Washing-Free Heterogeneous Immunosensor Using Proximity-Dependent Electron Mediation between an Enzyme Label and an Electrode

Abstract

Washing processes, essential in most heterogeneous labeled assays, have been a big hurdle in simplifying the detection procedure and reducing assay time. Nevertheless, less attention has been paid to washing-free heterogeneous labeled assays. We report a purely washing-free immunosensor that allows fast, sensitive, and single-step detection of prostate-specific antigen in serum with low interference. Proximity-dependent electron mediation of ferrocenemethanol (Fc) between an indium-tin oxide (ITO) electrode and a glucose-oxidase (GOx) label allows us to discriminate between a bound and an unbound label: a bound label offers faster electron mediation than an unbound one. The electrooxidation of Fc at a low applied potential (0.13 V vs Ag/AgCl) and a low electrocatalytic ITO electrode and the oxidation of l-ascorbic acid by l-ascorbate oxidase minimize the effect of the interfering species. With a high concentration of glucose (200 mM), the signal and background levels are hardly dependent on the glucose-concentration variation in the sample. The washing-free immunosensor can detect a concentration of ca. 1 pg/mL for mouse IgG in phosphate-buffered saline and a concentration of ca. 10 pg/mL for prostate-specific antigen spiked in female serum after an incubation period of 10 min. The concentrations measured with actual clinical serum samples are in good agreement with the concentrations measured with a commercial instrument, which renders the washing-free heterogeneous immunosensor appealing for practical use.

Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins

“…24,25 In recent years, we have shown that electrochemical biosensors using ALP labels combined with indium–tin oxide (ITO) electrodes facilitate high signal-to-background ratios, i.e. , very low detection limits 25–28 because ITO electrodes show low and flat capacitive background currents.…”

A self-powered one-touch blood extraction system: a novel polymer-capped hollow microneedle integrated with a pre-vacuum actuator

“…The blood sample volumes extracted via this one-touch system were determined by the pre-vacuum PDMS chamber volumes, and successful in vivo blood sampling was demonstrated in a rabbit experiment. The self-powered mechanism, disposability, and simplicity of one-touch operation make this system wellsuited for further integration with other microsystems, such as microfluidic chips, 38,39 biosensors, 40,41 and electrode sensors, 42,43 which may be used to realize a real-time micro total analysis system for POC diagnosis. In vivo blood extraction using the self-powered one-touch blood-extraction system.…”

Electrochemical immunosensor for the prostate specific antigen detection based on carbon nanotube and gold nanoparticle amplification strategy

“…The three electrode system consisted of a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference, and a platinum wire as the auxiliary. Impedance measurements were performed by applying an ac voltage of 10 mV amplitude in the 0.01 to 10 5 Hz frequency range in a 5.0 mM K 3 [Fe(CN) 6 6 ] redox probe solution with 0.1 M KCl.…”

“…A number of methods and strategies have been reported for the determination of PSA such as fluorescent labeling [2], chemiluminescence [3,4], enzyme labels [5,6], and gold nanoparticle-and carbon nanotube-based biosensors [7]. As point-of-care systems have being increasingly attracted interest in recent years, electrochemical immunoassay seems to be an excellent candidate for the rapid and inexpensive diagnosis of pathogenic biological species of clinical interest [8].…”