基于铁氰化镍的非标记型核酸适配体电化学传感器检测凝血酶

在玻碳电极（GCE）表面首先用增敏作用的多壁碳纳米管(MWCNTs)夹心于两层电沉积的铁氰化镍（NiHCF）氧化还原电化学探针之间,然后以金纳米粒子为固定核酸适配体的载体,构建了检测凝血酶的非标记型核酸适配体生物传感器。 利用扫描电子显微镜(SEM)对MWCNTs和NiHCF的形貌进行了表征。 利用电化学阻抗谱对传感器的组装过程进行了监测,用循环伏安法（CV）和差分脉冲伏安法（DPV）对传感器的电化学行为进行了研究。 以铁氰化镍为探针的传感器对凝血酶的检测在1.0 ng/L~1.0 mg/L范围内呈良好的线性关系,相关系数为0.998,检测限为0.2 ng/L(S/N=3)。     

Development of impedimetric and optical calcium biosensor by using modified gold electrode with porcine S100A12 protein

We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100A12 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption; we observed an increase of 1184.32m° in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66m° after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications.     

The behaviour of an electrochemical detector used in liquid chromatography and continuous flow voltammetry: Part 2. Evaluation of low-temperature isotropic carbon for use as an electrode material

Unalloyed Pyrolyte, a low-temperature isotropic carbon (LTIC) material is evaluated electrochemically for use as an electrode material in h.p.l.c. thin-layer electrochemical detection cell systems. LTIC is compared to carbon paste electrode material in all systems studied, and to glassy carbon impregnated with ceresin wax in the hexacyanoferrate(II)/(III) redox system. Electrode areas, residual currents, and potential windows are described. Quantitative kinetic studies are reported for the hexacyanoferrate(II)/(III) redox pair in 2.0 M KCl, and rate constants are given. Quantitative rate constant studies are also reported for o-dianisidine in 1.0 M H₂SO₄. Finally, qualitative rate comparisons are made by cyclic voltammetric techniques between carbon paste and LTIC electrode materials for maetanephrine, normetanephrine, adrenaline, noradrenaline, dopamine, 3,4-dihydroxyphenylalanine, 5-hydroxytryptophan, and chlorpromazine in chemical systems reflecting previous applications of h.p.l.c. with electrochemical detection.     

Indirect electrochemical determination of antioxidant capacity with hexacyanoferrate(III) reduction using a gold nanoparticle-coated o-phenylenediamine-aniline copolymer electrode

In spite of the many unstandardized literature methods for the determination of the antioxidant activity/capacity (AOA/AOC) of food extracts, there are a very limited number of documented voltammetric nanosensors, despite the fact that commercial electrochemical devices for rapid AOA estimation are on the rise. The mechanism of the developed sensor is based on the chemical reduction of hexacyanoferrate(III) to hexacyanoferrate(II) by antioxidants, followed by the decrement of the cathodic current intensity of hexacyanoferrate(III) in proportion to antioxidant concentration. During voltammetric measurements, the surface of the glassy carbon electrode was coated with an o-phenylenediamine-aniline copolymer and gold nanoparticles were accumulated on this electrode surface to increase the conductivity. It was shown that the developed electrode gave a reversible voltammogram for the hexacyanoferrate(III)/(II) redox couple, and that the cathodic peaks due to strong antioxidants having a standard redox potential less than that of this couple (E^o < 0.36 V) continuously emerged at very close peak potentials. Single antioxidants as well as binary–ternary mixtures were analyzed with this electrode using square wave voltammetry. The trolox-equivalent antioxidant capacities of selected antioxidants were evaluated with this electrode. The modified voltammetric sensor allowed precise measurement of the total antioxidant capacity of plant tea samples such as green tea, lime, and coral moss, and was not interfered by the food preservative sulfite. The results of the developed voltammetric sensor were statistically compared with those of a reference differential pulse voltammetry-cupric reducing antioxidant capacity electrochemical method established in literature.     

Highly sensitive lable-free electrochemical aptasensor for thrombin detection with cobalt hexacyanoferrate as the electrochemical probe

A highly sensitive label-free electrochemical aptasensor has been constructed for the electrochemical detection of thrombin (TB), where two layers of cobalt hexacyanoferrate (CoHCF) redox probes sandwiched with carbon nanotubes–Nafion were directly immobilized on the electrode surface by electrodeposition. Through the strong interaction between CN^? (CoHCF) and gold nanoparticles (GNPs), GNPs were assembled on the CoHCF-modified electrode for the immobilization of thiolated thrombin aptamers (TBA). In the presence of target TB, TBA on the electrode surface could catch TB to form TBA–TB complex, which made a barrier for the electron transfer, resulting in a greater decrease in CoHCF redox probe signals. Thus, the proposed aptasensor showed a high sensitivity and a much wider linearity to TB in the range of 1.0 pg/mL?～?1.0 μg/mL with a detection limit of 0.28 pg/mL.     

Electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of surface plasmon resonance signal

Sandwich immunoassay was conducted on a thin gold film set in a surface plasmon resonance (SPR) cell. Monochronal antibody (anti-IgG) was immobilized onto the gold film via 4,4′-dithiodibutyric acid (DDA) and avidin-biotin bonding. Next, IgG sample and alkaline phosphatase-conjugated anti-IgG (ALP anti-IgG) were introduced into the cell successively. Finally, p-aminophenyl phosphate (PAPP) was injected as an enzyme substrate, and the produced p-aminophenol (PAP) was electrochemically measured. Flow did not need to be stopped for incubation for the enzyme reaction, because of the thinness of the cell. In these processes, all the antigen-antibody reactions took place on the gold film. Therefore, the immobilization was performed quickly, and each process could be confirmed by SPR signal. This system had the advantage that the middle of the complicated process could be monitored. For example, the amount of antibody immobilized, which affected on the final electrochemical signal, could be confirmed in the course of immobilization. It was also convenient to investigate process conditions, such as removal of used antigens and labeled antibodies. Good correlation was obtained between the electrochemical current and the SPR signals due to the adsorption of IgG and ALP anti-IgG, and the sensitivity of the electrochemical measurement was much higher than the SPR’s.     

Monolayers of 3-mercaptopropyl-amino acid to reduce the nonspecific adsorption of serum proteins on the surface of biosensors

Monolayers prepared with polar or ionic amino acids with short side chains have a reduced nonspecific adsorption of serum proteins compared to that of hydrophobic amino acids and organic monolayers immobilized on the gold surface of surface plasmon resonance (SPR) biosensors. Proteins contained in biological samples adsorb on most surfaces, which in the case of biosensors causes a nonspecific response that hinders the quantification of biomarkers in these biological samples. To circumvent this problem, self-assembled monolayers (SAM) of N-3-mercaptopropyl-amino acids (3-MPA-amino acids) were prepared from 19 natural amino acids. These SAM were investigated to limit the nonspecific adsorption of proteins contained in biological fluids and to immobilize molecular receptors (i.e., antibodies) that are necessary in the construction of biosensors. SPR and Ge attenuated total reflection (GATR) FTIR spectroscopy were employed to characterize the formation of the amino acid SAMs. Monolayers of 3-MPA-amino acids densely packed on the surface of the SPR biosensors result in a surface concentration of approximately 10 (15) molecules/cm (2). SPR also quantifies the surface concentration of serum proteins nonspecifically adsorbed on 3-MPA-amino acids following the exposure of the biosensor to undiluted bovine serum. The concentration of nonspecifically bound proteins ranged from approximately 400 ng/cm (2) with polar and ionic amino acids to approximately 800 ng/cm (2) with amino acids of increased hydrophobicity. The nonspecific adsorption of serum proteins on the 3-MPA-amino acids increases in the following order: Asp < Asn < Ser < Met < Glu < Gln < Thr < Gly < His < Cys < Arg < Phe < Trp < Val < Pro < Ile < Leu < Ala < Tyr. The analysis of the adsorption and desorption curves for serum proteins on the SPR sensorgram has demonstrated the strong irreversibility of the protein adsorption on each surface. The effective hydrophilicity of the SAMs was measured from the contact angle with a saline buffer and has demonstrated that surfaces minimizing the contact angle with PBS performed better in serum. The antibody for beta-lactamase was immobilized on a 3-MPA-glycine SAM, and beta-lactamase was detected in the nanomolar range. The presence of beta-lactamase is an indicator of antibiotic resistance.     

Development of an impedimetric immunosensor based on electropolymerized polytyramine films for the direct detection of Salmonella typhimurium in pure cultures of type strains and inoculated real samples

The development of a faradic impedimetric immunosensor based on electropolymerized polytyramine (Ptyr) films for the detection of S. typhimurium in milk is described for the first time. Polyclonal anti-Salmonella was cross-linked, in the presence of glutaraldehyde vapors, on Ptyr-modified gold electrodes. The dielectric behaviour of Ptyr films was evaluated with capacitance measurements, while their stability in neutral aqueous solutions was examined with impedimetric measurements. The effect of the concentration of tyramine in the forming solution on both the sensitivity and the dynamic range of the resulted immunosensors was also investigated. The alteration of the interfacial features of the electrodes due to different modification or recognition steps, was measured by faradic electrochemical impedance spectroscopy in the presence of a hexacyanoferrate(II)/(III) redox couple. At samples containing a low initial concentration of 10 cfu mL⁻¹S. typhimurium, that actually defines the LOD of the immunosensors, signal changes of 33% and 88% were achieved after 3 and 10 h incubation, respectively. To achieve the working simplicity expected by a biosensor, immunoreaction was performed directly in cultures. This resulted in the elimination of various centrifugation and washing steps, which are used for the isolation of bacteria cells from the culture, thus making the proposed immunosensors promising candidates for on-site applications. Finally, the proposed immunosensors were successfully used for the detection of S. typhimurium in experimentally inoculated milk samples.     

The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.     

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

Properties of gold electrode modified with dimercaptosuccinic acid and electrochemical behavior of copper histidine complex

A self-assembled monolayer of meso-2,3-dimercaptosuccinic acid was prepared on the surface of gold disc electrode. The modified electrode was characterized using cyclic voltammetry in copper(II) solution and cyclic voltammetry and electrochemical impedance spectroscopy in the presence of potassium hexacyanoferrate( II)/(III) and hexaammineruthenium (II)/(III) chloride. Binding of copper(II) histidine complex (Cu–His) onto the electrode was successfully achieved for a wide range of tested concentrations, as shown with adsorption transfer stripping voltammetry. Electrode response (logΔI_p) was linearly proportional to logc(Cu–His) with correlation coefficient R³² = 0.9839.     

Surface functionalization with redox active molecule-based imidazolium via click chemistry

In this study, the redox active molecule N-ferrocenylmethyl-N-propargylimidazolium bromide was immobilized onto the surface of an electrode. The surface modification was performed by coupling the electrochemical reduction of the 4-azidophenyldiazonium generated in situ with a copper(I) catalyzed click chemistry reaction. Surface and electrochemical investigations suggest the attachment of a monolayer of redox active molecules containing an ionic liquid framework onto the electrode surface. Furthermore, scanning electrochemical microscopy studies revealed the conductive behavior of the attached ferrocenyl moieties on the ITO surface.     

Effect of Gold Nanoparticles on the Structure and Electron‐Transfer Characteristics of Glucose Oxidase Redox Polyelectrolyte‐Surfactant Complexes

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox‐active polyelectrolyte–surfactant complex containing [Os(bpy)₂Clpy]²⁺ (bpy=2,2′‐bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron‐transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing‐incidence small‐angle X‐ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz‐crystal microbalance with dissipation (QCM‐D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron‐transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five‐fold increase in current response to glucose compared with analogous supramolecular AuNP‐free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron‐transfer process.     

Quantitation of intracellular recombinant human superoxide dismutase using surface plasmon resonance

An immunosensor assay for the quantitation of intracellular recombinant human superoxide dismutase (rhSOD) in Escherichia coli cultivations based on detection with surface plasmon resoance (SPR) is described. A monoclonal antibody for rhSOD was immobilized on a SPR dextran gold chip. Bacterial samples were sonicated and centrifugated prior to injection over the antibody chip for SPR detection. The assay time was 7 min and allowed quantitation in the range of 1-64 nM SOD in lysate samples with a precision of 1.1-3.4%. The assay was applied to monitor the concentration of rhSOD during E. coli bioreactor cultivations where the rhSOD production was induced by iso-propyl-b-d-thiogalactoside (IPTG). The assay allowed accurate monitoring of the production of rhSOD where the important phases in the product formation were possible to see. The report also discusses influence from sample preparation, SPR selectivity and sensitivity and quantitation limits. The assay proved to be fast, sensitive and accurate with low background effects from the dextran matrix of the SPR chip.     

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid-thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) and non-specific (Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10⁴ and 10³ cfu mL⁻¹ of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10⁻³ due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.     

Surface plasmon resonance spectroscopy and electrochemistry study of 4-nitro-1,2-phenylenediamine: a switchable redox polymer with nitro functional groups

Electrochemistry and electrochemical surface plasmon resonance (SPR) spectroscopy have been applied to study the electrochemical deposition and the redox transition of poly(4-nitro-1,2-phenylenediamine) (P4NoPD) on gold disk. It was shown that SPR can be the signal transducer for the different redox states of P4NoPD. Using a model biomolecular system, involving streptavidin, biotinylated DNA, and its complementary target DNA, it was found that the presence of nitro groups in P4NoPD allows the biorecognition events to be modulated by voltages. There is minimal nonspecific binding of biomolecules on oxidized (+0.2 V) or as-prepared P4NoPD, and binding occurs more significantly on the reduced P4NoPD (-0.2 to -0.6 V) with the presence of amine groups. The electrochemical deposition of P4NoPD film was also conducted on boron-doped diamond (BDD) electrode. The stability of the reduced P4NoPD film on gold and BDD was comparatively evaluated by electrochemical impedance spectroscopy (EIS). The result showed that BDD allows the electrochemical reduction of the P4NoPD film at wider cathodic limits than gold.     

Investigation of the redox property of a metalloprotein layer self-assembled on various chemical linkers

Myogloblin, a well-known metalloprotein, was immobilized on a gold surface using various chemical linkers to investigate the length effect of chemical linker on the electron transfer in protein layers, because chemical linkers play roles in the pathway that transfers the electron from the protein to the gold substrate and act as protein immobilization reagents. Chemical linkers with 2, 6, 11, and 16 carbons were utilized to confirm length-effects. The immobilization of protein and chemical linker was validated with surface plasmon resonance (SPR) and atomic force microscopy (AFM). The electrochemical property was evaluated by cyclic voltammetry (CV) and chronocoulometry (CC). In those results, redox peaks of immobilized protein were controlled via the length of chemical linkers, and it could be directly applied to the realization of bioelectronic device.     

Performance of a Faradaic impedimetric immunosensor for blood group antigen A

The performance is described of a label-free Faradaic impedimetric immunosensor based on immobilized monoclonal IgM antibodies to blood group antigen A (anti–A) for blood typing. Anti–A was directly immobilized onto gold electrodes modified with an amine-reactive self assembled monolayer of dithiobis(succinimidylundecanoate). The alteration of the interfacial features of the electrodes due to different modification or recognition steps was probed by Faradaic impedance spectroscopy and cyclic voltammetry in the presence of a hexacyanoferrate(II)/(III) redox couple. Various optimization studies were undertaken with respect to the construction and potential use of the immunosensors as a diagnostic tool for blood typing.     

High Sensitive Detection of Prostate Specific Antigen by Using Ferrocene Cored Asymmetric PAMAM Dendrimer Interface Screen Printed Electrodes

In this study, electrochemical immunosensors were developed for the detection of prostate specific antigen (PSA) using ferrocene (Fc) and polyamidoamine dendrimer (PAMAM) constructs. The biosensor fabrication was designed by modifying the screen‐printed gold electrode (Au) with ferrocene cored dendrimers (FcPAMAM) synthesized in three different generations. The self‐assembled monolayer principle was followed, to obtain sensitive, selective and disposable electrodes. Therefore, the Au electrodes were modified with cysteamine (Cys) to obtain a functional surface for FcPAMAM dendrimers to bind. Dendrimer generations were attached to this surface using a cross‐linker (glutaraldehyde) so that a suitable surface was obtained for binding of biological components. The Monoclonal PSA antibody (anti‐PSA) was immobilized on the Au electrode surface which coated with dendrimer, and (Au/Cys/FcPAMAM/anti‐PSA) biosensing electrode was obtained. The PSA detection performances of electrochemical impedance spectroscopy (EIS) and Amperometry based immunosensors exhibited very low detection limits; 0.001 ng mL^?1 and 0.1 pg mL^?1, respectively. In addition, EIS and Amperometry based biosensors using Au/Cys/FcPAMAM/anti‐PSA sensing electrode were represented excellent linear ranges of 0.01 ng mL^?1 to 100 ng mL^?1 and 0.001 ng mL^?1 to 100 ng mL^?1. In order to determine the applicability recovery and selectivity tests were performed using three different proteins in human serum.     

Ultrasensitive aptamer-based on-off assay for lysozyme using a glassy carbon electrode modified with gold nanoparticles and electrochemically reduced graphene oxide

The authors describe an electrochemical aptamer based assay for the determination of the serine protease lysozyme in very low (pM) concentrations. The method is based on the formation of a complex between anti-lysozyme aptamer fragments and lysozyme, and on electrochemical detection by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The surface of a glassy carbon electrode was modified with a nanocomposite consisting of gold nanoparticles and electrochemically reduced graphene oxide nanosheets (AuNPs/erGO), and the thiolated aptamer was then linked to the AuNPs by self-assembly through Au-S bonds. The interaction of immobilized aptamers with lysozyme leads to the decreased peak current in DPV and increased charge transfer resistance (R_ct) in EIS when using hexacyanoferrate or Methylene Blue as a redox probe. The calibration plot, when applying EIS and working at a typical voltage of ?0.22 V (vs. SCE), is linear over 1.0 to 104.3 pM concentration range, with a detection limit of 0.06 pM (at a signal-to-noise ratio of 3). The respective data for DPV are a 9.6–205.5 pM linear range with a detection limit of 0.24 pM. Depending on the redox marker applied, the method works in the “signal-off” or “signal-on” mode in DPV and EIS protocols, respectively. The sensing interface is high specific for lysozyme and not affected by other proteins. The method was applied to the determination of lysozyme in spiked diluted human serum, and the results agreed well with data obtained with a standard ELISA.

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Graphical abstract The surface of a glassy carbon electrode was modified with a nanocomposite consisting of gold nanoparticles and electrochemically reduced graphene oxide nanosheets (AuNPs/erGO). Then, the thiolated aptamer was linked to the AuNPs by self-assembly through Au-S bonds. The modified electrode was applied to the determination of lysozyme with “signal off” and “signal on” strategies.