Electrochemical Aptasensor Based on ZnO Modified Gold Electrode

We developed an electrochemical thrombin aptasensor based on ZnO nanorods functionalized by electrostatically adsorption of 30‐mer DNA aptamers. The sensor surface was characterized by AFM and SEM. The surface layer assembling was optimized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with ferricyanide ions as redox markers. The peak current of the ferricyanide and the charge transfer resistance gradually decreased with increasing concentration of thrombin in the range from 3 pM to 100 nM due to formation of aptamer‐thrombin complexes and slower diffusion of the marker ions through the surface layer. At optimal conditions, a limit of detection (LOD) of 3 pM for EIS measurements and 10 pM for CV response was calculated from the S/N=3.     

Electrochemical Aptasensor Based on Poly(Neutral Red) and Carboxylated Pillar[5]arene for Sensitive Determination of Aflatoxin M1

Aptasensor for highly sensitive determination of aflatoxin M1 (AFM1) was developed on the base of glassy carbon electrode (GCE) covered with polymeric Neutral red (NR) dye obtained by electropolymerization in the presence of polycarboxylated pillar[5]arene derivative. Aptamer against AFM1 and NR label were then covalently linked to the carboxylic groups of the carrier by carbodiimide binding. At presence of AFM1 the cathodic peak current related to the NR conversion decreases. AFM1 induced also an increase of the charge transfer resistance measured by electrochemical impedance spectroscopy. In optimal conditions, this make it possible to determine from 5 to 120 ng/L AFM1 in standard solutions with limit of detection (LOD) of 0.5 ng/L. The aptasensor was validated on the spiked samples of cow and sheep milk as well as in kefir after their methanol dilution. Reliable detection of the 40–160 ng/kg of mycotoxins was reached. This is below limited threshold value (50 μg/kg) established in EC.     

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).     

基于石墨烯化学修饰电极的适体传感器

采用石墨烯(RGO)作载体,凝血酶适体(TBA)作探针,凝血酶为目标蛋白,电化学阻抗谱(EIS)为检测技术,建立了检测蛋白质的新方法。由于RGO可增大电极有效表面积并提高电极表面电子传输速率以及TBA的特异性识别能力,此方法具有较高的灵敏度和良好的选择性。采用本方法检测凝血酶的线性范围为0.3～10 fmol/L,检出限为0.26 fmol/L。本研究将RGO应用于电化学适体传感器,证实了RGO修饰电极在电化学适体传感器领域中潜在的应用价值。     

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.     

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.     

Impedimetric Aptasensor Based on Disposable Graphite Electrodes Developed for Thrombin Detection

The impedimetric aptasensor for Thrombin (THR) was developed for the first time herein by measuring changes at the charge‐transfer resistance, R_ct upon to protein? aptamer complex formation. After covalent activation of pencil graphite electrode (PGE) surface using covalent agents, amino linked aptamer (APT) was immobilized onto activated PGE surface. Then APT‐THR interaction was explored by electrochemical impedance spectroscopy (EIS). After the optimization of experimental conditions (e.g., APT and THR concentration, immobilization and interaction times), the selectivity of impedimetric aptasensor was tested in the presence of other biomolecules: factor Va and bovine serum albumine (BSA) both in buffer media, or in diluted fetal bovine serum (FBS).     

Electrochemical Immunosensor Made with Zein-based Nanofibers for On-site Detection of Aflatoxin B1

A disposable electrochemical immunosensor for on-site detection of aflatoxin B1(AFB1), one of the most toxic mycotoxins in agri-food products, was fabricated through a low-cost cut-printing method and then modified with zein/polypyrrole(PPy) electrospun nanofibers onto which anti-AFB1 monoclonal antibodies were immobilized covalently. Fabrication was possible with an innovative and simple approach to adsorb nanofibers onto the working electrode during electrospinning. Electrochemical impedance spectroscopy was employed as the principle of detection, and the data collected with a portable potentiostat were treated with information visualization techniques. The nanostructured immunosensor showed a high sensitivity for AFB1 with a linear detection range from 0.25 to 10 ng mL⁻¹ and a theoretical limit of detection of 0.092 ng mL⁻¹, which is adequate to detect AFB1 in food, according to regulatory agencies.     

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.     

基于纳米ZnO/壳聚糖复合膜DNA电化学传感器用于HIV基因的免标记检测

以室温固相合成法制备纳米ZnO,通过壳聚糖(CHIT)的成膜效应将纳米ZnO固定在玻碳电极(GCE)表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。DNA的固定和杂交通过电化学交流阻抗进行表征。以电化学交流阻抗免标记法检测目标DNA,固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10-11~2.0×10-6mol/L,检出限为2.0×10-12mol/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.     

Polyphenothiazine Modified Electrochemical Aptasensor for Detection of Human α‐Thrombin

QCM aptasensor for detection human thrombin has been developed on the base of polymeric forms of phenothiazine dyes, Methylene Blue and Methylene Green. Electrostatic accumulation of the analyte in the polyphenothiazine layer made it possible to increase the sensitivity of QCM detection of thrombin in comparison with bare gold electrodes coated with avidin or neutravidin. The influence of nonspecific binding of human serum albumin and the optimal composition of the surface layers were determined. The aptasensors developed make it possible to detect 10–100 nM of thrombin.     

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.     

Label‐Free Electrochemical Aptasensor for Determination of Chloramphenicol Based on Gold Nanocubes‐Modified Screen‐Printed Gold Electrode

An ultrasensitive label‐free electrochemical aptasensor was developed for selective detection of chloramphenicol (CAP). The aptasensor was made using screen‐printed gold electrode modified with synthesized gold nanocube/cysteine. The interactions of CAP with aptamer were studied by cyclic voltammetry, square wave voltammetry (SWV) and electrochemical impedance spectroscopy. Under optimized conditions, two linear calibration curves were obtained for CAP determination using SWV technique, from 0.03 to 0.10 µM and 0.25–6.0 µM with a detection limit of 4.0 nM. The aptasensor has the advantages of good selectivity and stability and applied to the determination of CAP in human blood serum sample.     

A Simple Structure-Switch Aptasensor Using Label-Free Aptamer for Fluorescence Detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is one of the mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, and it causes contamination in foods and great risk to human health. Simple sensitive detection of AFB1 is important and demanded for food safety and quality control. Aptamers can specifically bind to targets with high affinity, showing advantages in affinity assays and biosensors. We reported an aptamer structure-switch for fluorescent detection of aflatoxin B1 (AFB1), using a label-free aptamer, a fluorescein (FAM)-labeled complementary strand (FDNA), and a quencher (BHQ1)-labeled complementary strand (QDNA). When AFB1 is absent, these three strands assemble into a duplex DNA structure through DNA hybridization, making FAM close to BHQ1, and fluorescence quenching occurs. In the presence of AFB1, the aptamer binds with AFB1, instead of hybridizing with QDNA. Thus, FAM is apart from BHQ1, and fluorescence increases with the addition of AFB1. This assay allowed detection of AFB1 with a detection limit of 61 pM AFB1 and a dynamic concentration range of 61 pM to 4 μM. This aptamer-based method enabled detection of AFB1 in complex sample matrix (e.g., beer and corn flour samples).     

Development of an Electrochemical Biosensor for the Rapid Detection of Carbofuran Based on Air Stable Lipid Films with Incorporated Calix[4]arene Phosphoryl Receptor

This work describes the preparation of a selective receptor for the rapid, selective and sensitive electrochemical flow injection analysis of carbofuran in foods using air stable lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated artificial receptor. The selective receptor was synthesized by transformation of the ? OH groups of resorcin[4]arene receptor into phosphoryl groups. These lipid films were supported on a methylacrylate polymer (i.e., methacrylic acid was the functional monomer for the polymerization, ethylene glycol dimethacrylate was used as the crosslinker and 2,2′‐azobis‐2‐methylpropionitrile as an initiator). A minisensor device was constructed for the electrochemical flow injection analysis of toxicants based on air stabilized lipid films supported on a polymer. The device can sense the analyte in a drop (50 μL) of sample. Carbofuran was injected into flowing streams of a carrier electrolyte solution. A host‐guest complex formation between the calix[4]arene phosphoryl receptor and carbofuran takes place through hydrogen bonding. This enhances the preconcentration of carbofuran at the lipid membrane surface which in turn causes dynamic alterations of the electrostatic fields and phase structure of membranes; as a result ion current transients were obtained and the magnitude of these signals was correlated to the substrate concentration. The response times were ca. 80 s and carbofuran was determined at concentration levels of nM. The effect of potent interferences included a wide range of compounds and other insecticides. The effect of interference of proteins and lipids was also examined. The reproducibility of the method was checked by recovery experiments in fruit and vegetable samples with satisfactory results.     

Impedimetric Aptasensor for Thrombin Recognition Based on CD Support

We report an aptamer biosensing array for thrombin detection by measuring the electrochemical impedance upon aptamer‐protein formation at the surface of CD‐trodes (GCDTs) in the presence of the redox couple [Fe(CN)₆]^3?/4?. GCDTs are fabricated from recordable compact discs that contain a fine gold layer. The biosensor is constructed by self‐assembling of a thiol‐modified thrombin binding aptamer (TBA) onto a GCDT surface. The sensor reveals good ligand specificity, recognition in a wide range of thrombin concentrations from 20 nM to 1 µM with a limit of detection of 5 nM.     

Electrochemical Properties of Multilayered Coatings Implementing Thiacalix[4]arenes with Oligolactic Fragments and DNA

Layer‐by‐layer assembling of polyelectrolyte complexes on glassy carbon electrode utilizing derivatives of p‐tert‐butylthiacalix[4]arene modified with oligolactic acid in cone, partial cone and 1,3‐alternate configurations has been performed and characterized by atomic force microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. Structure, surface morphology and permeability of the coatings depended on the number and nature of individual layers. Configuration of macrocyclic core influenced the charge distribution and flexibility of polyelectrolytes and the electrochemical properties of the coatings. Partial substitution of oligolactic derivatives with the DNA molecules allowed distinguishing native, thermally and oxidatively damaged DNA by ferricyanide ion signals. The relationships between the coating structure and the content can be further used for development of electrochemical sensors devoted to the detection of specific DNA interactions.     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

Fe3O4 Nanorods-RGO-ionic Liquid Nanocomposite Based Electrochemical Sensor for Aflatoxin B1 in Ground Paprika

A novel and sensitive method for the determination of aflatoxin B1 (AFA−B1) in ground paprika using a methyltrioctylammonium chloride ionic liquid (IL), iron oxide nanorods (Fe₃O₄ nanorods) and reduced graphene oxide (RGO) fabricated glassy carbon electrode (GCE) was developed. The synthesized nanoparticles, nanocomposites and modified electrode surfaces were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermogravimetric analysis (TGA/DSC) and x-ray diffraction (XRD) analyses. Moreover, the electrochemical performance of the developed sensor was determined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The obtained results demonstrate that the sensitivity of AFA−B1 is significantly enhanced on RGO-Fe₃O₄ nanorods-IL-GCE in comparison with bare GCE, RGO-GCE and RGO-Fe₃O₄ nanorods-GCE. The redox peak currents of AFA−B1 exhibited good linear relationship with its concentration in the range from 0.02 to 0.33 ng mL⁻¹ with detection limit of (LOD) 0.03 ng mL⁻¹ and limit of quantification (LOQ) 0.36 ng mL⁻¹ respectively (S/N=3). In addition, the fabricated electrode showed good stability and reproducibility. The proposed technique was effectively applied to identify the AFA−B1 in real ground paprika samples with acceptable results.