A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.     

Sensitive Electrochemical Aptasensor for Thrombin Detection Based on Graphene Served as Platform and Graphene Oxide as Enhancer

A sensitive electrochemical aptasensor was developed with conductive graphene served as platform and inert graphene oxide (GO) as enhancer. An electrodeposited nano-Au layer was firstly formed on conductive graphene modified glass carbon electrode surface for further immobilizing of electrochemical redox probe hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was formed for immobilizing of thrombin aptamer (TBA). In the presence of thrombin, the TBA on the electrode surface could bind with thrombin, which made a barrier for electrons and inhibited the electro-transfer, resulting in the decreased electrochemical signals of NiHCFNPs. Owing to the non-conductivity property of graphene oxide, further decreased electrochemical signals of NiHCFNPs could be obtained via the sandwich reaction with GO-labeled TBA. According to the signal changes before the thrombin recognition and after sandwich reaction, trace detection of thrombin could be achieved. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range from 0.005 nM to 50 nM with a detection limit of 1 pM.     

Highly sensitive electrochemical label-free aptasensor based on dual electrocatalytic amplification of Pt-AuNPs and HRP

In this work, a label-free electrochemical aptamer-based sensor (aptasensor) was constructed on account of the direct immobilization of redox probes on an electrode surface. For this proposed aptasensor, a gold nanoparticles (AuNPs)-coated electrode was firstly modified with redox probes-nickel hexacyanoferrates nanoparticles (NiHCFNPs) through chemisorption and electrostatic adsorption. Then, platinum-gold alloy nanoparticles (Pt-AuNPs) and horseradish peroxidase (HRP) were respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the electrochemical signal of NiHCFNPs and immobilizing thiolated thrombin aptamers (TBAs). In the presence of target thrombin, the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of NiHCFNPs amplified by the Pt-AuNPs and HRP toward H(2)O(2). The proposed method avoided the redox probes labeling process, increased the amount of redox probes, and further amplified the electrochemical signal. Thus, the approach showed a high sensitivity and a wider linearity to thrombin in the range between 0.01 nM and 50 nM with a detection limit of 6.3 pM.     

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

Label-free aptasensor for thrombin using a glassy carbon electrode modified with a graphene-porphyrin composite

We report on an electrochemical aptasensor for the ultrasensitive determination of thrombin. A glassy carbon electrode modified with a graphene-porphyrin nanocomposite exhibits excellent electrochemical activity and can be used as a redox probe in differential pulse voltammetry of the porphyrin on its surface. The thrombin aptamer is then immobilized via p-stacking interactions between aptamer and graphene and π-π stacking with porphyrin simultaneously. The resulting electrochemical aptasensor displays a linear response to thrombin in the 5–1,500 nM concentration range and with a limit of detection of 0.2 nM (at an S/N of 3). The sensor benefits from the synergetic effects of graphene (with its high conductivity and high surface area), of the porphyrin (possessing excellent electrochemical activity), and of the aptamer (with its high affinity and specificity). This kind of aptasensor conceivably represents a promising tool for bioanalytical applications.

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).     

G‐Quadruplex‐Based DNAzymes Aptasensor for the Amplified Electrochemical Detection of Thrombin

A novel G‐quadruplex‐based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine‐rich thrombin‐binding aptamer (TBA) to form horseradish peroxidase (HRP)‐mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10^–11 M. On the basis of results, this method could have broad applications in the detection of proteins and other biomolecules.     

Sandwich electrochemical thrombin assay using a glassy carbon electrode modified with nitrogen- and sulfur-doped graphene oxide and gold nanoparticles

Graphene oxide doped with nitrogen and sulfur was decorated with gold nanoparticles (AuNP-SN-GO) and applied as a substrate to modify a glassy carbon electrode (GCE). An aptamer against the model protein thrombin was self-assembled on the modified GCE which then was exposed to thrombin. Following aptamer-thrombin interaction, biotin-labeled DNA and aptamer 2 are immobilized on another AuNP-SN-GO hybrid and then are reacted with the thrombin/AuNP-SN-GO/GCE to form a sandwich. The enzyme label horseradish peroxidase (HRP) was then attached to the electrode by biotin–avidin interaction. HRP catalyzes the oxidation of hydroquinone by hydrogen peroxide. This generates a strong electrochemical signal that increases linearly with the logarithm of thrombin concentration in the range from 1.0?×?10^?13 M to 1.0?×?10^?8 M with a detection limit of 2.5?×?10^?14 M (S/N?=?3). The assay is highly selective. It provides a promising strategy for signal amplification. In our perception, it has a large potential for sensitive and selective detection of analytes for which appropriate aptamers are available.

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Graphic abstract A sandwich-type electrochemical aptasensor is fabricated for detection of thrombin using a glassy carbon electrode modified with nitrogen- and sulfur-doped graphene oxide and gold nanoparticles.

     

An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy

In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled “signal-on” and “signal-off” strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized “signal on” strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized “signal off” strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N = 3). In addition, this design strategy could be applied to the detection of other proteins and small molecules.     

Application of a sensitive nanocomposite-based electrochemical sensor for voltammetric determination of dicyclomine hydrochloride in real samples

In the present study a glassy carbon electrode, modified with nanocomposite of gold nanoparticles/multiwalled carbon nanotubes (GNPs/MWCNTs/GCE), was used for determination of dicyclomine hydrochloride (DcCl). The results showed that synergetic effects of GNPs and MWCNTs highly improved electrochemical response and sensitivity of the sensor. The electrochemical oxidation of DcCl was investigated by cyclic voltammetry and differential pulse voltammetry. Also, scanning electron microscopy and energy dispersive x-ray spectroscopy were used to evaluate microstructure of electrochemical sensor. The effect of various experimental parameters including pH and scan rate on the voltammetric response of DcCl were investigated. Under the optimal conditions linear response was observed in range of 1.0–1.2 × 10² µmol L^?1 for DcCl. The lower detection limit was found to be 0.40 µmol L^?1 for DcCl. The investigated method showed good stability, reproducibility and repeatability. The proposed sensor was successfully applied to the determination of DcCl in real samples.     

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。     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

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.     

Electrochemical aptasensor for the determination of bisphenol A in drinking water

We present an electrochemical aptasensor for rapid and ultrasensitive determination of the additive bisphenol A (BPA) and for screening drinking water for the presence of BPA. A specific aptamer against BPA and its complementary DNA probe were immobilized on the surface of a gold electrode via self-assembly and hybridization, respectively. The detection of BPA is mainly based on the competitive recognition of BPA by the immobilized aptamer on the surface of the electrode. The electrochemical aptasensor enables BPA to be detected in drinking water with a limit of detection as low as 0.284 pg?mL^?1 in less than 30 min. This extraordinary sensitivity makes the method a most powerful tool for on-site monitoring of water quality and food safety.

基于铁氰化镍的非标记型核酸适配体电化学传感器检测凝血酶

在玻碳电极（GCE）表面首先用增敏作用的多壁碳纳米管(MWCNTs)夹心于两层电沉积的铁氰化镍（NiHCF）氧化还原电化学探针之间,然后以金纳米粒子为固定核酸适配体的载体,构建了检测凝血酶的非标记型核酸适配体生物传感器。 利用扫描电子显微镜(SEM)对MWCNTs和NiHCF的形貌进行了表征。 利用电化学阻抗谱对传感器的组装过程进行了监测,用循环伏安法（CV）和差分脉冲伏安法（DPV）对传感器的电化学行为进行了研究。 以铁氰化镍为探针的传感器对凝血酶的检测在1.0 ng/L~1.0 mg/L范围内呈良好的线性关系,相关系数为0.998,检测限为0.2 ng/L(S/N=3)。     

A Label‐Free Electrochemical Aptasensor for Thrombin Using a Single‐Wall Carbon Nanotube (SWCNT) Casted Glassy Carbon Electrode (GCE)

An electrochemical aptasensor with a thrombin binding aptamer (TBA) was developed using a single‐wall carbon nanotube (SWCNT) casted GCE. The TBA was immobilized on SWCNTs through π‐stacking without any special modification, resulting in helical wrapping to the surface. In the presence of thrombin, the TBA binds with thrombin and the TBA concentration on the SWCNT surface decreases. The remaining amount of TBA can be analyzed by an electrochemical method without any label, because the guanine bases of the nucleic acid are measurable by electrochemical methods. The electrochemical oxidation of guanine nucleotides was enhanced by electrocatalytic mediation using Ru(bpy)₃²⁺ for higher sensitivity and reduction of the overpotential for electrochemical detection.     

Characterization and Electrocatalytic Property of Cobalt Hexacyanoferrate Film on Carbon Nanotubes Modified Gold Electrode

Abstract

Cobalt hexacyanoferrate (CoHCF) film was formed on multiwalled carbon nanotubes (MWNTs) modified gold electrode by electrodeposition from 0.5 M KCl solution containing CoCl₂ and K₃Fe(CN)₆. The electrochemical behavior and the electrocatalytic property of the modified electrode were investigated. Compared with CoHCF/gold electrode, the CoHCF/MWNTs/gold electrode exhibits greatly improved stability and enhanced electrocatalytic activity toward the oxidation of thiosulfate. A linear range from 5.0×10^?5 to 6.5×10^?3 M (r=0.9990) for thiosulfate detection at the CoHCF/MWNTs/gold electrode was obtained, with a detection limit of 2.0×10^?5 M (S/N=3).     

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.     

An intriguing electrochemical impedance aptasensor based on a porous organic framework supported silver nanoparticles for ultrasensitively detecting theophylline

Porous organic frameworks (POFs) are excellently stable porous materials, which can be employed as host platforms to support metal nanoparticles as functional composites for various applications. Herein, a novel POF is successfully prepared via Friedel-Crafts reaction. Silver nanoparticles (Ag NPs) are embedded in the prepared POF to generate an Ag@POF composite, which not only possesses high surface area, outstanding physicochemical stability and outstretched π-conjugation skeleton, but also exhibits preferable electrochemical stability and conductivity. This composite is able to immobilize a mass of aptamer strands to fabricate an intriguing electrochemical aptasensor. Electrochemical impedance spectroscopy (EIS) is a commonly used technology to analyze the electrochemical signal variation. The Ag@POF-based biosensor shows the excellent electrochemical detection behavior through analyzing EIS. For instance theophylline as a research mode, the Ag@POF based electrochemical aptasensor reveals ultra-sensitiveness, high selectivity, remarkable stability, good repeatability and simple operability even in various real samples. Notably, this aptasensor has the sensitive detection performance with the limit of detection of 0.191 pg/mL (1.06 pmol/L) in a wide concentration range of 5.0 × 10^-4 – 5.0 ng/mL (2.78 × 10^-3 – 27.8 nmol/L).     

A comparison of four protocols for the immobilization of an aptamer on graphite composite electrodes

We have performed a comparative study on four protocols for the immobilization of the thrombin aptamer on a graphite-epoxy composite electrode with the aim to identify the most practical method for designing the corresponding impedimetric aptasensor. The protocols included (a) physical adsorption, (b) avidin-biotin affinity interaction, (c) electrochemical activation and covalent bonding via amide groups, and (d) electrochemical grafting using 4-carboxybenzenediazonium coupling. The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the (ferri/ferro)hexacyanide redox couple. An increase in the interfacial charge transfer resistance (R_ct) was noted in all cases after the aptamer-thrombin interaction had occurred. The selectivity of the aptasensor over common serum proteins was also systematically investigated. Physical adsorption resulted in the lowest detection limit of the probe (4.5 pM), while avidin-biotin interaction resulted in highest selectivity and reproducibility exhibiting a 4.9 % relative standard deviation at pM thrombin concentration levels.