A method for ultrasensitive visual determination of Cd2+ was developed based on the oscillatory reaction of poly(toluidine blue O)-gold (PTBO–Au). PTBO–Au was fabricated using HAuCl4 as the oxidizing reagent to polymerize toluidine blue O (TBO), a dye utilized as an indicator for colorimetric analysis and detection of Cd2+. Thiosulfate was used to leach Au nanoparticles onto PTBO–Au to form Au+ ions in the presence of (1-hexadecyl)trimethylammonium bromide. After addition of NaBH4, PTBO–Au, Au+, and Cd2+ were reduced to form reduced PTBO–Au (rPTBO–Au) and Au–Cd nanoalloy. The color of the reaction solution changed from blue to red (the color of the Au–Cd nanoalloy). When rPTBO–Au was oxidized by dissolved oxygen, a critical point of color change occurred, and the solution color returned to blue as the reduction rate of PTBO–Au became lower than that of oxidating rPTBO–Au (determined by the concentration of NaBH4). The Au–Cd nanoalloy was found to have minimal catalytic effect on PTBO–Au reduction in the oscillatory reaction, which is indicative of the slowest consumption of NaBH4 and the longest cycle time. Further, the time of the transition from blue → red → blue has positive correlation with the concentration of Cd2+. The color transition can be effectively used as an indicative method to quantitatively detect Cd2+ with the naked eye in the concentration range 1 μM to 1 nM without expensive instrumentation. This highly sensitive and selective method was applied for the determination of Cd2+ in spiked river water samples and obtained satisfactory recoveries, which suggests its potential other environmental applications.Keywords: Au−Cd nanoalloy; Cadmium ion; Catalytic performance; Oscillatory reaction; Poly(toluidine blue O)-gold; Visual detection;

The authors describe an immunoassay for the determination of carcinoembryonic antigen (CEA) tumor markers by depositing a polydopamine-Pb(II) nanocomposite on the surface of a glassy carbon electrode. The nanocomposite acts as a redox system that displays a large specific surface and provides a strong current signal at −0.464 V (vs. Ag/AgCl). After the deposition of PDA-Pb2+ on glassy carbon electrode, the electrode was additionally coated with a chitosan-gold nanocomposite. The immunoassay platform was obtained by immobilization of antibodies against carcinoembryonic antigens by using glutaraldehyde and blocking with bovine albumin. Owing to its large surface, good electrical conductivity and powerful current response, the immunoassay has a wide linear range that extends from 1 fg·mL−1 to 100 ng·mL−1, with a detection limit as low as 0.26 fg·mL−1. The results obtained with this immunoassay when determining CEAs in human serum were found to be consistent with those obtained by ELISAs.

A conductive hydrogel acting as a redox-active species was synthesized by crosslinking phytic acid as a ligand and lead(II) as the metal ion. The resulting gelatine-like material displays excellent redox activity and facilitates the transport of electrons and ions. Gold nanoparticles were electrochemically deposited on the hydrogel film, and antibody against cytokeratin antigen was immobilized thereon. An amperometric immunosensor for cytokeratin antigen 21–1 (CYFRA21-1), a kind of biomarker of lung cancer, was obtained by deposition of the composite on a glassy carbon electrode. If operated at − 0.58 V (vs. Ag/AgCl), the sensor exhibits a linear detection range that extends from 50 fg mL−1 to 100 ng mL−1 CYFRA21-1, and a 38 fg mL−1 detection limit (at a signal to noise ratio of 3). The quantitation of CYFRA21-1 in (spiked) human serum samples showed satisfactory accuracy compared to an ELISA.

Conducting polymers possess good conductivity, can be easily modified, have a particular redox activity. Noble metal nanomaterials, in turn, possess high conductivity, catalytic properties and large surface-to-volume ratios. Synergistic materials consisting of both conducting polymer and metal nanomaterial therefore are most useful materials for use in electrochemical immunosensors with improved sensitivity and specificity. This review (with 75 references) gives an overview on advances in conducting polymer based noble metal nanomaterial hybrids for amperometric immunoassay of the 13 most common tumor markers. The review is divided into the following sections: (1) Polyaniline based noble metal nanomaterial hybrids; (2) Polyaniline derivative-based noble metal nanomaterial hybrids; (3) Polypyrrole-based noble metal nanomaterial hybrids. A final section covers future perspectives regarding challenges on the design of electrochemical immunoassays.

A new kind of hollow cadmium–organic coordination nanoparticle (CdOP) was prepared by connecting Cd2+ with 2-aminoterephthalic acid which exhibited strong electrochemical redox activity at −0.71 V. The obtained nanoparticles were used to construct a redox substrate for label-free voltammetric immunoassay of prostate specific antigen (PSA) by drop-coating CdOPs onto the surface of a glassy carbon electrode (GCE). Then, the CdOP film was covered with a mixture of Nafion and graphene oxide (GO) due to their good film-forming properties and excellent biocompatibility. Based on the electrostatic interaction between the membrane and anti-PSA, a label-free voltammetric immunosensor was fabricated simply by immobilizing anti-PSA on the modified GCE. The determination linear range for PSA was from 0.01 ng mL−1 to 100 ng mL−1 and a detection limit of 0.97 pg mL−1 (S/N = 3) was achieved. Importantly, the results of human blood serum samples by this biosensor were in good agreement with the enzyme linked immunosorbent assay results, indicating that the biosensor possessed high reliability for protein determination.

•An aerogel with superhigh-capacity, pH-sensitivity, selectivity and reusability was fabricated.•The maximum adsorption ability was 3059.2 mg g−1 for methyl orange and 2043.7 mg g−1 for amaranth.•The microcosmic interaction models of two anionic dyes and aerogel were outlined.Amino-functionalization is an effective and facile way to modify the surface of adsorbent. A facile strategy to fabricate polyethyleneimine-functionalized graphene aerogel (PFGA) integrating superhigh adsorption ability, selectivity, pH-sensitivity and reusability was developed. The maximum adsorption capacities of the PFGA for methyl orange and amaranth were as high as 3059.2 mg g−1 and 2043.7 mg g−1, respectively, the highest capabilities among the reported adsorbents for both dyes. The microcosmic interaction models of the two dyes and PFGA were outlined to describe the relationships between dye structures and adsorption behaviors. Owing to the different affinities of PFGA for anionic and cationic dye, anionic dye methyl orange was able to be selectively separated from the dye mixture. In the experiment of competitive adsorption of acid dyes with different structures, amaranth with more sulphonate groups was preferentially adsorbed. In addition, PFGA showed meaningful pH-sensitive adsorption ability, enabling dye-adsorbed PFGA to be fully regenerated as soon as it was exposed to the solution of pH 11. The study on polyethyleneimine-functionalization is meaningful for the development of high adsorption capacity adsorbents and amino-functionalization or other functionalized materials.Download high-res image (175KB)Download full-size image

A glassy carbon electrode was modified with a redox-active nanocomposite consisting of polyresorcinol, gold nanoparticles (NPs) and platinum NPs. This nanocomposite possesses the following outstanding advantages: (1) strong electrochemical signal towards H2O2 at 0.92 V (vs Ag/AgCl); (2) good conductivity; and (3) ease of immobilization of antibody without the use of coupling agents. Based on these properties, a label-free amperometric immunoassay for the tumor marker neuron-specific enolase (NSE) was developed. The immunoelectrode has a linear response in the 10 pg·mL−1 to 100 ng·mL−1 NSE concentration range and a 7.8 pg·mL−1 detection limit (at an SNR of 3). The method was applied to the quantitation of NSE in human serum samples and showed satisfactory accuracy compared to an electrochemiluminescence immunoassay, a commonly used method in clinical applications.

The authors describe a method for the ultrasensitive optical determination of Pb(II). It is based on the formation of a Pt-Pb nanoalloy from Pt nanoparticles and Pb that is formed by reduction with NaBH4. The nanoalloy accelerates an oscillatory reaction involving Toluidine Blue (TB), NaBH4 and oxygen. In this reaction, TB is reduced by NaBH4 to form reduced TB (rTB), and this is accompanied by a color change from blue to colorless. However, rTB can be oxidized by dissolved oxygen and the color turns back to blue. The Pt-Pb nanoalloy exerts a strong catalytic effect on this reaction. If the concentration of NaBH4 is low enough, the rate of reducing TB is lower than that of the oxidation of TB. The time of the transition from blue → colorless → blue is inversely correlated to the concentration of Pb(II). The methods works in the 1 to 1000 nM Pb(II) concentration range, with a 0.63 nM detection limit, This highly sensitive and selective method was applied to the determination of Pb(II) in spiked river water samples.

•Conductive catalytic redox hydrogel composed of aniline and vinyl-ferrocene was used as electro-immunosensing substrate.•Catalytic activity of ferrocene toward ascorbic acid was used to amplify electrochemical signal.•A 0.54 pg mL−1 limit of detection was obtained in the detection of PSA.In this work, a conductive catalytic redox hydrogel composed of aniline and vinyl-ferrocene was prepared which showed good conductivity and strong electrochemical redox signal at 0.04 V (vs. Ag/AgCl). The hydrogel was simply generated in situ on the glassy carbon electrode to fabricate an amperometric immunosensor. The redox hydrogel provided a three dimensional porous structure, which improved the specific surface area of modified electrode. Based on the catalytic activity of ferrocene toward the oxidation of ascorbic acid, the electrochemical signal can be amplified. Gold nanoparticles were modified on the redox hydrogel by electrochemical deposition to promote electron transfer, which also can immobilize antibody. Prostate-specific antigen (PSA), a significant indicator of prostate cancer, had been measured as a model tumor marker. Under the optimized conditions, a linear range of the prostate specific antigen concentrations from 0.001 to 200 ng mL−1 with a detection limit of 0.54 pg mL−1 ((S/N) = 3) was obtained. Importantly, the detection of PSA in the human blood serum samples showed satisfactory accuracy with the enzyme linked immunosorbent assay results, which showed the redox hydrogel possessed huge potential for the detection of other tumor markers.A conductive catalytic redox hydrogel composed of aniline and vinyl-ferrocene was prepared and used to develop a high sensitivity and a wide linear response range immunosnesor for PSA.Download high-res image (142KB)Download full-size image

•A novel fluorescent and colorimetric dual detection of Hg2+ was developed.•PtNPs were straightly used to detect Hg2+ without modification.•Present method exhibited high sensitivity and selectivity.Pt nanoparticles (Pt NPs) can catalyze the oxidative reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H2O2). In this study, o-phenylenediamine was oxidized to 2, 3-phenazinediamin, and the color changed to bright yellow and produced yellow fluorescence. However, the catalytic activity of the Pt NPs was inhibited by the citrate reduction of Hg2+ at the Pt NPs surface. When the concentrations of Pt NPs, H2O2, and OPD were constant, the color changed from colorless to bright yellow, which totally depended on the concentration of Hg2+. Inspired by these principles, we developed a colorimetric and fluorescent dual-readout method for the sensitive determination of Hg2+, thus effectively improving the accuracy of the detection. The fluorescence and colorimetric linear responses of the proposed method exhibited a wide linear range from 10 nM–2 μM with ultralow detection limits of 0.14 nM and 0.8 nM, respectively, values that are much lower than the maximum permitted level of Hg2+ in drinking water by the United States Environmental Protection Agency (EPA). The relative errors of real samples were from −2.54% to 2.36%, indicating the high accuracy of the proposed approach.Pt nanoparticles, of which peroxidase-like catalytic ability can be inhibited by Hg2+, can catalyze the oxidation of o-phenylenediamine to form 2,3-phenazinediamin by hydrogen peroxide. 2,3-Phenazinediamin exhibits a visible color and a fluorescence of yellow, can be dual-readout by the method of colorimetry and fluorescence.Download high-res image (55KB)Download full-size image

•BSA was firstly implemented as an effective sensitivity enhancer for the peptide-based amperometric biosensor.•Multiple amplification strategies were developed for the amperometric biosensor for PSA.•Chitosan-Pb2[Fe(CN)6]-PDDA-GO as a new redox species was used as substrate.Bovine serum albumin (BSA) was firstly implemented as an effective sensitivity enhancer for a peptide-based amperometric biosensor for the ultrasensitive detection of prostate specific antigen (PSA). A porous and conductive substrate of chitosan-lead ferrocyanide-(poly(diallyldimethylammonium chloride)-graphene oxide) was in-situ generated on a glassy carbon electrode (GCE), in which Pb2[Fe(CN)6] served as a novel redox species with strong current signal at −0.46 V (vs. Ag/AgCl). Poly(diallyldimethylammonium chloride)-graphene oxide was applied to improve conductivity of the substrate. After adsorbing Pb2+ for signal amplification, chitosan provided active sites to simultaneously immobilize peptides and 1-aminopropyl-3-methylimidazolium chloride by glutaraldehyde. To enhance the sensitivity, BSA was chemically linked to the immobilized peptide, behaving as a serious decrease of current signal for BSA hindering the electron transfer. The dramatic increase of current signal of the biosensor was obtained by PSA cleaving the immobilized BSA-peptide. The proposed biosensor exhibited a detection limit of 1 fg mL−1 for PSA and its sensitivity was seven-fold higher than previous works.

•Multiple functional strategies for amplifying sensitivity of amperometric immunoassay are reviewed.•Effects of conductive nanomaterials, catalytic nanomaterials hybrids, new redox materials on the sensitivity were discussed.•Development of three-dimensional porous materials, new receptors, and blocking agents in immunoassay was summarized.Multiple functional strategies have shown great potential in ultrasensitive amperometric immunoassays for tumor markers, which promote conductivity and signal multiple amplification. The sensitivity of amperometric immunoassays is significantly affected by the conductivity and specific area of the sensing interface as well as the electrochemical activity of redox species. Thus, these strategies are generally based on integrating various materials together and endowing immunosensing systems with many advantages, such as large specific area, high electrochemical activity, good conductivity, biocompatibility, and catalytic performance. Owing to the rapid development of functional materials (such as conductive hybrids, catalytic hybrids, enzyme-like materials, highly electrochemical active species, redox nanocomposites, porous materials, hydrogels, and metal-organic framework) and new bioactive substances (including new blocking agents and receptors like peptides and oligonucleotide chains), the sensitivity of related biosensors is usually higher than that of traditional ones, indicating that multiple functional strategies are promising in amperometric immunoassays. Herein, we provide an overview of recent advances in multiple functional strategies that have proven to dramatically enhance the sensitivity of amperometric immunoassays, which incorporate the following materials: (1) conductive nanomaterials hybrids; (2) catalytic nanomaterials hybrids; (3) new redox materials; (4) three-dimensional porous materials; (5) new receptors and blocking agents.

•A novel electrochemical redox composite PPPD-Au/Pt was synthesized by one-pot method.•PPPD-Au/Pt was used as sensing substrate for label-free electrochemical immunosensor.•The immunosensor showed wide detection range and ultralow detection limit for the detection of CA199.A novel electrochemical redox-active nanocomposite was synthesized by a one-pot method using N,N′-diphenyl-p-phenylediamine as monomer, and HAuCl4 and K2PtCl4 as co-oxidizing agents. The as-prepared poly(N,N′-diphenyl-p-phenylediamine)-Au/Pt exhibited admirable electrochemical redox activity at 0.15 V, excellent H2O2 electrocatalytic ability and favorable electron transfer ability. Based on these, the evaluation of the composite as sensing substrate for label-free electrochemical immunosensing to the sensitive detection of carbohydrate antigen 199 was described. This technique proved to be a prospective detection tool with a wide liner range from 0.001 U mL−1 to 40 U mL−1, and a low detection limit of 2.3 × 10−4 U mL−1 (S/N = 3). In addition, this method was used for the analysis of human serum sample, and good agreement was obtained between the values and those of enzyme-linked immunosorbent assay, implying the potential application in clinical research. Importantly, the strategy of the present substrate could be extended to other polymer-based nanocomposites such as polypyrrole derivatives or polythiophene derivatives, and this could be of great significance for the electrochemical immunoassay.

•Chitosan-poly(acrylic acid) nanospheres doped with Cu2+, Cd2+, Pb2+ or Zn2+ were synthesized.•These nanospheres were used as immunoprobes for simultaneous detection of four tumor markers of pancreatic cancer.•This proposed biosensor performed very well on the detection of serum specimens.In this work, a one-pot method was designed to synthesize copper ions, cadmium ions, lead ions and zinc ions doped chitosan–poly(acrylic acid) nanospheres. Those nanospheres can not only produce independent electrochemical signals, but also react with glutaraldehyde (GA) to immobilize different labeled antibodies. Using the modified nanospheres as immunoprobes, a sandwich-type immunosensor was fabricated to simultaneous detection of four tumor markers (CEA, CA199, CA125 and CA242) of pancreatic cancer. This designed immunosensor exhibited good linear relationships in range from 0.1 to 100 ng mL−1 for CEA, 1 to 150 U mL−1 for CA199, CA125 and CA242, corresponding detection limits 0.02 ng mL−1, 0.4 U mL−1, 0.3 U mL−1 and 0.4 U mL−1, respectively. Meanwhile, the immunosensor was applied in analysis of clinical serum samples, whose results were well agreed with the enzyme-linked immunosorbent assay (ELISA), indicating that the proposed immunosensor gave a hope for the identification and validation of specific early cancer.

•Azide modified Au@SiO2 and alkyne group on carbon dots were successfully prepared.•As-prepared sensing system exhibited selective and sensitive of Cu2+ ions.•This sensing system exhibited accurate detection results for river water samples.Pursuing for a simple, sensitive and inexpensive method of detecting copper ions is very essential for both environment and human health. Herein, a new strategy for fluorescence detection of Cu2+ was designed which is based on the reduction of Cu2+ to Cu+ by sodium ascorbate to get cycloaddition reaction between 4-azidobenzoic acid-tagged Au@SiO2 and propiolic acid-tagged carbon dots (CDs). The proposed approach not only possesses a good selectivity, but also has a linear relationship between the fluorescence intensity and the concentrations of Cu2+ in the range of 0.1–100 nM with the detection limit of 0.08 nM (5.12 × 10−9 mg mL−1), which is far below the limit value of 1.3 mg mL−1 defined by U.S. Environmental Protection Agency in drinkable water. In the actual sample, the standard deviation ranges from 97.6% to 101.8%, indicating the high precision of present approach.A new strategy for fluorescence detection of Cu2+ was designed which is based on the reduction of Cu2+ to Cu+ by sodium ascorbate to get cycloaddition reaction between 4-azidobenzoic acid-tagged Au@SiO2 and propiolic acid-tagged carbon dots.

Four kinds of redox species including three kinds of polymerized dye-gold composites and one kind of a polyaniline-gold composite were synthesized by a one-pot method at room temperature. Gold in these composites plays the roles of improving the conductivity and fixing the detection antibodies. The four composites together with hexacyanocobaltate were used as redox active species for simultaneous detection of five biomarkers of lung cancer including the carcinoembryonic antigen (CEA), neuron specific enolase (NSE), carbohydrate antigen 125 (CA125), cytokeratin 19 fragment antigen 21–1 (Cyfra21–1), and squamous cell carcinoma antigen (SCCA). The immunoassays display good linear relationship between the current response and the logarithm of the analyte. The analytical ranges extend from 1 to 150 ng⋅mL-1 for CEA, NSE and Cyfra21–1, from 1 to 150 U⋅mL-1 for CA125, and from 0.1 to 100 ng⋅mL-1 for SCCA. The corresponding detection limits are 0.2 ng⋅mL-1, 0.9 ng⋅mL-1, 0.4 ng⋅mL-1, 0.9 U⋅mL-1 and 30 pg⋅mL-1, respectively. The immunoassay was applied to the analysis of human serum samples, and the results were found to agree well with those of the enzyme-linked immunosorbent assay.

•Hollow ZnS were directly synthesized via a facile etching reaction.•The etching reaction was extended to the synthesis of hZnS@Au.•hZnS@Au was used for the determination of hydrazine with an ultra-wide linear range between 2 μM and 24.22 mM.New strategies of hollow nanoparticles fabrication are of fundamental significance in synthesizing varieties of nanoparticles with certain property. A novel etching reaction has been established to synthesize hollow ZnS (hZnS) happening in the branched polyethyleneimine (bPEI) and 1,4-dibromobutane mixed solution dissolved in ethanol heated at 80 °C. Besides, the etching reaction was extended to the synthesis of hollow ZnS decorated with gold nanoparticles (hZnS@Au) where HAuCl4 as gold precursor. The synthesized core-satellite hZnS@Au with high specific surfaces, uniform morphology and chemically reactive facets was used for the detection of hydrazine. It was worth pointing that the resulting electrochemical sensor exhibited good performance toward hydrazine oxidation with ultra-wide linear response ranging from 2 μM to 24.22 mM (R2 = 0.994) at 0.7 V and low limit of detection of 0.667 μM at a signal-to-noise of 3.

•A novel poly(MB)-Au nanocomposites with uniform morphology was synthesized by one-pot method.•Poly(MB)-Au nanocomposites exerted innate redox peak.•The electrochemical immunosensor with double signal amplification was fabricated.•The electrochemical immunosensor had a low detection limit and wide linear range.In this work, a kind of novel redox-active species, metal-organic nanocomposites with uniform morphology and good conductivity, was synthesized by one-pot method using HAuCl4 as oxidant and methylene blue as monomer. With excellent biocompatibility of Au, the immunosensing probe was fabricated by means of immobilization of antibody on the nanocomposites for the purpose of signal amplification. Meanwhile, graphene-Au modified electrode was used as the substrate. A new electrochemical immunosensing platform was designed and simply fabricated. With the use of α-fetoprotein (AFP) as a model analyte, good performance was obtained with a wide linear range from 1 pg mL−1 to 100 ng mL−1, and low detection limit of 19.6 fg mL−1. The assay results of clinical serum with this immunoassay were well consistent with those obtained from ELISA method. The present work suggested an electrochemical immunosensing platform for tumor biomarker.

Two kinds of novel redox-active species, gold-decorated polyaniline derivatives (Au-PANI derivatives) including gold-poly(o-aminophenol) (Au-PoAP) and gold-poly(p-phenylenediamine) (Au-PpPD), which can produce oxidation peaks at −0.2 V and 0.25 V, respectively, were synthesized by a one-pot method using o-aminophenol and p-phenylenediamine as monomers, and chloroauric acid as the oxidant. The gold nanoparticles in the composite make a prominent contribution to its conductivity. These two Au-PANI derivatives together with gold-poly(o-phenylenediamine) (Au-PoPD, oxidation peaks at −0.5 V) were used as redox-active species for simultaneous electrochemical detection of three biomarkers, namely carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and neuron-specific enolase (NSE), which are typical biomarkers of lung cancer. The present immunosensor exhibited a wide linearity range of 0.01 to 100 ng mL−1 and limits of detection 6.3 pg mL−1 for CEA, 8.5 pg mL−1 for CYFRA21-1 and 7.9 pg mL−1 for NSE. In addition, for the clinical serum sample, the data from this immunosensor are consistent with those from enzyme-linked immunosorbent assay (ELISA).

A new sensitive electrochemical sensor for the detection of copper ions based on the copper ion assisted etching of Au@Ag nanoparticles was developed in this work. Since copper ions could greatly catalyze the etching process of the silver shell of Au@Ag nanoparticles in the presence of thiosulfate solutions, leading to an obvious decrease of the linear sweep voltammetry (LSV) signals of silver, the concentration of the copper ions, therefore, can be measured. Under the optimized conditions, the electrochemical sensor exhibited excellent sensitivity and selectivity for Cu2+, with wide linear ranges of 1 nM to 100 μM, and the detection limit of 0.3 nM. In addition, this method was successfully applied for the analysis of Cu2+ in river water and exhibited good analytical performance.

A new strategy for simultaneous detection of carcinoembryonic antigen and alpha-fetoprotein is introduced. In this work, an ionic liquid–reduced graphene oxide composite was combined with two different redox probes, thionine and ferricyanide, respectively, through electrostatic adsorption. The resulting nanocomposites were then attached to gold nanoparticles in order to supply a large, accessible surface for the immobilization of anti-carcinoembryonic antigen and anti-alpha-fetoprotein as immunosensing probes. A chitosan–AuNP modified glassy carbon electrode was used as a substrate to immobilize capture antibodies. As such, a sandwich-type electrochemical immunosensor for the simultaneous detection of carcinoembryonic antigen and alpha-fetoprotein was successfully developed. The electric potentials and voltammetric peaks measured using square wave voltammetry reflected the identity and concentrations of the corresponding antigen. Under optimized conditions, the immunosensor exhibited good sensitivity and selectivity with linear ranges of 0.05–100 ng mL−1, and detection limits for carcinoembryonic antigen and alpha-fetoprotein of 0.02 ng mL−1 and 0.01 ng mL−1 (S/N = 3), respectively. The reliability of the proposed immunosensor has been proven by the detection of carcinoembryonic antigen and alpha-fetoprotein in clinical serum samples and the results were in good agreement with a standard enzyme-linked immunosorbent assay.

Novel hollow AuPt alloy nanoparticles (hAuPt NPs) with a rough surface are prepared by a simple one-step galvanic displacement reaction between Co nanoparticles and a mixture of HAuCl4 and K2PtCl4 under mild conditions. The synthesized hAuPt NPs are decorated with graphene oxide reduced by thionine or thiol functionalized ferrocene, which are utilized as electrochemical immunosensing probes for simultaneous detection of multiple analytes in a signal run. Using carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes, this proposed immunosensing shows wide linear range and low detection limits to 0.011 and 0.015 ng mL−1 for CEA and AFP, respectively. The proposed immunosensor for the detection of serum samples is consistent with ELISA. These results suggest that the hAuPt NPs can be used as an immunosensing platform for detection of multiple analytes.

•The PPy/Pt nanocomposite was prepared by surfactant-free microwave-assisted polyol process.•The PPy/Pt nanocomposite possessed a good electrocatalytic activity toward the reduction of H2O2.•A nonenzyme electrochemical sensor of H2O2 based on PPy/Pt nanocomposite was constructed.In this work, we report a facile strategy for the preparation of polypyrrole/platinum nanocomposite (PPy/Pt nanocomposite), namely small and surface-clean Pt nanoparticles (Pt NPs) densely dispersed on polypyrrole nanoparticles (PPy NPs) by a simple, ultrafast, and microwave-assisted polyol process. The as-made nanocomposite was demonstrated to possess a good electrocatalytic activity toward the reduction of H2O2 by obviously enhancing the detection sensitivity and decreasing H2O2 reduction overpotential at −0.175 V (vs. Ag/AgCl). Accordingly, a selective, stable, repeatable and reproducible non-enzymatic electrochemical sensor of H2O2 based on the nanocomposite was constructed.

Redox probe modified reduced graphene oxide was fabricated through directly reducing graphene oxide using redox probes (toluidine blue (TB) or amino-functionalized ferrocene (NH2-Fc)). Self-assembling gold nanoparticles to fabricate redox probe modified reduced graphene oxide–Au nanocomposites via electrostatic interaction was then carried out. A sandwich-type electrochemical immunosensor, for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes), was designed. Redox probe modified reduced graphene oxide–Au nanocomposites are used as immunosensing probes and an ionic liquid functionalized reduced graphene oxide–Au (IL-rGO–Au) nanocomposite is used as an immunosensing substrate. The resulting immunosensor exhibited high selectivity and sensitivity during the simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100 ng mL−1 for both CEA and AFP and the detection limits reached 0.003 ng mL−1 for CEA and 0.002 ng mL−1 for AFP. Importantly, the proposed immunosensor was evaluated for the analysis of clinical serum samples, producing a good correlation with the data determined by the ELISA. This indicates that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.

•IL-rGO-Au-PDDA-PB nanocomposites were fabricated and used as a signal tag.•Ionic liquid functionalized reduced graphene oxide was used as a substrate.•An immunosensor was designed for AFP based on signal amplification.•This method performed very well on the detection of clinical serum specimens.In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H2O2 and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01–100 ng mL−1. The detection limit for AFP was 4.6 pg mL−1. The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result.

A simple and novel method for the colorimetric detection of Co2+ was developed based on controlling the oxidation level of methylene blue (MB). After a complex was formed between MB, 2-aminothiophenol (ATP) and copper nitrate (MB-ATP-Cu2+), the sensing of Co2+ showed high selectivity. The mechanism of sensing has also been discussed.

•The label-free immunosensor with good electrochemical performance simplified the operation.•The immunosensor avoided the cross-talk between the different analytes.•This method performed very well on the detection of clinical serum specimens.•The method could be modified and extended to the detection of other multiple targets.Here we prepared a label-free electrochemical immunosensor employing indium tin oxide (ITO) sheets as working electrodes and graphene nanocomposites as supporting matrix for simultaneous determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Reduced graphene oxide/thionine/gold nanoparticles nanocomposites were synthesized and coated on ITO for the immobilization of anti-CEA while reduced graphene oxide/Prussian Blue/gold nanoparticles were used to immobilize anti-AFP. The immunosensor determination was based on the fact that due to the formation of antibody–antigen immunocomplex, the decreased response currents of thionine and Prussian Blue were directly proportional to the concentrations of corresponding antigens. Experimental results revealed that the multiplexed immunoassay enabled the simultaneous determination of CEA and AFP with linear working ranges of 0.01–300 ng mL−1. The limit of detections for CEA is 0.650 pg mL−1 and for AFP is 0.885 pg mL−1. In addition, the methodology was evaluated for the analysis of clinical serum samples and received a good correlation with the enzyme linked immunosorbent assay.

•Au–ionic liquid functionalized reduced graphene oxide nanocomposite was prepared.•Ionic liquid functionalized reduced graphene oxide was densely covered with AuNPs.•The prepared nanocomposite was used to fabricate electrochemical immunosensor.•The simultaneous detection of multiple targets of cancer biomarker was realized.In this work, an Au–ionic liquid functionalized reduced graphene oxide nanocomposite (IL-rGO–Au) was fabricated via the self-assembly of ionic liquid functionalized reduced graphene oxide (IL-rGO) and gold nanoparticles (AuNPs) by electrostatic interaction. The IL-rGO can be synthesized and stabilized by introducing the cations of the amine-terminated ionic liquids (IL-NH2) into the graphene oxide (GO). With the assistance of IL-NH2, AuNPs were uniformly and densely absorbed on the surfaces of the IL-rGO. The proposed IL-rGO–Au nanocomposite can be used as an immunosensing platform because it can not only facilitate the electrons transfer of the electrode surface but also provide a large accessible surface area for the immobilization of abundant antibody. To assess the performance of the IL-rGO–Au nanocomposite, a sandwich-type electrochemical immunosensor was designed for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes). The chitosan (CS) coated prussian blue nanoparticles (PBNPs) or cadmium hexacyanoferrate nanoparticles (CdNPs) and loaded with AuNPs were used as distinguishable signal tags. The resulting immunosensor exhibited high selectivity and sensitivity in simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100 ng mL−1 for both CEA and AFP. The detection limits reached 0.01 ng mL−1 for CEA and 0.006 ng mL−1 for AFP, respectively. No obvious nonspecific adsorption and cross-talk was observed during a series of analyses to detect target analytes. In addition, for the detection of clinical serum samples, it is well consistent with the data determined by the ELISA, indicating that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.

•Electrochemical simultaneous detection of AFP and CEA was successfully performed.•CHIT-PB-AuNPs-Ab2,1 and CHIT-Fc-AuNPs-Ab2,2 were used as signal tags.•The detection limit was 0.03 ng mL−1 for AFP and 0.02 ng mL−1 for CEA.•This method performed very well on the detection of clinical serum specimens.In this work, a novel and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using functionalized chitosan composites. The immunosensing platform was prepared via immobilizing capture anti-AFP and anti-CEA on chitosan-Au nanoparticles (AuNPs) through EDC/NHS linking. The signal tags were fabricated by immobilizing electroactive redox probes – Prussian blue (PB) and ferrocenecarboxylic acid (Fc) on chitosan (CHIT), following by absorbing AuNPs to immobilize labeled anti-AFP and anti-CEA, respectively. A sandwich-type immunoassay format was employed for the simutaneous detection of AFP and CEA. The assay was based on the electrochemical oxidation/reduction of the redox species in signal tags, which has a relationship with the concentration of analytes. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simutaneous monitoring of AFP and CEA with a wide range of 0.05–100 ng mL−1 for both AFP and CEA. The detection limits (LOD) was 0.03 ng mL−1 for AFP and 0.02 ng mL−1 for CEA (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from standard ELISA method. And the negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.

•The multiple tumor biomarkers were sensitively detected.•The signals of different metal ions were distinct without the signal amplification.•Present method performed well on the detection of clinical serum specimens.In this work, a sandwich-format electrochemical immunosensor has been fabricated in the aim of simultaneous detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) using metal ions tagged immunocolloidal gold nanocomposites as signal tags. The capture anti-CEA and anti-AFP were immobilized onto the chitosan–Au nanoparticles (CHIT–AuNPs) membrane modified glassy carbon electrode through glutaraldehyde (GA). The metal ion labels could be detected directly through differential pulse voltammetry (DPV) without metal pre-concentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. Under the optimized conditions, the multiplexed immunoassay exhibited good sensitivity and selectivity for the simultaneous determination of CEA and AFP with linear ranges of 0.01–50 ng mL−1. The detection limits for CEA and AFP are 4.6 pg mL−1 and 3.1 pg mL−1, respectively. The method was successfully applied for the determination of AFP and CEA levels in clinical serum samples, and the results were in good agreement with standard enzyme linked immunosorbent assay (ELISA). This approach gives a promising simple and sensitive immunoassay strategy for clinical applications.

•Cd3[Co(CN)6]2 and Cu3[Co(CN)6]2 nanocubes (CdNCs and CuNCs) were fabricated.•CdNCs and CuNCs can produce obviously distinctive electrical signals.•Multiple cancer biomarkers can be simultaneously detected in a single run.In this work, Cd3[Co(CN)6]2 and Cu3[Co(CN)6]2 (CdNCs and CuNCs) nanocubes were synthesized simply by a one-step process at room temperature in the presence of chitosan (CS). It was found that CdNCs and CuNCs produced obviously distinctive anodic peak currents at −0.7 V and −0.1 V (vs. Ag/AgCl), whose separation enabled differentiation between two analytes. They were used as novel electrochemical probes in multiplex electrochemical detection for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) in a single run. The good performance of the new electrochemical probes was obtained. The linearity range was from 0.025 to 250 ng mL−1 for both CEA and AFP. The detection limit of CEA was 0.0175 ng mL−1 and that of AFP was 0.0109 ng mL−1 at a signal-to-noise of 3. Analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). The novel electrochemical probes could be generally used in multiplex protein detections.

•The Au nanostars (AuNSs) were synthesized using positively charged PDDA as stabilizer.•The size and morphology of the g AuNSs can be controlled by adjusting the amount of AgNO3.•The AuNSs exhibited an excellent catalytic effect toward the reduction of H2O2.•The AuNSs were used for the non-enzymatic detection of H2O2.Gold nanostars (AuNSs) with tunable morphology were synthesized by a seed-mediated growth method using poly(diallyldimethylammonium chloride) (PDDA) as stabilizer. The number and length of the branches of the nanostars can be controlled by adjusting the amount of silver nitrate. In addition, the AuNSs exhibit an excellent catalytic effect toward the reduction of hydrogen peroxide (H2O2) in deoxygenated phosphate buffer (pH 7.0). Thus, the PDDA-protected AuNSs were utilized to modify glassy carbon electrode directly for fabricating non-enzymatic H2O2 sensor. The fast amperometric response, low detection limit, wide linear range, good selectivity and long-term stability for the electrochemical detection of H2O2 imply a good performance of the AuNSs based sensor.

•Poly(thionine)-Au nano-network and nanowires were prepared by one-step approach.•Poly(thionine)-Au nano-network was used for H2O2 determination.•Fast detection of H2O2 with wide linear range and low detection limit was achieved.•The sensor exhibited good stability, reproducibility and selectivity.We describe the preparation of novel poly(thionine)-Au materials, where the poly(thionine)-Au nano-network and nanowires have been synthesized in aqueous solution via the polymerization of thionine using HAuCl4 as the oxidant in a single reaction setup. The synthesis process does not require templates, nor does it require large amounts of organic solvents or electrochemical methods. The morphology of the nanocomposites can be controlled by varying the thionine/HAuCl4 ratio. The resulting poly(thionine)-Au network was used to fabricate a novel non-enzyme hydrogen peroxide (H2O2) biosensor. In pH 7.0 phosphate buffer, almost interference-free determination of H2O2 was realized at − 0.1 V versus Ag/AgCl with a linear of 1 × 10− 4 to 5 × 10− 2 M, a correlation coefficient of 0.998 and a response time of < 2 s. The developed biosensor showed a detection limit of 0.2 μM (S/N = 3) with very good stability, reproducibility and high selectivity.

•PAMAM-CDs/Au nanocrystal nanocomposites were prepared.•PAMAM-CDs/Au was used to fabricate electrochemical immunosensor.•The detection limit for AFP reached 0.025 pg mL−1.•This method performed very well on the detection of clinical serum specimens.In this work, polyamidoamine dendrimers capped-carbon dots (PAMAM-CDs) were fabricated by one-step microwave assisted pyrolysis of citric acid (CA) and PAMAM, where the formation of CDs and the surface passivation were accomplished simultaneously. The obtained graphitic PAMAM-CDs, with abundant amine groups, were employed as reducing and capping agents for the formation of PAMAM-CDs/Au nanocrystal nanocomposites. The resulting nanocomposites exhibited excellent conductivity, stability and biocompatibility on the surface of electrode and were designed as an immobilized matrix for sensitive immunosensing of alpha-fetoprotein (AFP). The proposed immunosensor showed a wide linear detection range from 100 fg mL−1 to 100 ng mL−1. The detection limit for AFP was 0.025 pg mL−1. Importantly, the immunosensor was evaluated for the analysis of clinical serum samples, obtaining a good correlation with enzyme-linked immunosorbent assay (ELISA). The results indicated that the immunosensor provided a possible application for the detection of AFP in clinical diagnosis.

•A novel conductive hydrogel was synthesized by one-pot method.•The hydrogel was used as sensing substrate for label-free electrochemical immunosensor.•The immunosensor exhibited an ultralow detection limit of 0.26 pg mL− 1 for neuron-specific enolase.In this work, a new conductive hydrogel was prepared by a simple cross-linking coordination method using 1,3,5-benzenetricarboxylic acid as the ligand and Fe3 + as the metal ion. The hydrogel film was formed on a glassy carbon electrode (GCE) by a drop coating method, which can dramatically facilitate the transport of electrons. A sensitive label-free electrochemical immunosensor was fabricated following electrodeposition of gold nanoparticles (AuNPs) on a hydrogel film and immobilization of an antibody. Neuron-specific enolase (NSE), a lung cancer biomarker, was used as the model analyte to be detected. The proposed immunosensor exhibited a wide linear detection range of 1 pg mL− 1 to 200 ng mL− 1 and a limit of detection of 0.26 pg mL− 1 (the ratio of signal to noise (S/N) = 3). Moreover, the detection of NSE in human serum samples showed satisfactory accuracy compared with the data determined by enzyme-linked immunosorbent assay (ELISA), indicating good analytical performance of the immunoassay.

•SA-Pb2+-GO was synthesised and used to fabricate a novel redox substrate.•A multiple signal amplification method was developed for the label-free immunoassay.•The sensitivity of immunosensor for CA242 reached 32.98 μA (log10C)−1.In this work, multiple signal amplification strategies for ultrasensitive label-free electrochemical immunoassay for carbohydrate antigen 24-2 (CA242) were developed using redox sodium alginate-Pb2+-graphene oxide (SA-Pb2+-GO) hydrogel. The SA-Pb2+-GO hydrogel was synthesised by simply mixing SA, GO, and Pb2+ and then implemented as a novel redox species with a strong current signal at −0.46 V (vs. Ag/AgCl). After the three-dimensional and porous SA-Pb2+-GO hydrogel was in situ generated on a glassy carbon electrode (GCE), chitosan was adsorbed on the obtained electrode to further enrich Pb2+. When chitosan-Pb2+/SA-Pb2+-GO/GCE was incubated with anti-CA242 using glutaraldehyde and blocked by bovine serum albumin, the immunoassay platform for CA242 was obtained. Owing to the addition of GO, the obtained conductive SA-GO/GCE was beneficial for signal amplification. After incubating SA-GO/GCE with excessive amounts of Pb2+, the resistance of SA-Pb2+-GO/GCE further decreased and a strong redox signal was obtained. The chitosan fixed by electrostatic adsorption resulted in further adsorption of Pb2+, behaving as further amplifying the signal and improving conductivity. In this case, multiple signal amplification strategies were involved in the proposed immunosensor for the ultrasensitive detection of CA242. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.005 U mL−1 to 500 U mL−1 with an ultralow detection limit of 0.067 mU mL−1. In comparison to previous works, the sensitivity of this method was 32.98 μA (log10CCA242)−1, which was a five-fold increase from the previous works.

•A new peptide-based biosensor was fabricated for the ultrasensitive detection of prostate specific antigen.•Au-poly(methylene blue) as a novel redox species were synthesized by oxidative polymerization.•Polydopamine-Au-horse radish peroxidase nanocomposites were used to accelerate an enzyme-catalyzed precipitation reaction to enhance sensitivity.In general, current difference (ΔI) due to immunoreactions is significant in determining biosensor sensitivity. In this work, a new strategy of triple sensitivity amplification for ultrasensitive electrochemical detection of prostate specific antigen (PSA) was developed. Au-poly(methylene blue) (Au-PMB) was implemented as a redox species with strong current signal at −0.144 V and used to fabricate the substrate of the biosensor. Conductive reduced graphene oxide-Au nanocomposites (Au-rGO) were coated on the Au-PMB modified glassy carbon electrode (GCE) to amplify current signal. After peptides (CEHSSKLQLAK-NH2) were fixed on the Au-rGO/Au-PMB/GCE, the fixed peptides reacted with glutaraldehyde to immobilize polydopamine-Au-horse radish peroxidase nanocomposites (PDA-Au-HRP). The electrochemical sensing interface for PSA was realized. Due to smaller resistance compared to antibodies, the peptides which can be cleaved specifically by PSA were employed. After the incubation of PSA, a large ΔI was obtained and behaved as the decrease of current signal. Then the remaining PDA-Au-HRP accelerated an enzyme-catalyzed precipitation reaction between 4-chloro-1-naphthol and H2O2. A further decrease in current signal (namely the increase in ΔI) resulted from the poorly conductive precipitation adhering onto the biosensor. The designed biosensor presented a wide linear range from 1.0 fg mL−1 to 100 ng mL−1 with an ultralow detection limit of 0.11 fg mL−1.

Two kinds of novel redox-active species, gold-decorated polyaniline derivatives (Au-PANI derivatives) including gold-poly(o-aminophenol) (Au-PoAP) and gold-poly(p-phenylenediamine) (Au-PpPD), which can produce oxidation peaks at −0.2 V and 0.25 V, respectively, were synthesized by a one-pot method using o-aminophenol and p-phenylenediamine as monomers, and chloroauric acid as the oxidant. The gold nanoparticles in the composite make a prominent contribution to its conductivity. These two Au-PANI derivatives together with gold-poly(o-phenylenediamine) (Au-PoPD, oxidation peaks at −0.5 V) were used as redox-active species for simultaneous electrochemical detection of three biomarkers, namely carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and neuron-specific enolase (NSE), which are typical biomarkers of lung cancer. The present immunosensor exhibited a wide linearity range of 0.01 to 100 ng mL−1 and limits of detection 6.3 pg mL−1 for CEA, 8.5 pg mL−1 for CYFRA21-1 and 7.9 pg mL−1 for NSE. In addition, for the clinical serum sample, the data from this immunosensor are consistent with those from enzyme-linked immunosorbent assay (ELISA).

Redox probe modified reduced graphene oxide was fabricated through directly reducing graphene oxide using redox probes (toluidine blue (TB) or amino-functionalized ferrocene (NH2-Fc)). Self-assembling gold nanoparticles to fabricate redox probe modified reduced graphene oxide–Au nanocomposites via electrostatic interaction was then carried out. A sandwich-type electrochemical immunosensor, for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes), was designed. Redox probe modified reduced graphene oxide–Au nanocomposites are used as immunosensing probes and an ionic liquid functionalized reduced graphene oxide–Au (IL-rGO–Au) nanocomposite is used as an immunosensing substrate. The resulting immunosensor exhibited high selectivity and sensitivity during the simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100 ng mL−1 for both CEA and AFP and the detection limits reached 0.003 ng mL−1 for CEA and 0.002 ng mL−1 for AFP. Importantly, the proposed immunosensor was evaluated for the analysis of clinical serum samples, producing a good correlation with the data determined by the ELISA. This indicates that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.