Potentiometric Immunoassay Research Articles

Ion-selective electrode-based potentiometric immunoassays for the quantitative monitoring of alpha-fetoprotein by coupling rolling cycle amplification with silver nanoclusters.

Potentiometric immunoassays have been utilized for the quantitative detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma, but most of them involve low sensitivity and enzyme labels, and thus are unfavorable for routine use. In this work, we report the proof-of-concept of a sensitive and powerful ion-selective potentiometric sensing method for AFP detection with an in situ amplified signal readout. This potentiometric immunoassay mainly contains a silver nanocluster-functionalized single-stranded DNA (AgNC-DNA), a short DNA primer and two antibodies. A sandwich-type immunoreaction is employed for AFP determination on an anti-AFP capture antibody-coated microplate using a biotinylated human AFP secondary antibody. Coupling with a typical biotin-avidin system, the biotinylated DNA initiator strands are conjugated on the microplate in the presence of AFP to induce the rolling cycle amplification (RCA) reaction, followed by AgNC-DNA hybridization. Upon addition of HNO3, the hybridized AgNCs are dissolved into numerous Ag(I) ions, which can be readily determined on a portable handheld silver-ion selective electrode (Ag-ISE). Under optimal conditions, the electrode potential increases with an increase in AFP concentration and exhibits a good linear range of 0.01-100 ng mL-1 at a detection limit of 7.9 pg mL-1. Moreover, the Ag-ISE-based potentiometric immune assay also shows good reproducibility, high specificity and long-term storage stability. Importantly, 18 human serum specimens containing the AFP analyte are screened using the potentiometric immunoassay, giving well-matched experimental results relative to the referenced enzyme-linked immunosorbent assay (ELISA) method.

New Insights on Potentiometric Immunosensor at Carbon Fiber Microelectrode for Alpha‐Fetoprotein in Hepatocellular Carcinoma

AbstractHerein, we investigated the analytical features of potentiometric immunosensors for detection of alpha‐fetoprotein (AFP) in hepatocellular carcinoma at different electrodes, such as carbon fiber microelectrode (CFME) and carbon‐disk electrode (CDE), respectively. To construct such an immunosensor, anti‐AFP capture antibodies were first conjugated covalently onto the activated electrodes through typical carbodiimide coupling. Thereafter, one‐step immunoreaction protocol was successfully introduced to develop a new potentiometric immunoassay upon addition of AFP. Accompanying the antigen‐antibody reaction, the surface charges of the modified electrodes were changed for the readout of electric potential. Results indicated that the linear range of CDE‐based immunosensor was 0.1–100 ng mL−1 AFP, whereas the assay sensitivity by using CFME could be further increased to 3.2 pg mL−1 with the linear range from 0.01 to 500 ng mL−1 AFP. Meanwhile, CFME‐based immunosensor showed high sensitivity, good reproducibility and specificity, and could be utilized for the analysis of human serum specimens with consistent results relative to commercialized ELISA kit.

A one-step potentiometric immunoassay for plasma cardiac troponin I using an antibody-functionalized bis-MPA-COOH dendrimer as a competitor with improved sensitivity.

Herein, we have reported a new one-step potentiometric immunoassay for the sensitive and specific detection of human plasma cardiac troponin I (cTnI), a biomarker of cardio-cerebrovascular diseases. Initially, the cTnI biomolecules were immobilized on the surface of a gold nanoparticle-functionalized screen-printed graphite electrode (SPGE). Thereafter, rabbit polyclonal antibodies to cTnI were covalently conjugated to the bis-MPA-COOH dendrimers through typical carbodiimide coupling. The introduction of the target analyte caused a competitive immunoreaction between the immobilized cTnI on the electrode and the conjugated antibody on the dendrimers. The potentiometric measurement was mainly derived from the change in the surface charge on the surface of the modified electrode due to the negatively charged bis-MPA-COOH dendrimers after the immunoreaction. On increasing target cTcI, the number of charged dendrimers on the immunosensor decreased, resulting in a change in the electric potential. Under optimum conditions, the potentiometric immunosensor exhibited good potentiometric responses for the detection of cTcI and allowed the determination of the target analyte at a concentration as low as 7.3 pg mL-1. An intermediate precision of ≤8.7% was accomplished with batch-to-batch identification. Meanwhile, the potentiometric immunosensor showed good anti-interfering capacity and selectivity against other proteins and biomarkers. Importantly, our system displayed high accuracy for the analysis of human plasma serum samples containing target cTcI relative to commercial human cTcI enzyme-linked immunosorbent assay (ELISA) kits.

Enzyme-loaded liposome with biocatalytic precipitation for potentiometric immunoassay of thyroid-stimulating hormone in thyroid carcinoma

A simple and feasible potentiometric immunosensing platform based on enzymatic biocatalytic precipitation technique was designed for the sensitive detection of thyroid-stimulating hormone (TSH; a typical kind of biomarkers for thyroid carcinoma), using horseradish peroxidase (HRP)-loaded liposome for the signal amplification. To construct such an assay system, a sandwich-type immunoreaction was readily carried out on monoclonal anti-TSH capture antibody-coated electrode by using polyclonal anti-TSH secondary antibody-conjugated HRP-loaded liposome. Accompanying the formation of sandwich-type immunocomplex, the carried liposome was lysed through the added Triton X-100 to release the entrapped HRP molecules, which catalyzed the oxidation of 4-chloro-1-naphthol to produce an insoluble and uncharged organic precipitation on the electrode surface, thereby causing the change of the local electrical potential. Two labeling protocols with and without the liposome were investigated for detection of target TSH, improved analytical features were achieved with HRP-entrapped liposome. Under optimal conditions, the potentiometric immunosensor had good responses for TSH detection within the linear range of 0.01–30 μIU/mL at a detection limit of 0.0067 μIU/mL. Good reproducibility, high specificity and long-time stability were acquired during the assay procedure. Importantly, a well-matched accuracy between the potentiometric immunosensor and commercial human TSH ELISA kit was gave for the analysis of human serum samples.

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen (SCCA; cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction (HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle (AuNP). Then, the HCR reaction was readily executed between two glucose oxidase (GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.

Copper nanocluster-labeled hybridization chain reaction for potentiometric immunoassay of matrix metalloproteinase-7 in acute kidney injury and renal cancer

A simple and portable potentiometric immunoassay was designed for the quantitative detection of matrix metalloproteinase-7 (MMP7) in acute kidney injury and renal cancer on a copper ion-selective electrode (Cu-ISE).

Phytic acid-based copper(ii) ion-selective electrode on graphene oxide for potentiometric immunoassay of breast cancer antigen 15-3

A potentiometric immunoassay based on a copper(ii) ion-selective electrode (Cu-ISE) was designed for the quantitative determination of carbohydrate antigen 15-3 (CA 15-3; a tumor marker associated with breast cancer).

A novel potentiometric immunoassay for carcinoma antigen 15-3 by coupling enzymatic biocatalytic precipitation with a nanogold labelling strategy.

Methods based on potentiometric measurement have been developed for immunoassays, but most exhibit low sensitivities and are unsuitable for early diagnosis of disease. Herein we design a new potentiometric immunosensing platform for the sensitive detection of carcinoma antigen 15-3 (CA 15-3) by coupling with enzymatic biocatalytic precipitation and a nanogold labeling technique. The sandwich-type immunoreaction is carried out on a monoclonal anti-CA 15-3 capture antibody-modified working electrode, using horseradish peroxidase (HRP) and polyclonal anti-CA 15-3 detection antibody-labeled gold nanoparticles. Upon the introduction of target CA 15-3, the carried HRP molecules with an immunocomplex catalyze the oxidation of 4-chloro-1-naphthol (4-CN) into the insoluble benzo-4-chlorohexadienone. The formed product coated on the surface of the modified electrode results in a change of the electrical potential. Under optimal conditions, the shift in the electrical potential relative to the background signal increases with the increasing target CA 15-3 concentration, and exhibits a good linear relationship within the dynamic range of 0.01-30 U mL-1 at a detection limit of 7.8 mU mL-1. Good precision and reproducibility, and high specificity were acquired for the analysis of 15 human serum specimens, giving well matched results with those obtained from a human CA 15-3 ELISA kit.

A potentiometric immunosensor for enterovirus 71 based on bis-MPA-COOH dendrimer-doped AgCl nanospheres with a silver ion-selective electrode.

Herein a new potentiometric immunoassay for the point-of-care detection of enterovirus 71 (EV71) was developed by using a silver (Ag+) ion-selective electrode (ISE). Initially, the carboxylated dendrimer-doped AgCl nanospheres were synthesized by the reverse micelle method. Then the synthesized nanospheres were used to label a polyclonal mouse anti-EV71 antibody via a typical carbodiimide coupling method. The immunoreaction was executed on a monoclonal anti-EV71 antibody-coated microplate by using biofunctional AgCl nanospheres as the detection antibody. With a sandwich-type immunoassay format, the carried AgCl nanospheres could be dissolved in the presence of NH3·H2O, and the released silver ions were determined with an external silver ion-selective electrode. Under optimal conditions, the shift in the potential increased with the increase in the EV71 concentration, in a wide linear range of 0.3-300 ng mL-1, with a detection limit of 0.058 ng mL-1. Intra- and inter-assay relative standard deviations with identical batches were less than 4.15% and 6.15%, respectively. By validating the spiked serum samples, our system shows consistency with the enzyme-linked immunosorbent assay (ELISA) kit.

Potentiometric competitive immunoassay for determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles

The authors report on a competitive potentiometric immunoassay for aflatoxin B1 (AFB1) in food that displays distinctly improved sensitivity. Gold nanoparticles (AuNPs; 16 nm i. d.) were functionalized with polyclonal anti-AFB1 antibody (pAb), whilst the sensor electrode was prepared by immobilizing AFB1-bovine serum albumin conjugate (AFB1-BSA) on a glassy carbon electrode. Upon addition of target AFB1, competitive immunobinding occurs between the analyte and AFB1-BSA for the labeled pAb on the AuNPs. The change in the surface charge as a result of the antigen-antibody reaction causes a shift in the electrical potential. With increasing concentrations of analyte (AFB1), the quantity of pAb-AuNP captured by the electrode decreases. The shift in the output potential is linearly proportional to the logarithm of AFB1 concentration in the 0.1 to 5.0 μg · kg−1 range, with a detection limit (LOD) of 87 ng · kg−1 (87 ppt). An intermediate precision of 10.9 % was accomplished in batch-to-batch identification. The selectivity over AFB2 with similar chemical structure is acceptable. The method accuracy was evaluated by analyzing naturally contaminated and spiked peanut samples, giving consistent results (with RSD values of <12 %) between this immunoassay and the commercial ELISA.

Novel Planar Chip Biosensors for Potentiometric Immunoassay of Acid Phosphatase Activity Based on the Use of Ion Association Complexes as Novel Electroactive Materials

Novel Planar Chip Biosensors for Potentiometric Immunoassay of Acid Phosphatase Activity Based on the Use of Ion Association Complexes as Novel Electroactive Materials

Competitive-type displacement reaction for direct potentiometric detection of low-abundance protein

Prostate-specific antigen (PSA), one of the indications of possible prostate malignancy, is used as a biomarker for the diagnosis and prognosis of prostate cancer. Herein, we develop a new homogeneous potentiometric immunoassay for sensitive detection of low-concentration PSA without the need of sample separation and washing step. Two nanostructures including positively charged polyethyleneimine-poly(styrene-co-acrylic acid) (PEI-PSAA) nanospheres and negatively charged gold nanoparticles conjugated with anti-PSA antibody (Ab-AuNP) were first synthesized by using mulsifier-free emulsion copolymerization and wet chemistry method, respectively. Thereafter, the as-prepared PEI-PSAA was used as a pseudo hapten for the construction of immunosensing probe based on an electrostatic interaction between PEI-PSAA and Ab-AuNP. Upon target introduction, the added PSA competed with PEI-PASS for Ab-AuNP based on a specific antigen–antibody interaction, and displaced Ab-AuNP from PEI-PASS. The dissociated PEI-PASS was captured through the negatively charged Nafion- modified electrode, thereby resulting in the change of membrane potential. The fabrication process was characterized by using high-resolution transmission electron microscope (HRTEM), scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX), surface plasmon resonance (SPR) and dynamic laser scattering (DLS) technique. Under optimal conditions, the output signal was indirectly proportional to the concentration of target PSA in the sample and exhibited a dynamic range from 0.1 to 50ng/mL with a detection limit (LOD) of 0.04ng/mL. Intra- and inter-assay coefficients of variation (CVs) were 6.8 and 7.5%, respectively. In addition, the methodology was evaluated for analysis of 12 clinical serum samples and showed good accordance between the results obtained by the developed immunosensing protocol and a commercialized enzyme-linked immunosorbent assay (ELISA) method.

Pulsed Galvanostatic Control of a Polymeric Membrane Ion-Selective Electrode for Potentiometric Immunoassays

Pulsed galvanostatic control of ion fluxes across polymeric membrane ion-selective electrodes (ISEs) is an emerging field for potentiometric sensing. Herein we report a novel potentiometric enzyme immunoassay based on current-controlled release of an enzyme substrate, which eliminates the addition of marker ions in the sample solution. In this method, the carboxylated poly(vinyl chloride) matrix at the outer layer of the ISE membrane is employed to attach a primary antibody. A sandwich immunoassay with an alkaline phosphatase labeled antibody (ALP-Ab) as the reporter is used for the determination of human IgG (as a model protein). The large difference between the lipophilicity of the substrate ion and that of the product ion allows p-nitrophenyl phosphate to be used as the enzyme substrate for potentiometric immunosensors. After the immunoreactions, the captured ALP-Ab catalyzes the hydrolysis of the substrate ions released at the sample-membrane interface by using the pulsed galvanostatic technique. This process can be potentiometrically determined by measuring the open circuit potential of the ISE. Under optimal conditions, the potential response of the proposed immunosensor is proportional to the concentration of human IgG in the range of 50-1000 ng/mL with a detection limit of 30 ng/mL (3σ). Owing to simplicity and independence of sample volume and sample turbidity, the proposed potentiometric immunoassay offers a viable alternative to those based on optical absorbance.

Poly(amidoamine) Dendrimer-Functionalized Magnetic Beads as an Immunosensing Probe for Electrochemical Immunoassay for Carbohydrate Antigen-125 in Human Serum

The aim of this study is to develop a new potentiometric immunoassay for carbohydrate antigen-125 (CA125) in human serum by using poly(amidoamine) dendrimer-modified multifunctional magnetic beads conjugated with antibodies as immunosensing probes. With an external magnet, the developed immunosensor exhibited rapid potential response and wide linear range from 0.5 to 55 U/mL with a low detection limit of 0.1 U/mL CA125. The precision, selectivity, and stability of the developed immunosensor were acceptable. Analytical results obtained for the clinical serum specimen by the developed immunosensor were in accordance with those assayed by the enzyme-linked immunosorbent assay method.

Label-free electrochemical immunoassay of carcinoembryonic antigen in human serum using magnetic nanorods as sensing probes

A new potentiometric immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was designed by using a novel transparent immunoaffinity reactor. To construct such an affinity reactor, Fe3O4 nanorods with average diameter of 40 nm and length of 1.0 μm were initially synthesized through hydrolysis of FeCl3 and FeSO4 in the urea solution, and then the synthesized nanorods were used as the immobilized affinity support for the conjugation of anti-CEA antibodies (bionanorods). With an external magnet, the magnetic bionanorods were attached to the surface of a self-made carbon paste electrode. The performance and factors influencing the immunosensors were evaluated. Under optimal conditions, the binding of CEA in the samples onto the immobilized anti-CEA changed the potential response, a dynamic range from 1.5 to 80 ng·mL−1, and a detection limit of 0.9 ng·mL−1 CEA (at 3σ). The immunosensors show good precision, high sensitivity, acceptable stability and reproducibility. The CEA concentrations of the clinical serum specimens assayed by the immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay.

Potentiometric Immunoassay with Quantum Dot Labels

Potentiometric sensors based on polymer membrane electrodes, if properly optimized, are useful for measurements at trace levels. The expected independence of the electrochemical signal of the sample size makes them extremely attractive for measurements in small volumes. Here, we report on electrodes for the potentiometric detection of cadmium ions that reach a detection limit of 6 nM and utilize a Na(+)-selective electrode as pseudoreference in order to facilitate measurements in 150-microL samples. A potentiometric immunoassay of mouse IgG is performed via CdSe quantum dot labels on a secondary antibody according to a sandwich immunoassay protocol in a microtiter plate format. The CdSe quantum dots are found to be easily dissolved/oxidized in a matter of minutes with hydrogen peroxide, allowing us to maintain the pH at a near-neutral value. The potentiometric protein immunoassay exhibits a log-linear response ranging from 0.15 to 4.0 pmol of IgG, with a detection limit of <10 fmol in 150-microL sample wells.

Nonseparation Binding/Immunoassays Using Polycation-Sensitive Membrane Electrode Detection

Two novel homogeneous potentiometric immunoassay formats suitable for the detection of small molecules are described based on polycation-sensitive membrane electrode detection. One approach involves the use of synthetic polycationic-analyte conjugates (polycation labeled analyte) and a limited amount of binding protein sites (antibodies, etc.) to modulate the electrochemical response toward the conjugate. Upon conjugate binding to large binding proteins/antibodies in solution, the polycation label cannot be efficiently extracted into the transduction membrane of the polycation-sensitive device, greatly diminishing the observed EMF response. In the presence of analyte molecules, competitive binding frees up more of the conjugate, and an increase in EMF response is observed in proportion to the concentration of analyte present. With a 15 min reaction incubation period and 3 min electrochemical response time, the assay is demonstrated to function effectively for measurement of theophylline (model analyte) at sub-micromolar levels without any discrete washing or separation steps. The second approach is based on the use of trypsin as an enzymatic label in a homogeneous enzyme binding/immunoassay scheme. Trypsin-biotin conjugates (biotin used as model analyte) are shown to be inhibited nearly 100 % by avidin, but catalytic activity is regained as free biotin is present at increasing levels in the sample solution. The activity of trypsin can be conveniently monitored via polycation-sensitive membrane electrode detection by following the decrease in the EMF response to the polycationic protein protamine, as it is cleaved into smaller fragments by the enzyme. This new electrochemical homogeneous enzyme binding/immunoassay method is shown to respond to the analyte at ≥10 nM levels, again without need for washing or separation steps.

Electrochemical immunoassays.

Immunoassays are routinely used to detect, specifically, low levels of many antigens. The trend away from the use of radioisotopic labels has resulted in a proliferation of alternative labels, many of which have electrochemical activity. The more successful of these assays have used enzyme labels, coupled with amperometric or potentiometric methods of detection of the products. A number of assays have also been designed which are specifically electrochemical in origin, not simply adaptations of currently used spectrophotometric methods. Much effort has been expended in developing a potentiometric immunoassay that measures the change in potential that should occur when an antibody binds to its antigen. The use of electroactive labels has resulted in a number of assays for drugs. The advantages of an enzyme-linked mediated assay for lidocaine, an antiarrhythmic drug, are discussed.