Electrochemical Aptasensor with Layer‐by‐layer Deposited Polyaniline for Aflatoxin M1 Voltammetric Determination

Mycotoxins are highly toxic metabolites of some fungi that frequently contaminate water, food and feed and hence cause several human and animal diseases. In this work, a new approach to the fast and reliable determination of aflatoxin M1 (AFM1) in water and milk has been proposed with reagent free protocol of signal measurement. For this purpose, DNA aptamer selective to AFM1 was entrapped between two thin layers of polyaniline (PANI) electrodeposited on glassy carbon electrode. The incubation of the aptasensor in the AFM1 solution results in remarkable decrease of the PANI intrinsic activity monitored by direct current voltammetry or electrochemical impedance spectroscopy. Appropriate calibration curves were linear in the range from 3 to 90 ng/L with limit of detection (LOD) 1–5 ng/L depending on the measurement mode. Mechanism of signal generation involves shielding electrostatic interactions between the PANI and aptamer in the surface layer and variation of its redox activity attributed to the emeraldine form of PANI. Selectivity of the response was proved by similar experiments with aflatoxin B1 and ochratoxin A and by comparison of the results with those obtained with non‐specific aptamer in the sensing layer. Simple protocol for milk pretreatment has been proposed for reliable detection of AFM1 on the level of its threshold limited values (20 ng/L).     

Electrochemical Aptasensor Based on Polycarboxylic Macrocycle Modified with Neutral Red for Aflatoxin B1 Detection

Novel electrochemical aptasensors based on glassy carbon electrodes modified with electropolymerized Neutral red and polycarboxylated macrocyclic ligands onto which the DNA aptamers were covalently attached have been developed for detection of Aflatoxin B1 (AFB1). The interaction with an analyte resulted in the decrease of the cathodic peak current of the probe measured by CV and in the increase of the electron transfer resistance determined by EIS. The limit of detection was found to be 0.1 nM for CV and 0.05 nM for EIS methods, respectively. The aptasensor makes it possible to detect AFB1 in peanuts, cashew nuts, white wine and soy sauce with a recovery of 85–100 %.     

聚中性红膜修饰碳糊电极的电化学研究及其对维生素K3的检测

在一定温度下,以K2S2O8作引发剂,分别采用化学引发-电聚合和电引发-电聚合的方法制备了聚中性红膜修饰碳糊电极(PNR/CPE)。运用循环伏安法(CV)对修饰电极进行表征,并通过扫描电子显微镜(SEM)对聚合膜的表面形貌进行观察分析,推断了成膜的可能机理,探讨了最佳聚合条件。研究了维生素K3在该修饰电极上的电化学行为,并用差分脉冲伏安法(DPV)对其含量进行了测定。研究结果表明:经过化学引发后,中性红单体自由基增多,提高了成膜效率,此电极在NH3-NH4Cl溶液中对维生素K3有更好的电催化作用,其还原峰电流与浓度在2.0×10-6~1.2×10-4 mol/L范围内呈现出良好的线性关系,检出限为2.0×10-7 mol/L,回收率为94.7%~108.0%。     

Disposable Electrochemical Aptasensor for Ultrasensitive Determination of Aflatoxin B1 Using Copper Nanoparticles as Probes

An electrochemical aptasensor for aflatoxin B1 (AFB1) detection was constructed based on the copper nanoparticles (CuNPs) and gold nanoflowers modified screen-printed carbon electrodes as electrochemical probes and substrates, respectively. In the range of 100 aM to 100 pM, a good linear relationship between oxidation peak current of CuNPs and concentration of AFB1 was obtained. The high sensitivity could be ascribed to the amplified electrochemical signals by CuNPs. The high affinity of AFB1 with aptamer endowed its high selectivity. The above advantages and disposable traits made this aptasensor as an ideal platform for evaluation of AFB1 level in food samples.     

Electrochemical Aptasensor for the Determination of Ochratoxin A at the Au Electrode Modified with Ag Nanoparticles Decorated with Macrocyclic Ligand

An electrochemical aptasensor for ochratoxin A (OTA) detection has been developed on the base of a gold electrode covered with electropolymerized neutral red and silver nanoparticles obtained by chemical reduction with macrocyclic ligands bearing catechol fragments. Thiolated aptamers against OTA were covalently attached to silver nanoparticles via Ag? S bonding. The interaction with OTA induced the conformational switch of the aptamer, which caused increase of the charge transfer resistance measured by EIS in the presence of ferricyanide ions. The LOD achieved (0.05 nM) was comparable to other electrochemical aptasensors employing sophisticated assembling technique and enzyme amplification of the signal. The aptasensor was validated in spiked beer samples. The recovery of the OTA determination was found to be 66.3±14.1 % for light beer and 64.3±1.8 % for dark beer.     

一种简单灵敏的基于适配体的黄曲霉毒素B1电化学传感器

黄曲霉毒素B1（AFB1）以其高毒性和致癌性成为食品安全隐患而备受关注. 本文拟构建一种新颖、简单、快速、灵敏的传感器用于谷物食品中AFB1的痕量检测. 将介孔碳（CMK）修饰在工作电极表面来增大电极的表面积,再将工作电极恒电位沉积金纳米粒子（AuNPs）,提高电信号的同时,为下一步巯基化适配体的连接提供位点. 检测过程中,AFB1可以竞争性地去除吸附在适配体链上的亚甲基蓝（MB）引起电信号的变化,对AFB1进行定量检测. 修饰的工作电极导电性能得到改善,灵敏度大大提高,对AFB1的线性响应范围为0.1 ~ 75 μg·L^-1,检出限低至36 ng·L^-1. 在对不同谷物食品（大米、玉米、糯米）进行加标回收实验中,回收率在92.3% ~ 103.6%范围之间,实现对目标物的定量检测. 本文为食品中AFB1快速检测方法提供了一种新思路和新方法.     

A Novel Electrochemical Method for the Sensitive Determination of Aflatoxin B1 Using a Bivalent Binding Aptamer-cDNA Structure

In this study, a simple electrochemical sensing platform with the employment of a bivalent binding aptamer-cDNA probe (BBA-cDNA) structure is constructed for the detection of aflatoxin B₁ (AFB₁) as a mycotoxin. The BBA-cDNA structure is composed of two strands of aptamer (Apts) and their complementary strand (CS). Using a simple but accurate design, the presented measurement approach showed enhanced sensitivity and selectivity for AFB₁ detection with a LOD of 0.1 ng/mL. The approach presented in this study can be applied to the development of biosensors for the measurement of various toxins by substituting the proper aptamers and complementary strands.     

Impedimetric Aptasensors Based on Carbon Nanotubes – Poly(methylene blue) Composite

The effect of aptamer structure and immobilization platform on the efficiency of thrombin binding and its detection using electrochemical impedance spectroscopy (EIS) characteristics was investigated with aptasensors based on glassy carbon electrodes covered with multiwalled carbon nanotubes (MWNTs). Aptamers with one or two binding sequences GGTTGGTGTGGTTGG specific for thrombin and poly(dA) and poly(dT) tags able to form dimeric products (aptabodies) were used to establish significance of steric and electrostatic factors in aptasensor performance. We have shown that electropolymerization of methylene blue onto MWNTs significantly improved electrochemical characteristics and sensitivity of thrombin detection against bare MWNTs. Charge transfer resistance and capacitance of the surface layer were measured in the presence of redox probe [Fe(CN)₆]^3?/4?. Aptasensors make it possible to detect thrombin in the concentration range 1 nM–1 µM with the limit of detection of 0.7 nM (monitoring resistance changes) and 0.5 nM (capacitance changes), respectively.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Impedimetric DNA Sensor Based on Poly(proflavine) for Determination of Anthracycline Drugs

Electropolymerization is often used as a tool for immobilization of charged biopolymers and their electric wiring in the biosensor assembly. In this work, electropolymerization of proflavine has been for the first time used for the physical adsorption of DNA and measurement of anthracycline drugs (doxorubicin and daunorubicin) able to intercalate DNA. Redox properties of the proflavine polymers on the glassy carbon electrode and DNA deposition were characterized with cyclic voltammetry, scanning electron microscopy and electrochemical impedance spectroscopy. It was shown that DNA decreased the peak currents on voltammogram but increased the charge transfer resistance. The latter effect is pronounced with the following drug application. The impedimetric response regularly increased with the drugs concentration in the range 1 nM to 0.1 μM for doxorubicin and 1 pM–10 nM for daunorubicin (limit of detections 0.3 and 0.001 nM, respectively). The biosensor was tested on pharmaceutical preparations and spiked solution simulating the plasma electrolytes and possible interference of serum proteins.     

Development of Novel Glucose and Pyruvate Biosensors at Poly(Neutral Red) Modified Carbon Film Electrodes. Application to Natural Samples

Amperometric biosensors based on the corresponding oxidase enzyme with poly(neutral red) redox mediator have been developed for the determination of glucose and pyruvate. The enzymes have been immobilized on top of poly(neutral red) modified carbon film electrodes with glutaraldehyde as the cross‐linking agent. The biosensors were characterized by cyclic voltammetry and by electrochemical impedance spectroscopy. The glucose biosensor exhibited a linear response in the range 90 μM to 1.8 mM with a detection limit of 22 μM and the pyruvate biosensor in the range 90 to 600 μM with a detection limit of 34 μM. The relative standard deviations were found to be 2.1% (n=3) and 2.8% (n=4) respectively. The interference effects of various compounds were also studied. The glucose content of several types of wine and the amount of pyruvate in onion and garlic were determined and the results were compared with those obtained by standard spectrophotometric methods.     

Impedimetric Detection of DNA Damage with the Sensor Based on Silver Nanoparticles and Neutral Red

Novel electrochemical DNA‐sensor based on glassy carbon electrode (GCE) modified with Ag nanoparticles, Neutral red covalently attached to its surface and native DNA adsorbed on modifier coating was developed for the estimation of DNA damage on example of model system based on Fenton reagent. As was shown, the oxidation process resulted in synchronous increase of electron transfer resistance and capacitance measured by electrochemical impedance spectroscopy (EIS). The contribution of each sensor component on the signal was specified and sensitivity estimated against similar surface coatings. The shift of EIS parameters was found to be higher than that of similar biosensors reported. The DNA sensor was tested on the estimation of antioxidant capacity of green tea infusions again the results of coulometric titration with electrogenerated bromine.     

Electrochemical impedance spectroscopy of poly{pyrrole-trans-[(RuCl2(pmp)4]} copolymer films deposited on platinum electrodes

The present study focuses on the electronic and electrochemical features of a copolymer electrochemically grown from pyrrole and trans-[RuCl₂(pmp)₄] monomers, where pmp = 3-(pyrrol-1-ylmethyl)pyridine. The results from electrochemical impedance spectroscopy analyses revealed the redox behavior of the poly{pyrrole-trans-[RuCl₂(pmp)₄]} compound as well as the non-homogeneous nature of the extremely thin polymeric layers. An equivalent circuit is proposed for an optimized film produced under the scope of the work. Received: 4 May 1998 / Accepted: 24 August 1998     

Electroconductive Hydrogels: Electrical and Electrochemical Properties of Polypyrrole‐Poly(HEMA) Composites

Composites of inherently conductive polypyrrole (PPy) within highly hydrophilic poly(2‐hydroxyethyl methacrylate)‐based hydrogels (p(HEMA)) have been fabricated and their electrochemical properties investigated. The electrochemical characteristics observed by cyclic voltammetry suggest less facile reduction of PPy within the composite hydrogel compared to electropolymerized PPy, as shown by the shift in the reduction peak potential from ?472 mV for electropolymerized polypyrrole to ?636 mV for the electroconductive composite gel. The network impedance magnitude for the electroconductive hydrogel remains quite low, ca. 100 Ω, even upon approach to DC, over all frequencies and at all offset potentials suggesting retained electronic (bipolaronic) conductivity within the composite. In contrast, sustained application of +0.7 V (vs. Ag/AgCl, 3 M Cl^?) for typically 100 min. (conditioning) to reduce the background amperometric current to <1.0 μA, resulted in complete loss of electroactivity. Nyquist plots suggest that sustained application of such a modest potential to the composite hydrogel results in impedance characteristics that resembles p(HEMA) without evidence of the conducting polymer component. PPy composite gels supported a larger ferrocene monocarboxylate diffusivity (D_appt=7.97×10^?5 cm² s^?1) compared to electropolymerized PPy (D_appt=5.56×10^?5 cm² s^?1), however a marked reduction in diffusivity (D_appt=1.01×10^?5 cm² s^?1) was observed with the conditioned hydrogel composite. Cyclic voltammograms in buffer containing H₂O₂ showed an absence of redox peaks for electrodes coated with PPy‐containing membranes, suggesting possible chemical oxidation of polypyrrole by the oxidant     

基于酶催化沉积放大的电化学阻抗法用于人免疫球蛋白M的检测

提出了一种基于酶催化沉积放大的电化学免疫分析方法。先通过免疫夹心反应,将酶标抗体(羊抗人IgM-HRP)固定到电极表面。通过HRP催化双氧水氧化3,3′-二氨基联苯胺在电极表面形成不溶性沉积物,从而放大电化学检测信号。实验通过蛋白A实现抗体的定向固定。考察了包被抗体的浓度和包被溶液的pH对抗体固定的影响,以及酶标抗体的用量和沉积时间对免疫分析的影响。传感器信号响应与人免疫球蛋白M(IgM)浓度在2.1~670μg/L范围内具有良好的线性关系;检出限达0.08μg/L。     

DNA Electrochemical Aptasensor for Detecting Fumonisins B1 Based on Graphene and Thionine Nanocomposite

In this paper, a novel aptasensor was designed by with the dual amplification of Au nanoparticles (AuNPs) and graphene/thionine nanocomposites (GS‐TH) for sensitive determination of fumonisins B₁ (FB₁). AuNPs is modified at the electrode surface to increase the electrical conductivity and fabricate specific recognition interface for FB₁ through the hybridization of capture DNA and its aptamer. Large number of TH molecules were loaded at the surface of graphene sheet to served as electrochemical probe and increase its electrochemical signal due to the excellent conductivity and large surface area of graphene sheet. This type of nanocomposites is then assembled to the single strand section of FB₁ aptamer at electrode surface by π–π stacking interactions between them, leading to an enhanced electrochemical signal. After the specific combination between FB₁ aptamer and its target (FB₁) in solution, GS–TH was released from electrode surface, resulting in a decreased electrochemical signal. The result demonstrated that the decreased currents were proportional to the FB₁ concentration in the range of 1–10⁶ pg/mL with a detection limit of 1 pg/mL. Besides, the developed aptasensor was also applied successfully for the determination of FB₁ in feed samples. The result shows this aptasensor has a higher sensitivity and selectivity.     

An Electrochemical Impedance Immunosensor Based on Gold Nanoparticle‐Modified Electrodes for the Detection of HbA1c in Human Blood

A label‐free immunosensor for the detection of HbA1c was developed based on gold nanoparticle (AuNP)‐aryl diazonium salt modified glassy carbon (GC) electrode where transduction is achieved using electrochemical impedance spectroscopy (EIS). GC electrodes were first modified with 4‐aminophenyl (Ph‐NH₂) layers to which AuNPs were attached. Thereafter an oligo(ethylene glycol) (OEG‐COOH) species were covalently attached to the remaining free amine groups on the Ph‐NH₂ surface. The AuNP surfaces were further modified with Ph‐NH₂ followed by attachment of a glycosylated pentapeptide (GPP), an analogon to HbA1c. Exposure of this interface to anti‐HbA1c IgG resulted in a change in charge transfer resistance (R_ct) due to the anti‐HbA1c IgG selectively complexing to the surface bound GPP. To detect the amount of HbA1c, a competitive inhibition assay was employed where the surface bound GPP and HbA1c in solution compete for the anti‐HbA1c IgG antibodies. The higher the concentration of HbA1c, the less antibody binds to the sensing interface and the lower the change of R_ct. The response of the immunosensor is linear with the HbA1c% of total haemoglobin in the range of 0%–23.3%. This competitive inhibition assay can be used for the detection of HbA1c in human blood. The performance of the immunosensor for detection of HbA1c in human blood is comparable to the clinical laboratory method.     

Electrochemical Sensor Based on Gold Nanoparticles Stabilized in Poly(Allylamine hydrochloride) for Determination of Vanillin

Gold nanoparticles stabilized in poly(allylamine hydrochloride) (AuNP‐PAH) were synthesized, characterized and applied in the development of a new sensor for the determination of vanillin by square‐wave voltammetry. Under optimized conditions, the calibration curve showed a linear range for vanillin of 0.90 to 15.0 µmol L^?1, with a limit of detection of 55 nmol L^?1. The sensor demonstrated acceptable selectivity and stability, as well as good intra‐day and inter‐day repeatability and electrode‐to‐electrode repeatability (with relative standard deviations of 3.5, 4.5 and 3.9 %, respectively). The sensor was successfully applied in the determination of vanillin in different commercial samples.     

A Highly Sensitive Electrochemical Impedance Spectroscopy Immunosensor for Determination of 1‐Pyrenebutyric Acid Based on the Bifunctionality of Nafion/Gold Nanoparticles Composite Electrode

A simple, highly sensitive and label‐free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano‐Au/Nafion) composites for the determination of 1‐pyrenebutyric acid (PBA). Under the optimal conditions, the amount of immobilized antibody was significantly improved on the nano‐Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano‐Au/Nafion composite electrode for PBA detection were much better than those of nano‐Au modified glassy carbon electrode (nano‐Au/GCE). The plot of increased electron transfer resistances (R_ets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng·mL^?1 with the detection limit of 0.03 ng·mL^?1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.     

AC Impedance Studies on the Relationship between the Fractal Characteristics and Electrochemical Properties of Poly(Para‐Phenylene) Film

The effects of experimental conditions on the fractal structure of electrosynthetic polyparaphenylene films were studied by electrochemical impedance spectroscopy. The results indicate that, at the potential range (0‐0.6 V), the film surface fractal dimension rises while the corresponding charge transfer resistances R_ct decrease as charge increases. This is consistent with the oxidized behavior of the conducting polymer. As for the degradation of electrolytes, scanning electron microscopy observations of poly(para‐pheneylene) (PPP) film provide evidence of the close relationship between the degradation of electrolytes and the film morphology, also in good agreement with the electrochemical impedance results interpretation. The X‐ray photoelectron spectroscopic analysis results present that the relative oxygen content ratio (O/C) of the films increases with the times the electrolyte is used, which reveals that the degradation of the electrolyte may result in a compact and passivation PPP film.