Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A

An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 10⁷ redox events, leading to a substantial increase in charge-transfer resistance (R_ct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of R_ct to the increase of OTA concentrations over a wide range of 1 pg mL⁻¹–50 ng mL⁻¹ and could detect extremely low OTA concentration, namely, 0.3 pg mL⁻¹ or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method.     

Label‐free Electrochemical Bisphenol A Aptasensor Based on Designing and Fabrication of a Magnetic Gold Nanocomposite

The present study focuses on designing and fabricating an electrochemical aptasensor for the label free detection of bisphenol A (BPA) using gold nanoparticles (Au NPs) immobilized on functional cupper magnetic nanoparticles (CuFe₂O₄‐SH) and multiwall carbon nanotubes (MWCNTs) modified with aptamer and 6‐mercapto‐1‐hexanol (MCH). A number of analysis techniques were used to characterize the nanocomposite, including Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometer, elemental mapping analysis and energy dispersive x‐ray diffraction. The results of the analyses revealed that the fabricated aptasensor had an acceptable linearity index (0.05‐9 nM) with an ultralow detection limit (25.2 pM) when used to determine BPA. Electrochemical experiments were conducted using a [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ redox system. The results of the electrochemical tests indicated that the existence of Au NPs along with magnetic nanoparticles and MWCNTs in nanocomposite led to a synergistic augmentation on the surface of the modified electrode, thus facilitating the efficient sensing of BPA. This method is highly selective, sensitive and environmentally friendly. Moreover, proposed aptasensor has valuable potential applications in medical diagnostics and food industries where a fast and reliable detection of BPA is of paramount importance for the health of the public.     

Electrochemiluminescence Aptasensor for the MUC1 Protein Based on Multi‐functionalized Graphene Oxide Nanocomposite

The electrochemiluminescence (ECL) aptasensor was prepared for the detection of Mucin 1 based on its specific recognition by aptamer immobilized on multi‐functionalized graphene oxide nanocomposite, which was prepared with N‐(4‐aminobutyl)‐N‐ethylisoluminol (ABEI) and aptamer chemically bound to the surface of magnetic GO (nanoFe₃O₄@GO). ABEI and aptamer acted as the electrochemiluminophore and the capture device for Mucin 1 respectively. NanoFe₃O₄@GO brought multi‐functionalized graphene oxide nanocomposite attracted on the surface of magnetic glass carbon electrode through magnetism, enabled all the ABEI immobilized electrochemically active due to its good conductivity and thus then facilitated the sensitive detection of Mucin 1. In addition, the ECL aptasensor can be prepared through a one‐step process. Under optimal conditions, the ECL intensity of the aptasensor decreased proportionally to the logarithmic concentrations of Mucin 1 in the range of 0.005–1000 ng mL^?1. This aptasensor displays good specificity, stability, reproducibility and application. This method has a large potential because such a multi‐functionalized graphene oxide nanocomposite also may be applied to other ECL‐based aptasensors.     

Preparation of Epirubicin Aptasensor Using Curcumin as Hybridization Indicator: Competitive Binding Assay between Complementary Strand of Aptamer and Epirubicin

The present work explains the fabrication of a novel electrochemical aptasensor for identifying and measuring the epirubicin (Epi) by using curcumin (Cur) as an anticancer electrochemical indicator. The aptasensor prepared by immobilizing the thiolated aptamer on the surface of graphite screen‐printed electrode modified with gold nanoparticles/functionalized multiwall carbon nanotubes, ionic liquid and chitosan nanocomposite (AuNPs/FMWCNTs‐IL‐Chit/SPE). To evaluate the willingness of aptamer to interaction with Epi in the presence of complementary strand DNA, competitive binding assay between the complementary strand of aptamer and Epi were used. Cur tends to bound to the grooves of two strands DNA. With increasing the concentration of Epi in the range of 0.007–7.0 μM, the peak current of Cur decreased, due to the formation of aptamer‐Epi complex and decreasing the amount of complementary strand DNA. Through the control experiments, we examined the response of fabricated aptasensor for some anticancer drugs, which have a structure similar to the Epi. The results showed that using the thiol‐terminated aptamer as a recognition layer led to a sensor with a high tendency for Epi compared to other anticancer drugs.     

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.     

Aptamer-based highly sensitive electrochemical detection of thrombin via the amplification of graphene

Herein, we successfully fabricated a highly sensitive label-free electrochemical aptasensor for thrombin based on the amplification of graphene (Gra). The excellent electrochemical probe of nickel hexacyanoferrate nanoparticles (NiHCFNPs) was introduced to form Nafion-Graphene-NiHCFNPs (Nf-Gra-NiHCFNPs) nanocomposites membrane on the gold electrode. The employment of graphene not only enhanced the surface area of the electrode with increased NiHCFNPs immobilization, but also improved the conductivity of the electrode, which further effectively improved the sensitivity of this proposed aptasensor. Subsequently, AuNPs layer was formed to immobilize the thrombin aptamer (TBA) and enhance the stability of the composite monolayer mentioned above. Then, thiol-modified TBA was assembled onto the AuNPs layer. Thereafter, hexanethiol (HT) was employed to block the possible remaining active sites. With the dual amplification of Gra and AuNPs, the resulting aptasensor exhibited good current response to target thrombin with a wide linear range extended from 1 pM to 80 nM (the detection limit was 0.3 pM). Additionally, the morphologies of bare Au substrate, nickel hexacyanoferrate nanoparticles (NiHCFNPs) and nanocomposites were successfully characterized by atomic force microscopy (AFM).     

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.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

Development of Electrochemical Impedance Spectroscopy Based Malaria Aptasensor Using HRP‐II as Target Biomarker

Current demand for a stable, low cost and sensitive malaria sensor has prompted to explore novel recognition systems that can substitute widely used protein based labile biorecognition elements to be used in point of care diagnostic devices. Here, we report a novel ssDNA aptamer of 90 mer sequence developed by SELEX process against HRP‐II, a specific biomarker for Plasmodium falciparum strains. High stability of the secondary structure of the isolated aptamer was discerned from its free energy of folding of −20.40 kcal mole⁻¹. The binding constant (K_d) of the aptamer with HRP‐II analysed by isothermal titration calorimetry was ∼1.32 μM. Circular dichroism studies indicated B form of the aptamer DNA. The aptamer was chemically immobilized on a gold electrode surface through a self‐assembled monolayer of dithio‐bis(succinimidyl) propionate to produce the aptasensor. The step wise modification of the layers over the gold electrode during fabrication of the aptasensor was confirmed by cyclic voltammetry. The aptasensor was then challenged with different concentration of HRP‐II and analysed the interaction signals through electrochemical impedance spectroscopy. The impedance signal behaved reciprocally with the increasing concentrations of the target in the sample from which a dynamic range of 1 pM–500 pM (R²=0.99) and LOD of ∼3.15 pM were discerned. The applicability of the developed aptasensor to detect HRP‐II in mimicked real sample was also validated.     

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.

Electrochemical Aptasensor for the Determination of Cocaine Incorporating Gold Nanoparticles Modification

A novel electrochemical aptasensor incorporating a signal enhancement for the determination of cocaine was designed. Gold nanoparticles were self‐assembled onto the surface of a gold electrode through 1,6‐hexanedithiol. A bifunctional derivative of the 32‐base cocaine‐binding aptamer with a redox‐active ferrocene moiety and a thiol linker group at the termini of the strand was self‐assembled onto the surface of gold nanoparticles. The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0 μM with a detection limit of 0.5 μM. It was found that the sensitivity of the aptasensor with gold nanoparticles modification was ca. 10‐fold higher than that of the aptasensor without gold nanoparticles modification. This work demonstrates that gold nanoparticles‐assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity.     

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.     

Electrochemical aptamer sensor for highly sensitive detection of mercury ion with Au/Pt@carbon nanofiber‐modified electrode

In this paper, an electrochemical aptamer sensor was proposed for the highly sensitive detection of mercury ion (Hg²⁺). Carbon nanofiber (CNF) was prepared by electrospinning and high‐temperature carbonization, which was used for the loading of platinum nanoparticles (PtNPs) by the hydrothermal method. The Pt@CNF nanocomposite was modified on the surface of carbon ionic liquid electrode (CILE) to obtain Pt@CNF/CILE, which was further decorated by gold nanoparticles (AuNPs) through electrodeposition to get Au/Pt@CNF/CILE. Self‐assembling of the thiol‐based aptamer was further realized by the formation of Au‐S bond to get an electrochemical aptamer sensor (Aptamer/Au/Pt@CNF/CILE). Due to the specific binding of aptamer probe to Hg²⁺ with the formation of T‐Hg²⁺‐T structure, a highly sensitive quantitative detection of Hg²⁺ could be achieved by recording the changes of current signal after reacting with Hg²⁺ within the concentration range from 1.0 × 10^?15 mol/L to 1.0 × 10^?6 mol/L and the detection limit of 3.33 × 10^?16 mol/L (3σ). Real water samples were successfully analyzed by this method.     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

A Hairpin Electrochemical Aptasensor for Sensitive and Specific Detection of Thrombin Based on Homogenous Target Recognition

An electrochemical aptasensor was developed for sensitive and specific detection of thrombin by combining homogenous recognition strategy and gold nanoparticles (AuNPs) amplification. Streptavidin‐alkaline phosphatase was used as reporter molecule. Compared with the traditional hairpin aptasensor monitoring the distance of the redox molecule from the electrode surface, the proposed aptasensor successfully overcome the limitations of distance and improved the stability and high affinity of the aptamer hairpin through homogenous recognition, which enhanced the sensitivity and selectivity of the sensors effectively. Additionally, AuNPs were employed to increase the active area and conductivity of the electrode, thus, improving the sensitivity of the aptasensor. As a result, the designed thrombin detection sensor obtained a lower detection limit of 0.52 pM in buffer and 6.9 pM in blood serum.     

A sensitive, label free electrochemical aptasensor for ATP detection

A sensitive, label free electrochemical aptasensor for small molecular detection has been developed in this work based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA-AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH₃)₆]³⁺ (RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular reorganization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer-ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA-AuNPs) and release of abundant RuHex molecules trapped by RDNA-AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA-AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of 4 orders of magnitude (1 nM-10 μM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2 nM). Those results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecular aptasensors.     

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.     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.