A label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of Japanese B encephalitis vaccine

A label-free amperometric immunosensor for fast and sensitive assay of Japanese B encephalitis vaccine is presented. Antiserum of Japanese B encephalitis were immobilized on bilayer nano-Au/o-phenylenediamine polymer film with deposited Prussian blue as an electronic mediator on the Pt electrode. The electrochemical behavior of the biosensor was studied with Fe^2+/3+ as probe on the Pt surface using cyclic voltammetry technique. The variation of amperometric response to the concentration of Japanese B encephalitis vaccine, the target antigen, was evaluated by cyclic voltammetry in PBS. The immunosensor showed a specific response to Japanese B encephalitis vaccine in the range 1.1 × 10⁻⁸ to 1.9 × 10⁻⁶ lg pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of 6 × 10⁻⁹ lg pfu/ml. The correlation coefficient is 0.9955. The incubation time, incubation temperature, pH, reproducibility and stability of the immunosensor were also studied. The present work supplied a promising test method for biological products.     

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator

An amperometric biosensor has been developed for the determination of H₂O₂ in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H₂O₂ concentration linearly from 0 to 8×10^-6 M with a detection limit of 1.3×10^-7 M. On a wider concentration range of 8×10^-6 to 1.4×10^-4 M, the reciprocal of current response is linearly related to the reciprocal of H₂O₂ concentration. The apparent Michaelis-Menten constant (K_m^app) was calculated to be 0.0334 mM. The sensor has been tested by determining H₂O₂ concentration in plant leaf samples.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

Amperometric immunosensor for probing complement III (C3) based on immobilizing C3 antibody to a nano-Au monolayer supported by sol-gel-derived carbon ceramic electrode

An electrochemical immunosensor based on nano-size particulate gold (nano-Au) monolayer as sensing interface has been developed for probing complement III (C₃). The thiol functional group-derived carbon ceramic electrode (CCE) was firstly constructed using (3-mercaptopropyl) trimethoxy silane (MPTMOS) as sol-gel monomer. The stable nano-Au monolayer was obtained resulting from covalent combination between nano-Au and thiol group on the surface of CCE. The nano-Au monolayer formed was utilized as a sensing platform for the immobilization of C₃ antibody (anti-C₃) and subsequent immunoreaction. A competitive immunoassay format was adopted with horseradish peroxidase (HRP)-C₃ as a tracer, hydroquinone and hydrogen peroxide as the enzymatic substrates. The dynamic concentration range for C₃ is 0.08-5.6 μg ml⁻¹. The feasibility of regenerating nano-Au monolayer for consecutive assays was demonstrated by a simple chemical treatment after each determination. The high stability of formed nano-Au monolayer, readily adsorptive immobilization of antibody on nano-Au monolayer, efficient activity retention of loading immunoreactants as well as the simple operation for the formation of nano-Au monolayer make proposed methodology an attractive alternative for the designing new-type immunosensors.     

Reagentless amperometric carbohydrate antigen 19-9 immunosensor based on direct electrochemistry of immobilized horseradish peroxidase

A reagentless immunosensor for rapid determination of carbohydrate antigen 19-9 (CA19-9) in human serum was proposed. This strategy was based on the immobilization of antibody in colloidal gold nanoparticle modified carbon paste electrode and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to a CA19-9 antibody. The nanoparticles were efficient for preserving the activity of immobilized biomolecules. Thus, the immobilized HRP displayed its direct electrochemistry with a rate constant of 1.02 s⁻¹. The incubation of the immunosensor in phosphate buffer solution (PBS) including CA19-9 antigen leading to the formation of antigen-antibody complex, which made the block of electron transfer of HRP toward electrode and resulted in significant peak current decrease of HRP. Under the optimal conditions, the current decrease was proportional to CA19-9 concentrations ranging from 2 to 30 U/ml with a detection limit of 1.37 U/ml at a current decrease by 10%. The immunosensor showed an acceptable accuracy compared with those obtained from immunoradiometric assays, with intra-assay coefficient of 7.3 and 6.9% at CA19-9 concentrations of 5 and 15 U/ml, respectively, and inter-assay coefficient of 9.6% at a CA19-9 concentration of 20 U/ml. The storage stability was acceptable in a pH 7.0 PBS at 4 °C for 10 days. This method avoids the addition of electron transfer mediator, thus simplifies the immunoassay procedure and decreases the analytical time. It provides a new promising platform for clinical immunoassay.     

Potentiometric Immunosensor Based on Immobilization of Hepatitis B Surface Antibody on Platinum Electrode Modified Silver Colloids and Polyvinyl Butyral as Matrixes

A highly sensitive immunosensor based on immobilization of hepatitis B surface antibody (HBsAb) on platinum electrode (Pt) modified silver colloids and polyvinyl butyral (PVB) as matrixes has been developed for potentiometric immunoanalysis to detect hepatitis B surface antigen (HBsAg) in this study. HBsAb molecules were immobilized successfully on nanometer‐sized silver colloid particles associated with polyvinyl butyral on a platinum electrode surface. The modification procedure was electrochemically monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The HBsAb‐silver‐PVB‐modified electrode exhibited direct electrochemical behavior toward HBsAg. The factors influencing the performance of the resulting immunosensor were studied in detail. More than 94.7% of the results of human serum samples obtained by this method were in agreement with those obtained by enzyme‐linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited a sigmoid curve with log HBsAg concentration, high sensitivity (39.8 mV/decade), wide linear range from 16.0 to 800 ng mL^?1 with a detection limit of 3.6 ng mL^?1, fast potentiometric response (<3 min) and long‐term stability (>4 months). The response mechanism of the immunosensors was also studied with AC impedance techniques.     

Development of proof of concept for prostate cancer detection: an electrochemical immunosensor based on fullerene-C60 and copper nanoparticles composite film as diagnostic tool

Screening of Prostate-specific antigen (PSA) in human blood is the most common approach to diagnose prostate cancer. The joint application of biology and electrochemistry has shown a tremendous rise in research towards the development of electrochemical diagnostic tools for various diseases. The present study demonstrates the development of an effective immunosensing platform incorporating hydroquinone (HQ) immobilized, fullerene-C₆₀ and copper nanoparticles (CuNPs) composite film on glassy carbon electrode (HQ@CuNPs-reduced-fullerene-C₆₀/GCE) for the selective, quick and trace detection of PSA. In order to fabricate immunosensor sequential immobilization of primary antibody (Ab₁), blocking agent (bovine serum albumin (BSA)), antigen (prostate-specific antigen (PSA)) and secondary antibody (Ab₂) tagged with horseradish peroxide (HRP) was carried out on HQ@CuNPs-reduced-fullerene-C₆₀/GCE. Electrochemical characterization and the signal response of immunosensor were tested using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Due to the synergetic effect of fullerene-C₆₀ and CuNPs, the novel nanocomposite film exhibited excellent catalytic activity towards hydrogen peroxide (H₂O₂) reduction for greatly amplified immunosensing signals. HQ@CuNPs-fullerene-C₆₀/GCE exhibited a well-defined redox peak and accelerated electrochemical reduction of H₂O₂ without any interference of dissolved oxygen and false-positive result in phosphate buffer solution (PBS) at pH 7.0. The parameters influencing the electrochemical response were optimized. Under the optimized conditions, wide linearity between PSA concentrations and current responses ranging from 0.005 ng/mL to 20 ng/mL with the lower detection limit of 0.002 ng/mL was obtained at the proposed immunosensor. The clinical applicability of the proposed immunosensor was successfully tested in serum and urine samples. Results revealed that the proposed immunosensor may create new boundaries in the identification of PSA in human blood samples.     

基于电聚合作用的脂质体免疫传感器检测血液中2,4-二氯苯氧乙酸

利用旋蒸法和挤压法制备脂质体,通过脂质体包埋亚铁氰化钾,利用戊二醛交联2,4-二氯苯氧乙酸(2,4-D)兔抗制成免疫脂质体,制备快速检测血液中2,4-D的夹心型免疫传感器.实验结果表明:2,4-D在75 ng/mL～50 μg/mL浓度范围内呈现良好的线性关系,检测下限达到了36.42 ng/mL.该免疫传感器选择性强...     

Effect of the protonation level of poly(o-phenylenediamine) (PoPD) on the ac impedance of humidity-sensitive PoPD/poly(vinyl alcohol) composite film

A composite film consisting of poly(o-phenylenediamine) (PoPD) and poly(vinyl alcohol) (PVA) has been prepared, and the effect of the protonation level of PoPD on the response of the composite to humidity been investigated by ac impedance measurements. The electrochemical system of the PoPD/PVA composite in a humid atmosphere is represented by an electrochemical equivalent circuit containing film (R_film, C_film), Warburg (W) and interfacial (R_ct, C_dl) impedance. R_film was increased with decreasing protonation level of the PoPD in relative humidity regions higher than 40%, but it was almost independent of the protonation level in humidity regions lower than this percentage.     

A sensitive and highly stable electrochemical impedance immunosensor based on the formation of silica gel-ionic liquid biocompatible film on the glassy carbon electrode for the determination of aflatoxin B1 in bee pollen

The paper describes a sensitive and highly stable label-free electrochemical impedance immunosensor for the determination of aflatoxin B₁ (AFB₁), which is based on the formation of silica gel-ionic liquid biocompatible film on the glassy carbon electrode. The electrochemical performances of the sensor were investigated by electrochemical impedance spectroscopy using a Fe(CN)₆^3−/4− phosphate buffer solution as base solution for test. As new ionic liquid, 1-amyl-2,3-dimethylimidazolium hexafluorophosphate, offers a very biocompatible microenvironment for AFB₁ antibody, the sensor exhibits good repeatability (RSD = 1.2%), sensitive electrochemical impedance response to AFB₁ in the range of 0.1-10 ng ml⁻¹ and lowers the detection limit of AFB₁ (0.01 ng ml⁻¹)_. The electron-transfer resistance change of the sensor after and before incubation with AFB₁ of 2.0 ng ml⁻¹ can retain 95% over a 180-day storage period at 4 °C. The results present a remarkable improvement of sensitivity (2-fold) and long-term stability (190-fold) when compared to classical silica gel sensor. Moreover, proposed sensor has a high selectivity to AFB₁ alone with no significant response to AFB₂, AFG₁, AFG₂ and AFM₁ as single substrates, it has been successfully applied to the determination of trace AFB₁ in bee pollen samples with a spiked recovery in the range of 96.0-102.5%.     

Potentiometric analytical microsystem based on the integration of a gas-diffusion step for on-line ammonium determination in water recycling processes in manned space missions

The design, construction and evaluation of a versatile cyclic olefin copolymer (COC)-based continuous flow potentiometric microanalyzer to monitor the presence of ammonium ion in recycling water processes for future manned space missions is presented. The microsystem integrates microfluidics, a gas-diffusion module and a detection system in a single substrate. The gas-diffusion module was integrated by a hydrophobic polyvinylidene fluoride (PVDF) membrane. The potentiometric detection system is based on an all-solid state ammonium selective electrode and a screen-printed Ag/AgCl reference electrode. The analytical features provided by the analytical microsystem after the optimization process were a linear range from 0.15 to 500 mg L⁻¹ and a detection limit of 0.07 ± 0.01 mg L⁻¹. Nevertheless, the operational features can be easily adapted to other applications through the modification of the hydrodynamic variables of the microfluidic platform.     

Potentiometric membrane sensor based on 6-(4-nitrophenyl)-2,4-diphenyl-3,5-diaza-bicyclo[3.1.0]hex-2-ene for detection of Sn(II) in real samples

A Sn²⁺ ion-selective electrode which was prepared with a polymeric membrane based on 6-(4-nitrophenyl)-2,4-diphenyl-3,5-diaza-bicyclo[3.1.0]hex-2-ene (NDDBH) as a ionophore. Effects of experimental parameters such as membrane composition, nature and amount of plasticizer, the amount of additive and concentration of internal solution on the potential response of Sn²⁺ sensor were investigated. The electrode exhibited a Nernstian slope of 28.8 ± 1.1 mV/decade of Sn²⁺ over a concentration range of 1.0 × 10⁻⁵ to 1.0 × 10⁻¹ M of Sn²⁺ in an acidic solution (pH 1). The limit of detection was 4.0 × 10⁻⁶ M. The results show that this electrode can be used in ethanol media until 20% (v/v) concentration without interference. It can be used for more than 6 weeks without any considerable divergence in the potentials. The proposed membrane electrode revealed very good selectivity for Sn(II) ions over a wide variety of other cations and could be used in acidic media. The standard electrode potentials were determined at different temperatures and used to calculate the isothermal coefficient of the electrode. The stability constant (log K_s) of the Sn(II)-ionophore complex was determined at 25 °C by potentiometric titration in mixed aqueous solution. It was used as indicator electrode in potentiometric determination of Sn(II) ion in real samples.     

A novel approach based on ferricyanide-mediator immobilized in an ion-exchangeable biosensing film for the determination of biochemical oxygen demand

A novel biochemical oxygen demand (BOD) sensing method employing a ferricyanide (FC) mediator immobilized in an ion-exchangeable polysiloxane was developed. The ion-exchangeable polysiloxane containing alkylammonium groups (PAPS-Cl) was synthesized by sol-gel reaction of 3-(aminopropyl)trimethoxysilane (APTMOS) catalyzed by hydrochloric acid. FC was combined in PAPS-Cl via ion-association and the product was labeled as PAPS-FC, which was employed for a modified glassy carbon electrode. The apparent diffusion coefficient (D_app) of FC on the modified glassy carbon electrode was 9.8 × 10⁻¹¹ cm² s⁻¹. In a three-electrode system, a linear relationship between the anodic current responses and glucose/glutamate (GGA) concentration was obtained up to 40 mg O₂ L⁻¹ (r = 0.994) when the reaction mixture was incubated for 30 min. Single sensor and piece-to-piece reproducibility were less than 3.8 and 7.7%, respectively. The effects of dissolved oxygen, pH, temperature and co-existing substances on the BOD responses were studied. The sensor responses to nine pure organic substances were compared with the conventional BOD₅ method and other biosensor methods. Detection results of seawater samples were compared with those obtained from the BOD₅ method and showed a good correlation (r = 0.988).     

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1–1.0 × 10⁴ ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.     

A novel solid-state electrochemiluminescence detector for capillary electrophoresis based on tris(2,2'-bipyridyl)ruthenium(II) immobilized in Nafion/PTC-NH2 composite film

A new electrochemiluminescence (ECL) detector for capillary electrophoresis (CE) based on tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) immobilized in Nafion/PTC-NH₂ (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA)) composite film was presented for the first time. The Nafion/PTC-NH₂ composite film could effectively immobilize tris(2,2′-bipyridyl)ruthenium(II) via ion-exchange and electrostatic interaction. Cyclic voltammetric and ECL behavior of Nafion/PTC-NH₂/Ru composite film was investigated compared to Nafion/Ru composite. The Nafion/PTC-NH₂/Ru composite film exhibited good ECL stability and simple operability. Then the CE with solid-state ECL detector system was successfully used to detect sophora - a quinolizidine type - alkaloids as sophoridine (SR) and matrine (MT). The CE-ECL parameters that affected separation and detection were optimized. Under the optimized conditions, the linear range was from 2.5 × 10⁻⁸ to 2 × 10⁻⁶ mol/L for SR, 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ mol/L for MT. The detection limit (S/N = 3) was estimated to be 5 × 10⁻⁹ and 10⁻⁹ mol/L for SR and MT, respectively. It was shown that the CE coupling with solid-state ECL detector system exhibited satisfying sensitivity of analysis.     

Glucose biosensor based on immobilization of glucose oxidase in platinum nanoparticles/graphene/chitosan nanocomposite film

The bionanocomposite film consisting of glucose oxidase/Pt/functional graphene sheets/chitosan (GOD/Pt/FGS/chitosan) for glucose sensing is described. With the electrocatalytic synergy of FGS and Pt nanoparticles to hydrogen peroxide, a sensitive biosensor with a detection limit of 0.6 μM glucose was achieved. The biosensor also has good reproducibility, long-term stability and negligible interfering signals from ascorbic acid and uric acid comparing with the response to glucose. The large surface area and good electrical conductivity of graphene suggests that graphene is a potential candidate as a sensor material. The hybrid nanocomposite glucose sensor provides new opportunity for clinical diagnosis and point-of-care applications.     

A novel amperometric immunosensor based on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> magnetic nanoparticles/chitosan composite film for determination of ferritin

A novel amperometric immunosensor was developed by immobilizing ferritin antibody (FeAb) on the surface of Fe₃O₄ magnetic nanoparticles/chitosan composite film modified glassy carbon electrode (GCE). This material combined the advantages of inorganic Fe₃O₄ nanoparticles with the organic polymer chitosan. The stepwise assembly procedure of the immunosensor was characterized by means of differential pulse voltammetry (DPV) and ac impedance. The K₃Fe(CN)₆/K₄Fe(CN)₆ was used as a marker to probe the interface and to determinate ferritin. The factors that could influence the performance of the resulting immunosensor were studied in detail. After the immunosensor was incubated with ferritin for 32 min at 35 °C, the DPV current decreased linearly with the logarithm of ferritin concentration in the range from 20 to 500 ng mL⁻¹ with a correlation coefficient of 0.995 and a detection limit of 7.0 ng mL⁻¹. This immunosensor was used to analyze ferritin in human serum samples. The analytical results showed that the developed immunoassay was comparable with the radioimmunoassay (RIA), and the studied immunosensor exhibited good accuracy, high sensitivity, and long-term stability for 3 weeks, which implies a promising alternative approach for detecting ferritin in clinical diagnosis.     

Chemiluminescence behavior based on oxidation reaction of rhodamine B with cerium(IV) in sulfuric acid medium

The chemiluminescence (CL) of the rhodamine B (RhB)-cerium(IV) system was investigated by flow-injection. Rhodamine B was suggested to be a suitable chemiluminescent reagent in acidic conditions. When the concentration of rhodamine B was 100 mg l⁻¹ and cerium sulfate was 1.6 mmol l⁻¹ in sulfuric acid, the chemiluminescent intensity was found to be highest by using 0.3 mol l⁻¹ sulfuric acid as a carrier solution. The particular chemiluminescent system could tolerate such distinct acidic environments that it was utilized for detecting many compounds that are stable in acidic solutions. Furthermore, by virtue of IR, UV-Vis and luminescence spectroscopic measurements, the chemiluminescent behavior of rhodamine B was studied and a possible mechanism for this chemiluminescent reaction was proposed. The emitter was affirmed to be a radical species due to one of the oxidation products of RhB; the chemiluminescent emissive wavelength was about 425 nm.     

基于磁珠-适配体的高效液相色谱-串联质谱法检测食品中的黄曲霉毒素B1

本文构建了特异性识别黄曲霉毒素B₁(AFB₁)的磁珠-适配体,并与高效液相色谱-串联质谱联用(LC-MS/MS),建立食品中AFB₁的定量检测方法。 利用碳二亚胺盐酸盐(EDC)活化法,将羧基磁珠进行活化。 活化后的羧基磁珠与5'端氨基修饰的适配体进行孵育结合,通过酰胺反应将适配体共价连接在羧基磁珠表面,固定在磁珠表面的适配体作为捕捉探针将样品提取液中的AFB₁分离,通过LC-MS/MS对AFB₁进行定性和定量分析。 检测结果表明:AFB₁在浓度0.25～25 ng/mL呈良好的线性关系,相关系数R²=0.999,定量检出限为0.25 ng/mL,回收率达到80.3%～92.5%,相对标准偏差(RSD)低于8%。 该方法操作简单、快速便捷、可痕量地检测AFB₁,所制备的磁珠-适配体可重复利用,为定量检测AFB₁提供了另一种技术支持。     

Highly selective fluorescent probe based on a rhodamine B and furan-2-carbonyl chloride conjugate for detection of Fe3+ in cells

A furan-2-carbonyl chloride modified rhodamine B derivative (RBFC) has been designed and synthesized. The probe RBFC exhibited excellent sensitivity and selectivity for detection of Fe³⁺ with a 1:1 stoichiometry over other tested metal ions in a MeOH/H₂O (1:1, v/v, pH 7.36, HEPES buffer, 1.3?mM) solution. The association constant and the detection limit were calculated to be 7.28?×?10³?M^?1 and 0.437?μM based on fluorescence titration analysis. Furthermore, the probe RBFC was successfully applied in living cells, and the results indicated that the probe could monitor intracellular Fe³⁺ in MCF-7 cells.