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Applied Biochemistry and Biotechnology

, Volume 179, Issue 1, pp 168–178 | Cite as

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

  • Satyendra Kumar
  • Narendra Kumar
  • Siddhartha PandaEmail author
Article

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.

Keywords

Enzymatic immunosensors Signal amplification Optimized buffer Sandwich immunoassay 

Notes

Acknowledgment

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

References

  1. 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.CrossRefGoogle Scholar
  2. 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.CrossRefGoogle Scholar
  3. 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.CrossRefGoogle Scholar
  4. 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.CrossRefGoogle Scholar
  5. 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.CrossRefGoogle Scholar
  6. 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.CrossRefGoogle Scholar
  7. 7.
    Szcs, J., Pretsch, E., & Gyurcsányi, R. E. (2009). Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection. Analyst, 134, 1601–1607.CrossRefGoogle Scholar
  8. 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.CrossRefGoogle Scholar
  9. 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.CrossRefGoogle Scholar
  10. 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.CrossRefGoogle Scholar
  11. 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.CrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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.CrossRefGoogle Scholar
  14. 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.CrossRefGoogle Scholar
  15. 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.CrossRefGoogle Scholar
  16. 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.CrossRefGoogle Scholar
  17. 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.CrossRefGoogle Scholar
  18. 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.CrossRefGoogle Scholar
  19. 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.CrossRefGoogle Scholar
  20. 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.CrossRefGoogle Scholar
  21. 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.CrossRefGoogle Scholar
  22. 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.CrossRefGoogle Scholar
  23. 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.CrossRefGoogle Scholar
  24. 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.CrossRefGoogle Scholar
  25. 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.CrossRefGoogle Scholar
  26. 26.
    Schoning, M. J., & Poghossian, A. (2002). Recent advances in biologically sensitive field-effect transistors (BioFETs). The Analyst, 127, 1137–1151.CrossRefGoogle Scholar
  27. 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. 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.CrossRefGoogle Scholar
  29. 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.Google Scholar
  30. 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.CrossRefGoogle Scholar
  31. 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.CrossRefGoogle Scholar
  32. 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.CrossRefGoogle Scholar
  33. 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.CrossRefGoogle Scholar
  34. 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.CrossRefGoogle Scholar
  35. 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.Google Scholar
  36. 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.Google Scholar
  37. 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.Google Scholar
  38. 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.Google Scholar
  39. 39.
    Chen, R. K. (1992). Method of manufacturing soybean curd, US Patent, Appl. No. US 07/688,013, Pub. No. US5087465A.Google Scholar
  40. 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. 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.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Satyendra Kumar
    • 1
    • 3
  • Narendra Kumar
    • 2
    • 3
  • Siddhartha Panda
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Chemical EngineeringIndian Institute of Technology KanpurKanpurIndia
  2. 2.Materials Science ProgrammeIndian Institute of Technology KanpurKanpurIndia
  3. 3.Samtel Centre for Display TechnologiesIndian Institute of Technology KanpurKanpurIndia

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