title: Signal Amplification in Field Effect-Based Sandwich Enzyme-Linked Immunosensing by Tuned Buffer Concentration with Ionic Strength Adjuster.
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.
authors.affiliation: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India.|Samtel Centre for Display Technologies, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
authors.forename: Satyendra
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Materials Science Programme, Indian Institute of Technology Kanpur, Kanpur, 208016, India.|Samtel Centre for Display Technologies, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
authors.forename: Narendra
authors.initials: N
authors.lastname: Kumar
authors.affiliation: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India. spanda@iitk.ac.in.|Materials Science Programme, Indian Institute of Technology Kanpur, Kanpur, 208016, India. spanda@iitk.ac.in.|Samtel Centre for Display Technologies, Indian Institute of Technology Kanpur, Kanpur, 208016, India. spanda@iitk.ac.in.
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Panda
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002021:Buffers; D055664:Electrochemical Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry; D005947:Glucose / Q000737:chemistry / Q000302:isolation & purification*; D005949:Glucose Oxidase / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D009994:Osmolar Concentration
chemical_list: D002021:Buffers; D004800:Enzymes, Immobilized; D006046:Gold; D005949:Glucose Oxidase; D005947:Glucose
keywords: Enzymatic immunosensors; Optimized buffer; Sandwich immunoassay; Signal amplification