Label‐free Electrochemical Aptasensor for Carcino‐embryonic Antigen Based on Ternary Nanocomposite of Gold Nanoparticles,Hemin and Graphene

In this report, a label‐free electrochemical aptasensor for carcino‐embryonic antigen (CEA) was successfully developed based on a ternary nanocomposite of gold nanoparticles, hemin and graphene nanosheets (AuNPs‐HGNs). This nanocomposite was prepared by decorating gold nanoparticles on the surface of hemin functionalized graphene nanosheets via a simple wet‐chemical strategy. The aptamer can be assembled on the surface of AuNPs‐HGNs/GCE (glassy carbon electrode) through Au‐S covalent bond to form the sensing interface. Hemin absorbed on the graphene nanosheets not only acts as a protective agent of graphene sheets, but also as an in situ probe base on its excellent redox properties. Gold nanoparticles provide with both numerous binding sites for loading CEA binding aptamer (CBA) and good conductivity to promote the electron transfer. The current changes, which are caused by CEA specifically binding on the modified electrode, are exploited for the label‐free detection of CEA in a very rapid and convenient protocol. Therefore, the method has advantages of high sensitivity, wide linear range (0.0001–10 ng mL^?1), low detection limit (40 fg mL^?1) and attractive specificity. The results illustrate that the proposed label‐free electrochemical aptasensor has a potential application in the biological or clinical target analysis for its simple operation and low cost.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

Photoelectrochemical immunoassay for microcystin-LR based on a fluorine-doped tin oxide glass electrode modified with a CdS-graphene composite

We report on a sensitive electrochemical aptasensor for the detection of human prostate specific antigen (PSA). It is based on the signal amplification of the biotin-avidin system using a sensing platform that is making use of a graphite electrode modified with gold nanoparticles that were covered with graphitized mesoporous carbon nanoparticles (AuNPs@GMCs). The AuNPs@GMCs hybrid was prepared by linking 1,6-hexanedithiol-functionalized GMCs and gold nanoparticles via Au-S groups. Then, streptavidin was immobilized on the electrode modified with the AuNPs@GMCs so to enlarge the amount of biotin-aptamer which led to enhanced detection sensitivity. If an PSA aptamer captures the target PSA on the electrode, the differential pulse voltammetric (DPV) signal of the hexacyanoferrate redox system decreases. Factors affecting the performance of the aptasensor were studied in detail. Under optimal conditions, the DPV signal changes could be used to quantitatively detect PSA in the concentration range from 0.25 to 200?ng?mL^?1, with a lowest limit of detection as small as 0.25?ng?mL^?1. The aptasensor is highly specific and displays acceptable precision, good stability and repeatability.     

Direct and Rapid Detection of Diphtherotoxin via Potentiometric Immunosensor Based on Nanoparticles Mixture and Polyvinyl Butyral as Matrixes

In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D‐Ag) has been developed by means of self‐assembly of monoclonal diphtheria antibody (D‐Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D‐Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol‐gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen‐antibody reaction in a phosphate buffer solution (pH 7.0). The immobilized D‐Ab exhibited direct potentiometric response toward D‐Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long‐term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0×10^?3 to 1.2 μg?mL^?1 with a detection limit of 1.1×10^?3 μg?mL^?1. Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2 mol?L^?1 glycine‐hydrochloric acid (Gly‐HCl) buffer solution and 0.25 mol?L^?1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis.     

Development of a Creatinine Sensor Based on a Molecularly Imprinted Polymer‐Modified Sol‐Gel Film on Graphite Electrode

An electrochemical creatinine sensor based on a molecularly imprinted polymer (MIP)‐modified sol‐gel film on graphite electrode was developed. The surface coating of MIP over sol‐gel was advantageous to obtain a porous film with outwardly exposed MIP cavities for unhindered selective rebinding of creatinine from aqueous and biological samples. A fast differential pulse, cathodic stripping voltammetric response of creatinine can be obtained after being preanodized the sensor in neutral medium containing appropriate amount of creatinine at +1.8 V versus SCE for 120 s. A linear response over creatinine concentration in the range of 1.23 to 100 μg mL^?1 was exhibited with a detection limit of 0.37 μg mL^?1 (S/N=3).     

Voltammetric Immunoassay for the Detection of Protein Biomarkers

A strategy for a fast (ca. 20 min), specific, electrochemical immunoassay for the cardiac biomarker creatine kinase (CK) and the human cytokine interleukin 10 (IL10) has been developed in this paper. The polyaniline modified gold surface formed from electrochemical reduction of diazonium salt supplies a solid substrate to link the activated carboxylic acid groups from the antibodies, which were labelled with ferrocene. The direct electrochemistry of ferrocene allows the analysis of protein markers with good sensitivity. The creatine kinase sensor demonstrates limit of detection of 0.5 pg mL^?1 in a physiological Krebs‐Henseleit solution. The anti‐IL10 antibody retained fluorescence activity after further coupling to ferrocene and covalent immobilization on to a gold electrode, showing a linear detection range for IL‐10 from 0.001 ng mL^?1 to 50 ng mL^?1 in PBS. We attribute the high sensitivity to the well‐controlled modified surface which results in end–on antibodies that can specifically capture the antigen with ease.     

First Electroanalytical Methodology for the Determination of Hordenine in Dietary Supplements using a Boron‐doped Diamond Electrode

The present work describes the first electrochemical investigation and a simple, rapid and modification‐free electroanalytical methodology for quantification of hordenine (a potent phenylethylamine alkaloid) using a boron‐doped diamond electrode. At optimized square‐wave voltammetric parameters, the observed oxidation peak current in 0.1 M HClO₄ at +1.33 V (vs. Ag/AgCl) increased linearly from 5.0 to 100 μg mL^?1 (3.0×10^?5–6.1×10^?4 M), with detection limit of 1.3 μg mL^?1 (7.8×10^?6 M). The applicability of the developed method was tested with the determination of hordenine in the commercial dietary supplement formulations.     

Beta‐lactoglobulin Electrochemical Detection Based with an Innovative Platform Based on Composite Polymer

In this work, we present a new electrochemical disposable platform based on poly(aniline‐co‐anthranilic acid) (PANI/PAA) composite polymer coupled with an aptamer for sensitive detection of β‐lactoglobulin. Firstly, PANI/PAA film was electrodeposited on the graphite screen‐printed electrode surface by cyclic voltammetry. The co‐polymer modified electrode was then employed as platform for the covalent immobilization of an amino‐modified aptamer. Various β‐lactoglobulin solutions, with a fixed amount of biotinylated oligonucleotide complementary sequence, were dropped onto the aptasensor surface. A streptavidin‐alkaline phosphatase conjugate was then employed to trace the affinity reaction. After the addition of 1‐naphthyl‐phosphate enzymatic substrate, 1‐naphthol electroactive product was detected by differential pulse voltammetry. A decrease in the signal was obtained when the target concentration was increased, in according to a signal‐off approach. After optimization of key experimental parameters, a dose‐response curve was obtained between 0.01–1.0 μg mL^?1 β‐lactoglobulin concentration range. The limit of detection of 0.053 μg L^?1 was obtained. Milk samples spiked with β‐lactoglobulin were analyzed.     

Electrochemical Detection of Vascular Endothelial Growth Factor by Molecularly Imprinted Polymer

In this study, we report the development of a sensitive label‐free impedimetric sensor based on molecularly imprinted polymer (MIP) as biomimetic receptor coupled with screen‐printed electrodes (SPEs) for the detection of vascular endothelial growth factor (VEGF). Firstly, electropolymerization of o‐phenylenediamine (o‐PD) in the presence of VEGF molecule by cyclic voltammetry was performed onto graphite screen‐printed electrodes. The solvent extraction of the target was then carried out. The MIP based sensor was characterized by electrochemical techniques and scanning electron microscopy (SEM). Using optimized experimental conditions, the single‐use MIP‐based sensor showed a good analytical performance for VEGF detection from 20 to 200 pg mL^?1 with limit of detection of 0.08 pg mL^?1. Finally, the developed MIP‐based sensor in human serum samples was also tested.     

Simultaneous determination of fifteen multiclass organic pollutants in human saliva and serum by liquid chromatography–tandem ultraviolet/fluorescence detection: A validated method

A quick and inexpensive validated method, based on sample treatment by liquid–liquid microextraction followed by liquid chromatography (LC) coupled with ultraviolet tandem fluorescence detection is proposed for the determination of 15 multiclass pollutants both in serum and in saliva, as a simple and easy to draw matrix. The method was set up and validated according to European guidelines. The compounds of interest include some endocrine‐disrupting chemicals (i.e. bisphenol A, bisphenol B, bisphenol E, bisphenol F, bisphenol AF, bisphenol A diglycidyl ether, bisphenol M, diethylhexyl phthalate, monoethylhexyl phthalate, triclosan and 4‐nonylphenol), as well as other pollutants belonging to the class of volatile organic compounds (2‐chlorophenol, 1,2 dichlorobenzene, 1,2,4,5‐tetrachlorobenzene). The limits of quantifications ranged from 2.28 × 10^?3 μg mL^?1 (bisphenol A diglycidyl ether) to 6.29 μg mL^?1 (diethylhexyl phthalate), while those of detection ranged from 0.068 × 10^?3 μg mL^?1 (bisphenol A diglycidyl ether) to 1.031 μg mL^?1 (diethylhexyl phthalate). To test method suitability, it was applied to real saliva and serum samples of healthy human volunteers and was found to meet the demands of the laboratories handling simple and relatively inexpensive equipment for screening oriented at rapid and reliable contamination assessment of a population.     

[Ru(bpy)2(dcbpy)NHS] Labeling/Aptamer‐Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)₂(dcbpy)NHS] labeling/aptamer‐based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)₂(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)₃]²⁺ biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0×10^?13–1.0×10^?8 mol L^?1 with a detection limit of 1.0×10^?13 mol L^?1. This strategy for the aptasensor opens a rapid, selective, and sensitive route for the detection of lysozyme and potentially other proteins.     

Electrochemical impedance spectroscopy detection of lysozyme based on electrodeposited gold nanoparticles

A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) aptasensor based on an anti-lysozyme-aptamer as a molecular recognition element, was developed for the detection of lysozyme. Improvement in sensitivity was achieved by utilizing gold nanoparticles (AuNPs), which were electrodeposited onto the surface of a gold electrode, as a platform for immobilization of the aptamer. To quantify the amount of lysozyme, changes in the interfacial electron transfer resistance (R_et) of the aptasensor were monitored using the redox couple of an [Fe(CN)₆]^3−/4− probe. The R_et increased with lysozyme concentration. The plot of R_et against the logarithm of lysozyme concentration is linear over the range from 0.1 pM to 500 pM with a detection limit of 0.01 pM. The aptasensor also showed good selectivity for lysozyme without being affected by the presence of other proteins.     

Self‐Assembled Monolayer of L‐Cysteine on a Gold Electrode as a Support for Fatty Acid. Application to the Electroanalytical Determination of Unsaturated Fatty Acid

This article describes the formation of a SAM with chemisorbed cysteine to a gold surface by the thiol group to obtain a surface electrode with an amino and an acid group free for later reaction and accumulation with other molecules on the electrode surface. We explore the accumulation of unsaturated fatty acid and the electrochemical response of the electrode after modification with cysteine. The electrochemical study confirmed the accumulation of linoleic acid on the modified electrode. The optimum conditions for the determination of linoleic acid by differential pulse voltammetry of linoleic acid are studied a detection limit (3σ) of 0.03 μg mL^?1 and a determination limit (10σ) of 0.10 μg mL^?1 were obtained and applied to determination in olive oil and ham from Iberian breed hams.     

Impedimetric aptasensor for Staphylococcus aureus based on nanocomposite prepared from reduced graphene oxide and gold nanoparticles

We describe here an aptasensor for the ultrasensitive detection of Staphylococcus aureus by electrochemical impedance spectroscopy (EIS). Single-stranded DNA was linked to a nanocomposite prepared from reduced graphene oxide (rGO) and gold nanoparticles (AuNP). Thiolated ssDNA was covalently linked to the AuNPs linked to rGO, and probe DNA was immobilized on the surface of an AuNP-modified glassy carbon electrode to capture and concentrate Staph. aureus. The probe DNA of the aptasensor selectively captures the target bacteria in its three-dimensional space, and these results in a dramatic increase in impedance. Scanning electron microscopy, cyclic voltammetry and EIS were used to monitor the single steps of the electrode assembly process. The effect was utilized to quantify the bacteria in the concentration range from 10 to 10⁶ cfu mL^?1 and with a detection limit of 10 cfu mL^?1 (S/N?=?3). The relative standard deviation of Staphylococcus aureus detection was equal to 4.3 % (10⁵ cfu mL^?1, n?=?7). In addition to its sensitivity, the biosensor exhibits high selectivity over other pathogens.

Aptamer-based Biosensor for Detection of Phenylalanine at Physiological pH

A simple, sensitive aptamer-based biosensor for the detection of phenylalanine is developed using the electrochemical transduction method. For this proposed aptasensor, a 5-thiol-terminated aptamer is covalently attached onto a gold electrode. At the first time, the electrode was evaluated as an electrochemical aptasensor for determination of phenylalanine in aqueous solutions. This sensor was tested in a Tris–HCl buffer with physiological pH?=?7.4 by cyclic voltammetry and differential pulse voltammetry. The detection limit and sensitivity of the modified electrode toward phenylalanine were estimated to be 1 nM (S/N?=?3) and 0.367 μA nM^?1, respectively. The linear range of the signal was observed between 1 and 10 nM of phenylalanine with 0.9914 correlation factor. The herein-described approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.     

Ultrasensitive Electrochemical Immunosensor for Detection of Tumor Necrosis Factor‐α Based on Functionalized MWCNT‐Gold Nanoparticle/Ionic Liquid Nanocomposite

A novel and highly sensitive electrochemical immunosensor was developed for the detection of protein biomarker tumor necrosis factor‐alpha (TNF‐α) based on immobilization of TNF‐α‐antibody (anti‐TNF‐α) onto robust nanocomposite containing gold nanoparticles (AuNP), multiwalled carbon nanotubes (MWCNTs) and ionic liquid (1‐buthyl‐3‐methylimidazolium bis (trifluoromethyl sulfonyl)imide). Functionalized MWCNT‐gold nanoparticle was produced by one‐step synthesis based on the direct redox reaction. The electrochemical properties of nanocomposite were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The anti‐TNF‐α was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti‐TNF‐α labeled with horseradish peroxidase as secondary antibody. Under optimum conditions, the immunosensor could detect TNF‐α in a linear range from 6.0 to 100 pg mL^?1 with a low detection limit of 2.0 pg mL^?1. The simple fabrication method, high sensitivity, good reproducibility, stability, as well as acceptable accuracy for TNF‐α detection in human serum samples are the main advantages of this immunosensor, which might have broad applications in protein diagnostics and bioassay.     

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.     

A Novel Label Free Electrochemical Magnetoimmunosensor for Human Interleukin‐6 Quantification in Serum

A novel magnetoimmunosensor, designed for sensitive and selective quantification of interleukin 6, is herein reported. The experimental design involves the covalent immobilization of anti‐interleukin 6 antibody through an amidic bond formed with the carboxyl functionalities provided at the surface of protein G‐functionalized magnetic microparticles, assuring a sandwich‐type immunoassay with electrochemical label free detection. All the experimental parameters involved in the elaboration and testing protocol were optimized. A linear calibration plot between the charge transfer resistance and the logarithmic concentration of interleukin‐6 was achieved in the 1 pg mL^?1 to 1 μg mL^?1 range. A limit of quantification of 1 pg mL^?1 and a detection limit of 0.3 pg mL^?1 were obtained. The optimized magnetoimmunosensor showed an excellent selectivity against some potentially interfering proteins and has been successfully applied for the determination of target protein in human serum, proving its clinical relevance.     

Electroanalytical Determination of Cd(II) and Pb(II) in Bivalve Mollusks using Electrochemically Reduced Graphene Oxide‐based Electrode

An electrochemical sensor for the simultaneous determination of Cd(II) and Pb(II) by square wave anodic stripping voltammetry (SWASV) in bivalve mollusks using a glassy carbon electrode modified with electrochemically reduced graphene oxide has been developed. The modified surface was characterized by cyclic voltammetry, high resolution scanning electron microscopy (HR‐SEM), and Raman spectroscopy. The optimum conditions were optimized and a linear range was observed from 15–105 μg L^?1 with a limits of detection of 15 μg L^?1 for Cd(II) and Pb(II). The methodology was validated and applied in different samples of commercial bivalve mollusks with satisfactory results. The high conductivity and greater surface area of the modifying agent improves the preconcentration capacity of the electrochemical sensor, allowing to develop a simple, rapid and sensitive analysis in the detection of lead and cadmium in marine resources.     

以聚吡咯和纳米金为基质基于电化学交流阻抗测试的乙肝表面抗原免疫传感器研究

A novel simple immunosensing strategy for fabrication of hepatitis B surface antigen detection has been developed via electrochemical impedance spectroscopy （EIS） as a platform. At first, the conductive polymer polypyrrole （PPy） film was electrodeposited on a platinum electrode surface to adsorb the gold nanoparticles （nano-Au） via the opposite-charged adsorption technique, and then hepatitis B surface antibodies were adsorbed onto the surface of nano-Au. The modification procedure was characterized by EIS. Such spectroscopy is attributed to the concomitant conductivity changes of the polymerized pyrrole film and gold nanoparticles. The factors influencing the performance of resulting immunoelectrode were studied in detail. The linear range of the resulting immunoelectrode is from 2.6 to 153.6 ng.mL^-1 with a detection limit of 1.3 ng·mL^-1 at 3σ. In addition, the experiment results indicate that antibody immobilized on this way exhibits a good sensitivity, selectivity, high stability and a long-term maintenance of bioactivity, implying a great promising alternative approach for reagentless immunosensing analysis in the clinical diagnosis.