F. J., Vettorazzi, N. R., Zón, M. A., & Fernández, H. (2012). Development of a very sensitive electrochemical magneto immunosensor for the direct determination of ochratoxin A in red wine. Sensors and Actuators B: Chemical, 162, 327–333.

    Article  CAS  Google Scholar 

  4. Yang, H., Liang, W., He, N., Deng, Y., & Li, Z. (2015). Chemiluminescent labels released from long spacer arm-functionalized magnetic particles: a novel strategy for ultrasensitive and highly selective detection of pathogen infections. ACS Applied Materials & Interfaces, 7, 774–781.

    Article  CAS  Google Scholar 

  5. Li, Z., Yang, H., He, N., Liang, W., Ma, C., Shah, M. A. A., Tang, Y., Li, S., Liu, H., Jiang, H., & Guo, Y. (2013). Solid-phase hybridization efficiency improvement on the magnetic nanoparticle surface by using dextran as molecular arms. Journal of Biomedical Nanotechnology, 9, 1945–1949.

    Article  CAS  Google Scholar 

  6. Fitzgerald, J., Leonard, P., Danaher, M., & O’Kennedy, R. (2015). Rapid simultaneous detection of anti-protozoan drugs using a lateral-flow immunoassay format. Appl Biochem Biotechnol, 176, 387–398.

    Article  CAS  Google Scholar 

  7. Szcs, J., Pretsch, E., & Gyurcsányi, R. E. (2009). Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection. Analyst, 134, 1601–1607.

    Article  Google Scholar 

  8. Jung, H. S., Song, K. S., & Kim, T. (2007). Electrochemical immunosensing of GOx-labeled CRP antigen on capture antibody monolayer immobilized on calixcrown-5 SAMs. Bulletin of the Korean Chemical Society, 28, 1792–1796.

    Article  CAS  Google Scholar 

  9. Singh, A., Park, S., & Yang, H. (2013). Glucose-oxidase label-based redox cycling for an incubation period-free electrochemical immunosensor. Analytical Chemistry, 85, 4863–4868.

    Article  CAS  Google Scholar 

  10. Horak, J., Dincer, C., Bakirci, H., & Urban, G. (2014). Sensitive, rapid and quantitative detection of substance P in serum samples using an integrated microfluidic immunochip. Biosensors and Bioelectronics, 58, 186–192.

    Article  CAS  Google Scholar 

  11. de Bellefontaine, C., Josse, F., Domurado, M., & Domurado, D. (1994). Immunoassay for native enzyme quantification in biological samples. Appl Biochem Biotechnol, 48, 117–123.

    Article  Google Scholar 

  12. Selvanayagam, Z. E., Neuzil, P., Gopalakrishnakone, P., Sridhar, U., Singh, M., & Ho, L. C. (2002). An ISFET-based immunosensor for the detection of β-Bungarotoxin. Biosensors and Bioelectronics, 17, 821–826.

    Article  CAS  Google Scholar 

  13. Kamahori, M., Ishige, Y., & Shimoda, M. (2008). Enzyme immunoassay using a reusable extended-gate field-effect-transistor sensor with a ferrocenylalkanethiol-modified gold electrode. Analytical Sciences, 24, 1073–1079.

    Article  CAS  Google Scholar 

  14. Jang, H.-J., Ahn, J., Kim, M.-G., Shin, Y.-B., Jeun, M., Cho, W.-J., & Lee, K. H. (2015). Electrical signaling of enzyme-linked immunosorbent assays with an ion-sensitive field-effect transistor. Biosensors and Bioelectronics, 64, 318–323.

    Article  CAS  Google Scholar 

  15. Elanchezhian, V. S., & Kandaswamy, M. (2009). A ferrocene-based multi-signaling sensor molecule functions as a molecular switch. Inorganic Chemistry Communications, 12, 161–165.

    Article  CAS  Google Scholar 

  16. Qiu, J. D., Zhou, W. M., Guo, J., Wang, R., & Liang, R. P. (2009). Amperometric sensor based on ferrocene-modified multiwalled carbon nanotube nanocomposites as electron mediator for the determination of glucose. Anal Biochem, 385, 264–269.

    Article  CAS  Google Scholar 

  17. Wu, Y., Zheng, J., Li, Z., Zhao, Y., & Zhang, Y. (2009). A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode. Biosensors & bioelectronics, 24, 1389–1393.

    Article  CAS  Google Scholar 

  18. Parkash, O., Yean, C., & Shueb, R. (2014). Screen printed carbon electrode based electrochemical immunosensor for the detection of dengue NS1 antigen. Diagnostics, 4, 165–180.

    Article  Google Scholar 

  19. Li, Y., Fang, L., Cheng, P., Deng, J., Jiang, L., Huang, H., & Zheng, J. (2013). An electrochemical immunosensor for sensitive detection of Escherichia coli O157:H7 using C60 based biocompatible platform and enzyme functionalized Pt nanochains tracing tag. Biosensors and Bioelectronics, 49, 485–491.

    Article  CAS  Google Scholar 

  20. Ren, J., Tang, D., Su, B., Tang, J., & Chen, G. (2010). Glucose oxidase-doped magnetic silica nanostructures as labels for localized signal amplification of electrochemical immunosensors. Nanoscale, 2, 1244–1249.

    Article  CAS  Google Scholar 

  21. Zhang, J., Pearce, M. C., Ting, B. P., & Ying, J. Y. (2011). Ultrasensitive electrochemical immunosensor employing glucose oxidase catalyzed deposition of gold nanoparticles for signal amplification. Biosensors and Bioelectronics, 27, 53–57.

    Article  Google Scholar 

  22. Dastidar, S., Agarwal, A., Kumar, N., Bal, V., & Panda, S. (2015). Sensitivity enhancement of electrolyte-insulator-semiconductor sensors using mesotextured and nanotextured dielectric surfaces. Sensors Journal IEEE, 15, 2039–2045.

    Article  CAS  Google Scholar 

  23. Kumar, N., Kumar, J., & Panda, S. (2015). Sensitivity enhancement mechanisms in textured dielectric based electrolyte-insulator-semiconductor (EIS) sensors. ECS Journal of Solid State Science and Technology, 4, N18–N23.

    Article  CAS  Google Scholar 

  24. Jang, H.-J., & Cho, W.-J. (2012). Fabrication of high-performance fully depleted silicon-on-insulator based dual-gate ion-sensitive field-effect transistor beyond the Nernstian limit. Applied Physics Letters, 100, 073701.

    Article  Google Scholar 

  25. Bae, T.-E., Jang, H.-J., Lee, S.-W., & Cho, W.-J. (2013). Enhanced sensing properties by dual-gate ion-sensitive field-effect transistor using the solution-processed Al2O3 sensing membranes. Japanese Journal of Applied Physics, 52, 06GK03.

    Article  Google Scholar 

  26. Schoning, M. J., & Poghossian, A. (2002). Recent advances in biologically sensitive field-effect transistors (BioFETs). The Analyst, 127, 1137–1151.

    Article  Google Scholar 

  27. Das Neves, L., & Vitolo, M. (2007). Use of glucose oxidase in a membrane reactor for gluconic acid production. Appl Biochem Biotechnol, 137–140, 161–170.

    Google Scholar 

  28. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., & Singh, R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry, 5, 467–477.

    Article  CAS  Google Scholar 

  29. Stoll, V. S., & Blanchard, J. S. (2009). Methods in enzymology, vol. 463. In R. B. Richard, P. D. Murray (Eds.), Bufferes: principles and practice (pp. 43–56). Academic Press.

  30. Schöning, M. J., Arzdorf, M., Mulchandani, P., Chen, W., & Mulchandani, A. (2003). Towards a capacitive enzyme sensor for direct determination of organophosphorus pesticides: fundamental studies and aspects of development. Sensors, 3, 119–127.

    Article  Google Scholar 

  31. Preetha, R., Rani, K., Veeramani, M. S. S., Fernandez, R. E., Vemulachedu, H., Sugan, M., Bhattacharya, E., & Chadha, A. (2011). Potentiometric estimation of blood analytes - triglycerides and urea: comparison with clinical data and estimation of urea in milk using an electrolyte-insulator-semiconductor-capacitor (EISCAP). Sensors and Actuators B: Chemical, 160, 1439–1443.

    Article  CAS  Google Scholar 

  32. Senillou, A., Jaffrezic-Renault, N., Martelet, C., & Cosnier, S. (1999). A miniaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip. Talanta, 50, 219–226.

    Article  CAS  Google Scholar 

  33. Kumar, N., Kumar, J., & Panda, S. (2015). Low temperature annealed amorphous indium gallium zinc oxide (a-IGZO) as a pH sensitive layer for applications in field effect based sensors. AIP Advances, 5, 067123.

    Article  Google Scholar 

  34. Kumar, S., Ch, R., Rath, D., & Panda, S. (2011). Densities and orientations of antibodies on nano-textured silicon surfaces. Materials Science and Engineering: C, 31, 370–376.

    Article  CAS  Google Scholar 

  35. Good, N. E., & Izawa, S. (1972). Methods in enzymology, vol. 24. In A. San Pietro (Ed.), Hydrogen ion buffers (pp. 53–68). Academic Press.

  36. Bright, H. J., & Appleby, M. (1969). The pH dependence of the individual steps in the glucose oxidase reaction. Journal of Biological Chemistry, 244, 3625–3634.

    CAS  Google Scholar 

  37. Tomotani, E., das Neves, L., & Vitolo, M. (2005). Twenty-sixth symposium on biotechnology for fuels and chemicals, vol. 121-124. In B. Davison, B. Evans, M. Finkelstein, J. McMillan (Eds.), Oxidation of glucose to gluconic acid by glucose oxidase in a membrane bioreactor (pp. 149–162). Humana Press.

  38. Hoxsey, J. A. (2013). Mineral-releasing compost and method of using the same for soil remediation. US Patent, Appl. No. 13/817,128, Pub. No. US 2013/0233036A1.

  39. Chen, R. K. (1992). Method of manufacturing soybean curd, US Patent, Appl. No. US 07/688,013, Pub. No. US5087465A.

  40. Kumar, S., & Panda, S. (2012). Correlation of kinetics and conformations of free and immobilized enzymes on non- and nanotextured silicon biosensor surfaces. Bio Nano Sci., 2, 171–178.

    Google Scholar 

  41. Kojima, K., Hiratsuka, A., Suzuki, H., Yano, K., Ikebukuro, K., & Karube, I. (2003). Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film. Analytical Chemistry, 75, 1116–1122.

    Article  CAS  Google Scholar 

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Acknowledgment

The authors gratefully acknowledge the financial support of the DST Science and Engineering Research Board, India (Grant No. SB/S3/CE/055/2013).

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    Authors and Affiliations

    1. Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India

      Satyendra Kumar & Siddhartha Panda

    2. Materials Science Programme, Indian Institute of Technology Kanpur, Kanpur, 208016, India

      Narendra Kumar & Siddhartha Panda

    3. Samtel Centre for Display Technologies, Indian Institute of Technology Kanpur, Kanpur, 208016, India

      Satyendra Kumar, Narendra Kumar & Siddhartha Panda

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    1. Satyendra Kumar
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    Correspondence to Siddhartha Panda.

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    Satyendra Kumar and Narendra Kumar contributed equally to this work.

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    Kumar, S., Kumar, N. & Panda, S. Signal Amplification in Field Effect-Based Sandwich Enzyme-Linked Immunosensing by Tuned Buffer Concentration with Ionic Strength Adjuster. Appl Biochem Biotechnol 179, 168–178 (2016). https://doi.org/10.1007/s12010-016-1986-y

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