Optimization of Phosphatase- and Redox Cycling-Based Immunosensors and Its Application to Ultrasensitive Detection of Troponin I

Abstract

The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.

Ascorbic acid-triggered electrochemical–chemical–chemical redox cycling for design of enzyme-amplified electrochemical biosensors on self-assembled monolayer-covered gold electrodes

“…It can remove a phosphate group from the substrate by hydrolyzing phosphoric acid monoesters into a phosphate ion and an electroactive molecule with a free hydroxyl group. Recently, the strategy for signal amplification using an ALP-based enzymatic reaction plus a redox-cycling reaction has been particularly popular in electrochemical immunoassays since it only requires the addition of more chemicals to the electrolyte solution and not a change in the detection procedure of conventional enzyme-based immunoassays [16][17][18][19][20][21][22][23][24]. In the system, the enzymatic product is regenerated after its electrochemical oxidization by a chemical reducing reagent, thus amplifying the electrochemical signal.…”

“…Moreover, the reducing reagent can also prevent the auto-oxidation of the enzymatic product. Among the commonly used ALP substrate/product couples, including 4-aminophenyl phosphate (p-APP)/4-aminophenol (p-AP), hydroquinone diphosphate (HQDP)/hydroquinone (HQ), L-ascorbic acid 2-phosphate (AAP)/L-ascorbic acid (AA), 4-amino-1-naphthyl phosphate (ANP)/4-amino-1-naphthol (AN) and 1-naphthyl phosphate (NPP)/1-naphthol (NP), AAP is better because of its low cost, the easy dissolution of AAP and AA in aqueous solutions, the high formal potential of AAP and low formal potential of AA and the high signal-to-background ratio [20]. Self-assembled monolayer (SAM) on gold has been used frequently for controlling the adsorption of biomolecules and developing electrochemical biosensors [1,[25][26][27].…”

Facile electrochemical detection of Escherichia coli using redox cycling of the product generated by the intracellular β-d-galactosidase

“…For example, hydrolysis of 4-aminopheyl-␤-d-galactopyranoside (APGP) by Gal generates an electroactive 4-aminophenol (AP) [21][22][23][24]. Some of the recent studies have shown that electrochemical-chemical (EC) redox cycling [25,26] and electrochemical-chemical-chemical (ECC) redox cycling [27][28][29][30] of the enzyme product allows for high signal amplification, thereby lowering the detection limits. The electrochemical redox cycling schemes can be combined with the enzymatic reaction simply by adding one or two chemical species to the solution without using an additional electrode or enzyme.…”

Electrogenerated chemiluminescence peptide-based biosensing method for cardiac troponin I using peptide-integrating Ru(bpy)3 2+-functionalized gold nanoparticles as nanoprobe

“…Cardiac troponin I (cTnI), a part of the troponin complex present in cardiac muscle tissues, is a reliable biomarker of cardiac muscle tissue injury and is widely used in the early diagnosis of acute myocardial infarctions [1,2]. Although an increase in cTnI concentration from 10 pg mL −1 to over 1 ng mL −1 into blood vessels within a few hours occurs during the damage to cardiac muscles, it is present at ultralow levels following the onset of acute myocardial infarction symptoms.…”