Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

FeS2−AuNPs Nanocomposite as Mimicking Enzyme for Constructing Signal‐off Sandwich‐type Electrochemical Immunosensor Based on Electroactive Nickel Hexacyanoferrate as Matrix

In this work, a novel sandwich‐type electrochemical immunosensor with electroactive nickel hexacyanoferrate nanoparticles (NiHCFNPs) as matrix was constructed for α‐fetoprotein (AFP) detection in a signal‐off manner by using FeS₂?AuNPs nanocomposite catalyzed insoluble precipitation to significantly inhibit the electrochemical signal. Initially, the NiHCFNPs with excellent electrochemical property was modified on the electrodeposited nano‐Au electrode to obtain a strong initial electrochemical signal. Subsequently, another nano‐Au layer was formed for immobilization of capture antibody (Ab₁). In the presence of target AFP, the prepared FeS₂?AuNPs‐Ab₂ bioconjugate could be specifically recognized and immobilized on electrode through the sandwich‐type immunoreaction. The FeS₂ with large specific surface areas were used as scaffolds to load abundant mimicking enzyme AuNPs. With the help of hydrogen peroxide (H₂O₂), FeS₂?AuNPs with peroxidase‐like activity accelerated the 4‐chloro‐1‐naphthol (4‐CN) oxidation with generation of insoluble precipitation on electrode, which would greatly hinder the electron transfer and thus caused the decrease of electrochemical signal for quantitative determination of AFP. This approach achieved a wide dynamic linear range from 0.0001 to 100 ng mL^?1 with an ultralow limit detection of 0.028 pg mL^?1. Especially, the proposed AFP immunosensor can be applied to detect human serum samples with satisfactory results, indicating a potential application in clinical monitoring of tumor biomarkers.     

基于酶标抗体和媒介体共吸附于金胶壳聚糖膜的PSA免疫传感器的制备

本文研制了一种用金胶壳聚糖仿生膜来同时固定四甲基联苯胺（TMB）和酶标抗体的新型电化学免疫传感器,用于检测血清肿瘤标志物前列腺特异性抗原（PSA）的含量。固定的TMB作为电子传递媒介体,在扫速小于45 mV/s时,电极表现为一个表面控制过程,而在扫速大于45 mV/s时则表现为一个扩散控制过程。将固定有酶标抗体和TMB的免疫传感器与待测PSA抗原一起培育,在该传感器上形成的免疫复合物通过TMB-H₂O₂-HRP电化学体系进行了测定。在优化实验条件下,PSA的线性检测范围为5-30 ng·mL^-1,检测限为1.0 ng·mL^-1。该PSA免疫传感器制备方法简单,成本低廉,具有较好的稳定性和重现性。     

Calcium Carbonate–Gold Nanocluster Hybrid Spheres: Synthesis and Versatile Application in Immunoassays

Fluorescent gold nanoclusters (AuNCs) were incorporated into porous calcium carbonate spheres through electrostatic interaction. The resulting CaCO₃/AuNCs hybrid material exhibited interesting properties, such as porous structure, excellent biocompatibility, good water solubility, and degradability. These properties make the CaCO₃/AuNCs hybrid material a promising template to assemble horseradish peroxidase/antibody conjugates (HRP‐Ab₂). By using CaCO₃/AuNCs/HRP‐Ab₂ bioconjugates as probes, a versatile immunosensor was developed for fluorescent and electrochemical detection of the cancer biomarker neuron‐specific enolase (NSE). The detection limits of the sensor were 2.0 and 0.1 pg mL^?1 for fluorescent and electrochemical detection, respectively. The immunosensor shows high sensitivity and offers an alternative strategy for the detection of other proteins and DNA.     

Signal amplification strategy for sensitive immunoassay of prostate specific antigen (PSA) based on ferrocene incorporated polystyrene spheres

A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab₂) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL⁻¹ to 20 ng mL⁻¹ with a detection limit of 1 pg mL⁻¹. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.     

A Photoelectrochemical Immunosensor for Prostate Specific Antigen Detection Based on Graphdiyne Oxide Conjugated with Horseradish Peroxidase

Graphdiyne (GDY) was a novel flat material with sp and sp² hybridized carbon atoms. It exhibited good biocompatibility. The application of GDY in PEC immunosensor was very limited. Thus, a novel photoelectrochemical sensor for the sensitive detection of prostate specific antigen (PSA) was proposed by using GDY oxide (GDYO) conjugated with horseradish peroxidase (HRP) and secondary antibody for photocurrent signal inhibition. GDYO was prepared by oxidation of honeycomb-like nanotubes composed of numerous GDY nanosheets. It showed high loading capacity for HRP and the catalytic activity of HRP could be remained. With reduced graphene oxide-CdS (rGO-CdS) as photoelectrochemical sensing platform, a sandwich-type photoelectrochemical (PEC) immunosensor was thus fabricated. The immunosensor presented a wide linear concentration range of 10 fg mL⁻¹–20.0 ng mL⁻¹ with a detection limit (LOD) of 3.5 fg mL⁻¹. Moreover, the PEC immunosensor displayed ideal reproducibility, stability, and selectivity, which was a promising platform for the detection of other important tumor targets.     

Highly Sensitive Electrochemical Immunosensor Based on Mesoporous PdPt Nanoparticles Anchored on a Three-dimensional PANI@CNTs Network as Nanozyme Labels

In this study, a novel strategy to amplify electrochemical signals by mesoporous PdPt nanoparticles with core-shell structures anchored on a three-dimensional PANI@CNTs network as nanozyme labels (PdPt/PANI@CNTs) was proposed for the sensitive monitoring of α-fetoprotein (AFP, Ag). First, the mesoporous PdPt nanoparticles prepared by a facile chemical reduction method had excellent biocompatibility with biomolecules, which could capture a large amount of AFP-Ab₂ (Ab₂) and exhibit plentiful pores to entrap more thionine (Thi) into mesoporous PdPt nanoparticles with enhanced loading and abundant active sites. Furthermore, the resulting mesoporous PdPt nanoparticles were abundantly dotted on the surface of a three-dimensional PANI@CNTs network with excellent conductivity and a high specific surface area through the bonding of the amino group to form PdPt/PANI@CNTs nanozyme labels. Most importantly, the as-prepared PdPt/PANI@CNTs nanozyme labels exhibited unexpected enzyme-like activity towards the reduction of hydrogen peroxide owing to the highly indexed facets, enhancing the current response to realize signal amplification. In view of the advantages of nanozyme labels and the involvement of gold nanoparticles (AuNPs, which behave as electrode materials) for the sensitive determination of AFP, the as-developed immunosensor could obtain a dynamic working range of 0.001 ng mL^−1–100.0 ng mL⁻¹ at a detection limit of 0.33 pg mL⁻¹ via DPV (at 3σ). Furthermore, the nanozyme-based electrochemical immunosensor exhibited remarkable analytical performance, which brought about feasible ideas for disease diagnosis in the future.     

Polystyrene microspheres based sandwich immunosensor using CdTe nanoparticles amplification and ultrasensitive flow-injection chemiluminescence detection

In this paper we propose a specific sandwich immunoassay method for human-immunoglobulin G (HIgG). This immunoassay protocol takes advantage of sandwich binding of primary and secondary antibodies for increased specificity. Polystyrene microspheres (PS) serve as immobilizing support, site for sandwich immunoassay and then subsequently used for chemiluminescence (CL) detections. In this sandwich immunoassay, PS microspheres were modified with the primary anti-HIgG (Ab₁) via electrostatic interaction, while CdTe nanoparticles (CdTeNPs) were modified with horseradish peroxidase labeled anti-HIgG (Ab₂) via covalent binding. Antigen HIgG (Ag) was specifically captured by the first and secondary antibody and form sandwich immunoassay format. Combination of the remarkable sensitivity of CL method and the use of CdTe NPs as anti-HIgG–HRP carrier for the enzymatic signal amplification, provide a linear response range of HIgG from 0.01 to 300 ng mL⁻¹ with an extremely low detection limit of 0.3 pg mL⁻¹. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. The assay results were compared with enzyme-linked immunosorbent assay (ELISA), and showed relatively good reliability. Significantly the new protocol may become quite promising technique for protein immune-detection as well as DNA analysis and other biological analyses.     

Label-free sandwich type of immunosensor for hepatitis C virus core antigen based on the use of gold nanoparticles on a nanostructured metal oxide surface

We report on the construction of a label-free electrochemical immunosensor for detecting the core antigen of the hepatitis C virus (HCV core antigen). A glassy carbon electrode (GCE) was modified with a nanocomposite made from gold nanoparticles, zirconia nanoparticles and chitosan, and prepared by in situ reduction. The zirconia nanoparticles were first dispersed in chitosan solution, and then AuNPs were prepared in situ on the ZrO₂-chitosan composite. In parallel, a nanocomposite was synthesized from AuNPs, silica nanoparticles and chitosan, and conjugated to a secondary antibody. The properties of the resulting nanocomposites were investigated by UV-visible photometry and transmission electron microscopy, and the stepwise assembly process was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. An sandwich type of immunosensor was developed which displays high sensitivity to the HCV core antigen in the concentration range between 2 and 512?ng?mL^?1, with a detection limit of 0.17?ng?mL^?1 (at S/N?=?3). This immunosensor provides an alternative approach towards the diagnosis of HCV.

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.     

Heparin‐gold Nanoparticles and Liquid Crystal Applied in Label‐free Electrochemical Immunosensor for Prostate‐specific Antigen

A label‐free electrochemical immunosensor based on the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br−Py), together with heparin‐stabilized gold nanoparticles (AuNP‐Hep) and Nafion is proposed for the determination of prostate‐specific antigen (PSA). The Br−Py liquid crystal presented redox properties and good film‐forming abilities on the electrode surface, and thus it is a suitable alternative as a redox probe for a label‐free electrochemical immunosensor, which could simplify the analysis methodology. The stepwise construction of the immunosensor and the incubation process (immunocomplex formation) were characterized by voltammetry and electrochemical impedance spectroscopy. The proposed immunosensor could directly detect PSA concentrations in the incubation samples, based on the suppression of the Br−Py redox peak (‘base peak’) current. After optimization, the immunosensor exhibited a linear response to PSA concentrations in the range of 0.1 to 50 ng mL⁻¹, with a calculated detection limit of 0.08 ng mL⁻¹. The reproducibility (coefficient of variance less than 3.0 %), selectivity and accuracy of the methodology were adequate. The immunosensor was satisfactorily applied in the quantification of PSA in human blood plasma samples.     

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high 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.     

A photoelectrochemical immunosensor based on Au-doped TiO2 nanotube arrays for the detection of α-synuclein

α‐Synuclein (α‐SYN) is a very important neuronal protein that is associated with Parkinson’s disease. In this paper, we utilized Au‐doped TiO₂ nanotube arrays to design a photoelectrochemical immunosensor for the detection of α‐SYN. The highly ordered TiO₂ nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au‐doped TiO₂ nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab₁) with high stability and bioactivity to bind target α‐SYN. The enhanced sensitivity was obtained by using {Ab₂‐Au‐GOx} bioconjugates, which featured secondary antibody (Ab₂) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H₂O₂, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO₂ nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α‐SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL^?1 to 100 ng mL^?1 with a detection limit of 34 pg mL^?1. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.     

Ultrasensitive electrochemiluminescent immunosensor based on dual signal amplification strategy of gold nanoparticles-dotted graphene composites and CdTe quantum dots coated silica nanoparticles

A facile and ultrasensitive electrochemiluminescent (ECL) immunosensor for detection of prostate-specific antigen (PSA) was designed by using CdTe quantum dots coated silica nanoparticles (SiO₂@QDs) as bionanolabels. To construct such an electrochemiluminescence immunosensor, gold nanoparticles-dotted graphene composites were immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies as well as improve the electronic transmission rate. The as-prepared SiO₂@QDs used as bionanolabels, showed good ECL performance and good ability of immobilization for secondary antibodies. The approach provided a good linear response ranging from 0.005 to 10 ng?mL^?1 with a low detection limit of 0.0032 ng?mL^?1. Such immunosensor showed good precision, acceptable stability, and reproducibility. Satisfactory results were obtained for determination of PSA in human serum samples. Therefore, the proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

Development of an Amperometric Immunosensor for the Quantification of Staphylococcus aureus Using Self‐Assembled Monolayer‐Modified Electrodes as Immobilization Platforms

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self‐assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A‐bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3‐mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H₂O₂, and it was co‐immobilized onto the MPA‐modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×10⁵ S. aureus cells mL^?1), as well as a better repeatability and reproducibility of the measurements.     

Complexation of dodecyl‐substituted poly(acrylate) by linked β‐cyclodextrin dimers and trimers in aqueous solution

Two‐dimensional NOESY ¹H NMR, isothermal titration calorimetric (ITC), and rheological studies of host–guest complexation by β‐cyclodextrin, β‐CD, and the β‐CD groups of the linked β‐CD dimers, β‐CD₂ur and β‐CD₂su and trimers, β‐CD₃bz and β‐CDen₃bz, of the dodecyl, C12, substituents of the 3.0% substituted poly(acrylate), PAAC12, in aqueous solution are reported. Complexations by β‐CD, β‐CD₂ur, β‐CD₂su, β‐CD₃bz, and β‐CDen₃bz of the C12 substituents of PAAC12 in 0.2 wt % solution exhibit complexation constants 10^?4K₁₁ (298.2 K) = 0.83, 5.80, 4.40, 15.0, and 1.50 dm³ mol^?1, respectively. (The corresponding ΔH₁₁ and TΔS₁₁ show a linear relationship.) The rheologically determined zero‐shear viscosities of 3.3 wt % aqueous solutions of PAAC12 alone and in the presence of β‐CD, β‐CD₂ur, β‐CD₂su, β‐CD₃bz, and β‐CDen₃bz (where the β‐CD groups and C12 substituents are equimolar) are 0.016, 0.03, 0.12, 0.25, 0.12, and 0.08 Pa s (298.2 K), respectively, and show PAAC12 to form interstrand cross‐links through complexation. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1278–1286     

Ultrasensitive electrochemical immunosensors for multiplexed determination using mesoporous platinum nanoparticles as nonenzymatic labels

An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab₂) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab₁) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab₂ was bound to immunosensor surface to catalyze the electro-reduction of H₂O₂ reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U mL⁻¹, 0.001 U mL⁻¹ and 7.0 pg mL⁻¹ for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics.     

A No-washing Point-of-Care Electrochemical Biosensor Based on CuS Nanoparticles for Rapid and Sensitive Detection of Neuron-specific Enolase

Electrochemical biosensors have made outstanding achievements in recent years. However, the single pursuit of sensitivity and accuracy sometimes cannot meet the detection requirements and achieve high-efficiency measurements. Therefore, no-washing biosensors have more practical advantages. In this work, a disposable point-of-care (POC) electrochemical biosensor was designed for the sensitive and fast detection of neuron-specific enolase (NSE). Fe₃O₄ and CuS nanoparticles were used as the substrate material for capturing Ab₁ and the signal probe for labeling Ab₂ respectively. The disposable syringe filter was introduced into the determination procedure for simple sample separation, which easily realized no-washing detection. Due to the syringe filters with 200 nm pore diameter could only allow the small nanoparticles of CuS−Ab₂ pass through, the large-sized immunocomplex of Fe₃O₄−Ab₁/NSE/CuS−Ab₂ were blocked on the membrane. The uncombined CuS−Ab₂ particles were pushed out from the syringe and would occur electron transfer between Cu²⁺ and Cu⁺ to generate a current signal detected by the Au electrode. Under optimal conditions, the no-washing biosensor shows a wide linear concentration range (100 fg mL⁻¹∼50 ng mL⁻¹) with the limit of detection of 33 fg mL⁻¹ (S/N=3). Additionally, the biosensor exhibited excellent selectivity, storage stability and reproducibility. The outstanding advantages of the no-washing biosensor make it more suitable for POC testing.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.