Enzyme-free Electrochemical Immunoassay Research Articles

Bioinspired synthesis of organic–inorganic hybrid nanoflowers for robust enzyme-free electrochemical immunoassay

Protein-inorganic nanoflowers have been extensively used for sensing and biosensing applications by virtue of the signal enhancement of protein component---enzyme. Can the inorganic component of protein-inorganic nanoflowers be employed to amplify signal transducing for enzyme-free detection? In this work, a new kind of BSA-antibodies-copper phosphate hybrid nanoflowers (BSA-Ab2-Cu3(PO4)2) has been prepared by one-pot biomimetic mineralization process as signal enhancer for enzyme-free electrochemical immunoassay. C-reactive protein (CRP) has been chosen as a model biomarker. To the best of our knowledge, it is the first trial using the inorganic component---phosphate ions of BSA-Ab2-Cu3(PO4)2 for transducing electrochemical readout, which features the following advantages: (1) the three-dimensional hierarchical porous nanoflower morphology with a high specific surface area could load more antibodies, and BSA for blocking non-specific sites, greatly increasing the sensitivity of the fabricated immunosensors, (2) the Cu3(PO4)2 hybrid nanoflowers can supply a huge amount of phosphate anions to react with molybdate yielding molybdophosphate precipitates and generating redox currents for more robust enzyme-free electrochemical signal readout. The fabricated immunosensor has exhibited good detection performance with a linear range of 5 pg/mL–1 ng/mL and a limit of detection of 1.26 pg/mL. Moreover, our method has presented good feasibility for clinical sample analysis.

Ag+-coordinated oligonucleotides on gold nanoparticles for anodic-stripping voltammetric immunoassay of cancer antigen 125 for cervical carcinoma

A simple and enzyme-free electrochemical immunoassay was developed for the sensitive screening of cancer antigen 125 using anodic-stripping voltammetry.

A nanohybrid composed of Prussian Blue and graphitic C3N4 nanosheets as the signal-generating tag in an enzyme-free electrochemical immunoassay for the neutrophil gelatinase-associated lipocalin.

An enzyme-free electrochemical immunoassay is described for the neutrophil gelatinase-associated lipocalin (NGAL; a biomarker of kidney disease). Prussian Blue (PB) nanoparticles with redox activity were deposited on graphitic C3N4 nanosheets (g-C3N4) by in-situ reduction. A screen printed electrode (SPCE) was modified with antibody against NGAL, and the PB-g-C3N4 nanohybrid was used as the signal-generating tag for the secondary antibody against NGAL. Upon addition of target NGAL and of secondary antibody, a sandwich is formed on the SPCE. At an applied potential of typically 0.13V (vs. Ag/AgCl), a well-defined voltammetric peak is observed that results from the presence of PB on the secondary antibody. Under optimal conditions, the peak current increases linearly in the 0.01 to 10ng·mL-1 NGAL concentration range, and the detection limit is 2.8pg·mL-1. An average precision of <12% was accomplished in the batch-to-batch mode. Other disease-related biomarkers do not interfere. The accuracy and inter-laboratory validation of this method were evaluated for target NGAL detection in spiked human serum by using a commercial ELISA. The results obtained by the two methods are in good accordance. Graphical abstract An enzyme-free electrochemical immunoassay was used for detection of neutrophil gelatinase-associated lipocalin by Prussian blut/graphitic-C3N4 nanohybrids as the signal-generation tags.

Robust enzyme-free electrochemical immunoassay of CEA enhanced by porous PdCu nanoparticles

The interferences of dissolved oxygen towards the electrochemical immunosensors occurred constantly, which will restrict the practical clinical application of electrochemical immunoassay. Herein, a new enzyme-free strategy has been proposed for ultrasensitive electrochemical immunoassay of carcinoembryonic antigen (CEA), which can completely avoid the interference of dissolved oxygen. Porous PdCu nanoparticles can catalyze aniline to produce polyaniline by the inherited peroxidase-like activity for generating electrochemical detection signal. More interesting, with the assistance of electropolymerization strategy, the electrochemical measurement signal could enhance sharply. Accordingly, we established a sandwich-type electrochemical immunosensor based on porous PdCu nanoparticles produced polyaniline with electropolymerization strategy for enzyme-free and dissolved oxygen interference-free detection of CEA, affording a satisfied performance with a broad linear detection range (0.1pg/mL∼10ng/mL) and low detection limit (0.08pg/mL). This method might be considered as a robust approach for clinical applications.

Enzyme-Free Ultrasensitive Electrochemical Immunoassay Based on the Strategy of Converting Titanium Phosphate Nanospheres into Redox Active Molybdophosphate

Enzyme-Free Ultrasensitive Electrochemical Immunoassay Based on the Strategy of Converting Titanium Phosphate Nanospheres into Redox Active Molybdophosphate

Redox active molybdophosphate produced by Cu3(PO4)2nanospheres for enhancing enzyme-free electrochemical immunoassay of C-reactive protein

Redox-active molybdophosphate produced by Cu3(PO4)2nanospheres has been directly employed for signal amplification of an enzyme-free electrochemical immunosensor.

A New Enzyme-Free Electrochemical Immunoassay for Escherichia coli Detection using Magnetic Nanoparticles

ABSTRACTThis paper reports a novel enzyme-free electrochemical immunoassay for Escherichia coli (ATCC 25922) that involves the formation of an immunocomplex between antibodies immobilized on the surface of a platinum electrode and bacteria-labelled magnetite nanoparticles. Magnetite nanoparticles modified with chitosan were used as the signal label in the immunoassay. The main novelty is the collection of an electrochemical signal from a magnetic nanolabel included within the immunocomplex on the electrode surface. The developed immunosensor is highly specific, enabling the determination of E. coli from 10 to 105 colony-forming units (CFU)/mL with the relative standard deviation below 10%. The limit of detection for the immunosensor was 9.3 CFU/mL. Bacteria were determined in model mixtures and real samples. The recovery was between 95 and 99%, comparable with results from enzyme-linked immunosorbent assays.

Mesoporous carbon-enriched palladium nanostructures with redox activity for enzyme-free electrochemical immunoassay of brevetoxin B

A new signal amplification strategy based on mesoporous carbon-enriched palladium nanostructure (MSC-PdNS) was designed for enzyme-free electrochemical immunoassay of brevetoxin B (BTB) in marine toxins. The assay was carried out on a BTB-bovine serum albumin-functionalized electrode by using monoclonal mouse anti-BTB-labeling MSC-PdNS as the signal-transduction tag. A competitive-type assay protocol was successfully introduced to develop a high-efficiency enzyme-free immunoassay accompanying the doped palladium nanostructure into MSC-PdNS toward reduction of H2O2. Under the optimal conditions, the catalytic current decreased with the increment of BTB concentration in the range from 0.01 to 10 ng mL−1 with a detection limit (LOD) of 5.0 pg mL−1 BTB at the 3sblank criterion. The selectivity and precision were acceptable. In addition, the methodology was further validated for assaying spiked seafood samples, and consistent results between the electrochemical immunoassay and the referenced enzyme immunoassay were obtained. Importantly, the enzyme-free electrochemical immunoassay provides a promising approach for rapid screening of marine toxin because of its simplicity, low cost, sensitivity, specificity and without the need of sample pretreatment.

Gold nanorods-paper electrode based enzyme-free electrochemical immunoassay for prostate specific antigen using porous zinc oxide spheres–silver nanoparticles nanocomposites as labels

Gold nanorods-modified paper electrode and porous zinc oxide spheres–silver nanoparticles nanocomposites were used to construct an enzyme-free immunosensor.

Paper-based electrochemical immunosensor for carcinoembryonic antigen based on three dimensional flower-like gold electrode and gold-silver bimetallic nanoparticles

An enzyme-free electrochemical immunoassay is introduced into microfluidic paper-based analytical devices for the first time using novel three dimensional flower-like Au nanoparticles modified paper working electrode (FLAuNPs-PWE) as sensor platform and gold (Au)-silver (Ag) bimetallic nanoparticles (Au-Ag BMNPs) as tracer. FLAuNPs with large surface area serve as an effective matrix for primary antibodies. Au-Ag BMNPs with good biological activity, such as mimicking natural peroxidases, are used for binding of signal antibodies (Ab2). Then the Ab2 labeled BMNPs is linked to the FLAuNPs-PWE via sandwich immunoreactions. Enhanced sensitivity is achieved by efficient catalysis of the Au-Ag BMNPs toward hydrogen peroxide. Under the optimized experimental conditions, the proposed paper-based electrochemical immunosensor exhibits excellent analytical performance for enzyme-free detection of carcinoembryonic antigen, ranging from 0.001-50ngmL−1 with a low detection limit of 0.3pgmL−1. The immunosensor shows good precision, acceptable stability and reproducibility. The proposed method provides a new promising platform for the design of the highly sensitive detection method, showing great promise for clinical application.

NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay

NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP–MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP–MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP–MWCNT. Using the NiCoBP–MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035ngmL−1. More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody.

Ultrasensitive enzyme-free electrochemical immunoassay for free thyroxine based on three dimensionally ordered macroporous chitosan–Au nanoparticles hybrid film

The measurement of free thyroxine concentration in serum is considered to be an essential indicator of thyroid function. Here, a novel enzyme-free sandwich electrochemical immunosensor for the detection of FT4 antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous chitosan–Au nanoparticles hybrid (3DOM CS–AuNPs) film electrode, and magnetic multiwall carbon nanotubes (MMWCNTs) were used as label of secondary antibody (Ab2). The 3DOM CS–AuNPs film electrode was constructed by one-step electrodeposition of CS–AuNPs composite onto Au electrode with silica opal template. MMWCNTs were prepared by chemical co-precipitation of Fe2+ and Fe3+ salts on carboxylated MWCNTs. Ru(bpy)32+ labeled anti-FT4 (Ru(bpy)32+–Ab2) was covalently attached to MMWCNTs through the formation of amide bond between the carboxylic groups of MWCNTs and the amine groups of antibody. Under the optimal conditions, FT4 was detected in a concentration range from 0.71fgmL−1 to 1.15pgmL−1 with a correlation coefficient of 0.998 and a detection limit of 0.20fgmL−1. Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.

Prussian blue-doped nanogold microspheres for enzyme-free electrocatalytic immunoassay of p53 protein

We report on a new enzyme-free electrochemical immunoassay for the sensitive detection of the p53 protein (p53; a model analyte) by using a screen-printed carbon electrode modified with monoclonal mouse anti-human p53 antibody tagged with gold nanoparticles. First, nanogold microspheres doped with Prussian Blue were synthesized by a reverse micelle method. The resulting microspheres were used to label polyclonal anti-p53 antibody which then was applied in a sandwich immunoassay in pH 6.5 buffer solution using the Prussian Blue in the particles as the redox-active reporter. The electrochemical signal of the immunosensor is shown to increase with the concentration of the analyte (p53 protein) in the range from 0.5 to 80 U mL−1, with a detection limit of 0.1 U mL−1. No non-specific adsorption was observed. Coefficients of variation for intra-assay and inter-assay were below 8.5 % and 11.5 %, respectively. In addition, the method was applied to the analysis of 15 human serum samples, and a good relationship was found between the new immunoassay and the referenced electro-chemiluminescence method.

Ultrasensitive enzyme-free electrochemical immunosensor based on hybridization chain reaction triggered double strand DNA@Au nanoparticle tag

An ultrasensitive enzyme-free electrochemical immunoassay was developed for detection of the fg/mL level carcinoembryonic antigen (CEA) by using a double strand DNA@Au nanoparticle (dsDNA@AuNP) tag and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator. The dsDNA@AuNP was synthesized by one-pot hybrid polymerization of dsDNA on initiator DNA modified AuNPs via hybridization chain reaction. The immunosensor was prepared by covalently cross-linking capture antibody on chitosan/AuNP nanocomposite modified glass carbon electrode. The AuNPs accelerated the electron transfer and led to high detection sensitivity. With a sandwich-type immunoreaction and a biotin-streptavidin affinity reaction, the dsDNA@AuNP tag was conjugated on the immunocomplex to bring a high amount of RuHex to the electrode surface via electrostatic interaction, resulting in an amplified electrochemical signal. Under optimal conditions, the proposed sensing platform showed a wide linear detection range from 10 fg/mL to 10 ng/mL along with a detection limit of 3.2 fg/mL for CEA. The immunosensor exhibited high sensitivity and good stability, showing a promising application in early cancer diagnosis and could be extended to sensitive electrochemical biosensing of other analytes.

Guanine-Decorated Graphene Nanostructures for Sensitive Monitoring of Neuron-Specific Enolase Based on an Enzyme-Free Electrocatalytic Reaction

A new and enzyme-free electrochemical immunoassay protocol was developed for the sensitive electronic monitoring of neuron-specific enolase (NSE) on a monoclonal mouse anti-human NSE antibody (mAb)-modified glassy carbon electrode, using guanine-decorated graphene nanostructures (GGN) as nanotags. To construct such an enzyme-free immunoassay format, guanine and polyclonal rabbit anti-human NSE antibody (pAb) were co-immobilized on the graphene nanostructures through the carbodiimide coupling. Based on a sandwich-type immunoassay mode, the assay was carried out in 0.1 M pH 7.4 PBS containing 5 μM Ru(bpy)3(2+) through the catalytic oxidation of Ru(bpy)3(2+) toward the guanine on the GGN. The presence of graphene nanostructures increased the immobilized amount of guanine, thus amplifying a detectable electronic signal. The covalent conjugation of guanine and pAb on the GGN resulted in a good repeatability and intermediate reproducibility down to 9.5%. Under optimal conditions, the dynamic concentration range of the developed immunoassay spanned from 0.005 to 80 ng mL(-1) NSE with a detection limit of 1.0 pg mL(-1) at the 3S(blank) level. In addition, the methodology was evaluated by assaying the spiking serum samples, and the relative standard deviation (RSD) between the electrochemical immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) were 2.8-7.0%.

Platinum nanoflower-based catalysts for an enzyme-free electrochemical immunoassay of neuron-specific enolase

Platinum nanoflowers (PtNFs) were for the first time employed as a label for a highly efficient, enzyme-free electrochemical immunoassay of neuron-specific enolase (NSE, as a model) by coupling a nanocatalyst-based electrocatalytic reaction with catalytic recycling of self-produced reactants.

Electrochemical stripping voltammetry of gold ions for development of ultra-sensitive immunoassay for chlorsulfuron

The paper reports a highly sensitive enzyme free electrochemical immunoassay (EFEIA) for the detection of herbicide chlorsulfuron. The assay is based upon oxidative gold nanoparticle (GNP) dissolution in an acidic solution. The consequent release of large amounts of gold (Au) metal ions after dissolution of gold nanoparticles tagged to antibody leads to the development of sensitive stripping voltammetry based immunoassay. The detection is made possible by the reduction of Au 3 + ions at the screen printed electrode surface followed by metal analysis by using the square wave voltammetry technique. The sensitivity of chlorsulfuron detection by competitive assay procedure was 6.7 pg mL − 1 for EFEIA in marked contrast to optical detection using Standard ELISA procedure that gives a sensitivity of 4.97 ng mL − 1 .

A novel strategy for ultra-sensitive electrochemical immunoassay of biomarkers by coupling multifunctional iridium oxide (IrOx) nanospheres with catalytic recycling of self-produced reactants

In this study, a novel and enzyme-free electrochemical immunoassay was developed for the detection of alpha-fetoprotein (AFP; as a model biomarker) by using a novel redox nanocatalyst, patterned gold-iridium oxide nanoparticle assemblies (Au-IrO(x)), on a Prussian blue (PB) functionalized glassy carbon electrode. The as-prepared Au-IrO(x) nanospheres were not only used for the labeling of secondary anti-AFP antibody but also executed for water oxidation. With a sandwich immunoassay format, the electrochemical measurement was carried out in 0.1 M KNO(3)-HCl, pH 3.2, containing 1.0 mM p-aminophenol (AP). Initially, the added AP was oxidized to p-quinone imine (QI) with the help of the immobilized PB on the electrode, and then the generated QI was reduced back to AP by hydrogenation reaction from water oxidation. The self-produced QI reactants were catalytically recycled and thus amplified the signal of electrochemical response. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.005 to 200 ng/mL with a low detection limit of 0.5 pg/mL AFP (at 3s(B)). Intra- and inter-assay coefficients of variation were below 10%. In addition, the methodology was validated with real serum samples, receiving a good agreement with the results obtained from commercially available electrochemiluminescence automated analyzer.

Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts

A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6 mM p-nitrophenol (NP) and 6 mM NaBH 4 after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH 4, then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH 4. The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0 × 10 −7–2.0 × 10 2 ng/mL with a lower detection limit (LOD) of 0.8 fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP.

Equilibrium values of short range order in gold-silver and gold-palladium alloys

An enzyme-free electrochemical immunoassay is introduced into microfluidic paper-based analytical devices for the first time using novel three dimensional flower-like Au nanoparticles modified paper working electrode (FLAuNPs-PWE) as sensor platform and gold (Au)-silver (Ag) bimetallic nanoparticles (Au-Ag BMNPs) as tracer. FLAuNPs with large surface area serve as an effective matrix for primary antibodies. Au-Ag BMNPs with good biological activity, such as mimicking natural peroxidases, are used for binding of signal antibodies (Ab2). Then the Ab2 labeled BMNPs is linked to the FLAuNPs-PWE via sandwich immunoreactions. Enhanced sensitivity is achieved by efficient catalysis of the Au-Ag BMNPs toward hydrogen peroxide. Under the optimized experimental conditions, the proposed paper-based electrochemical immunosensor exhibits excellent analytical performance for enzyme-free detection of carcinoembryonic antigen, ranging from 0.001-50 ng mL−1 with a low detection limit of 0.3 pg mL−1. The immunosensor shows good precision, acceptable stability and reproducibility. The proposed method provides a new promising platform for the design of the highly sensitive detection method, showing great promise for clinical application.