Electrochemical Aptasensor For Thrombin Research Articles

Hemin/G-quadruplex and AuNPs-MoS2 based novel dual signal amplification strategy for ultrasensitively sandwich-type electrochemical thrombin aptasensor

In this work, a novel sandwich-type electrochemical aptasensor based on the dual signal amplification strategy of hemin/G-quadruplex and AuNPs-MoS2 was designed and constructed, which realized the highly sensitive and specific detection of thrombin (TB). In this aptasensor, the 15-mer TB-binding aptamer (TBA-1) modified with thiol group was immobilized on the surface of AuNPs modified glassy carbon electrode (AuNPs/GCE) as capturing elements. Another thiol-modified 29-mer TB-binding aptamer (TBA-2) sequence containing G-quadruplex structure for hemin immobilization was designed. The formed hemin/G-quadruplex/TBA-2 sequence was further combined to the AuNPs decorated flower-like molybdenum disulfide (AuNPs-MoS2) composite surface via Au-S bonds, acting the role of reporter probe. In presence of the target TB, the sandwich-type electrochemical aptamer detection system could be formed properly. With the assistance of the dual signal amplification of AuNPs-MoS2 and hemin/G-quadruplex toward H2O2 reduction, the sandwich-type electrochemical aptasensor was successfully constructed for sensitive detection of TB. The results demonstrate that the fabricated aptasensor displays a wide linear range of 1.0 × 10−6 ∼ 10.0 nM with a low detection limit of 0.34 fM. This proposed aptasensor shows potential application in the detection of TB content in real biological samples with high sensitivity, selectivity, and reliability.

Target-Synergized Biologically Mediated RAFT Polymerization for Electrochemical Aptasensing of Femtomolar Thrombin.

The assay of thrombin levels is integral to the assessment of coagulation function and clinical screening of coagulation disorder-related diseases. In this work, we illustrate the ingenious use of the target-synergized biologically mediated reversible addition-fragmentation chain transfer (RAFT) polymerization (tsBMRP) as a novel amplification strategy for the electrochemical aptamer-based biosensing of thrombin at the femtomolar levels. Briefly, the tsBMRP-based strategy relies on the boronate affinity-mediated decoration of the glycan chain(s) of the target itself with RAFT agents and the subsequent recruitment of signal labels via BMRP, mediated by the direct reduction of RAFT agents by NADH into initiating/propagating radicals. Obviously, the tsBMRP-based strategy is biologically friendly, low-cost, and simple in operation. As thrombin is a glycoconjugate, its electrochemical aptasensing involves the use of the thrombin-binding aptamer (TBA) as the recognition receptor, the site-specific decoration of RAFT agents to the glycan chain of thrombin via boronate affinity, and further the recruitment of ferrocene signal labels via the BMRP of ferrocenylmethyl methacrylate (FcMMA). As boronate affinity results in the decoration of each glycan chain with tens of RAFT agents while BMRP recruits hundreds of signal labels to each RAFT agent-decorated site, the tsBMRP-based strategy allows us to detect thrombin at a concentration of 35.3 fM. This electrochemical aptasensor is highly selective, and its applicability to thrombin detection in serum samples has been further demonstrated. The merits of high sensitivity and selectivity, low cost, good anti-interference capability, and simple operation make the tsBMRP-based electrochemical thrombin aptasensor great promise in biomedical and clinical applications.

Enzyme‐free Recycling Amplification‐based Sensitive Electrochemical Thrombin Aptasensor

AbstractIn this work, an enzyme‐free recycling amplification‐based sensitive electrochemical thrombin aptasensor using catalytic hairpin assembly (CHA) and bisferrocene was proposed for electrochemical beacon. Thrombin triggered the CHA reaction that in turn formed a number of double‐stranded complexes,which are fixed on the surface of the gold electrode through potential‐assisted Au−S deposition, and a large number of labeled bisferrocene are placed close to the gold electrode to generate electrochemical signal. The linear range of the electrochemical sensor was 0.25 pM‐2.5 nM for thrombin with detection limit of 0.18 pM. This sensor can be employed to monitor the thrombin in human serum samples.

An electrochemical thrombin aptasensor based on the use of graphite-like C3N4 modified with silver nanoparticles.

An electrochemical aptasensor for thrombin is introduced that makes use of a nanohybrid composed of silver nanoparticles and graphite-like carbon nitride (Ag-g-C3N4). The material has a large surface and good biocompatibility. AgNPs are modified directly on the surface of g-C3N4 via chemical reduction. A glass carbon electrode (GCE) modified with Ag-g-C3N4 can immobilize a large number of amino-terminated thrombin binding aptamers (NH2-TBA) through strong Ag-N bonds. The electrochemical impedance signal of the aptasensor increases in the presence of thrombin. Under the optimal conditions and by using[Fe(CN)6]3-/4- as an electrochemical probe, the aptasensor shows a wide linear range of 100 fM - 20nM with a lower detection limit of 38 fM. The method was applied to the determination of thrombin in spiked human plasma and the recoveries fluctuated from 97.2% to 103%. Graphical abstractSchematic representation of an electrochemical aptasensor using graphite-like carbon nitride (C3N4) modified with silver nanoparticles as electrode substrate for thrombin (TB) detection.

An Ultrasensitive Electrochemical Aptasensor for Thrombin Detection Using MoS2 Nanoparticles Loaded Iron-Porphyrinic Metal-Organic Framework as Signal Amplifier

An ultrasensitive electrochemical aptasensor for thrombin (TB) was designed using MoS2 nanoparticles loaded iron-porphyrinic metal-organic framework (PCN-223-Fe) as the electrochemical tracer. Spindle-shaped PCN-223-Fe was synthesized with zirconium ion as node and iron-porphyrin as linker, and in situ loaded with MoS2 nanoparticles to obtain MoS2/PCN-223-Fe composite. MoS2/PCN-223-Fe was characterized by electron microscopies, X-ray diffraction and X-ray photoelectron spectroscopy. Due to the synergistic effect of MoS2 and PCN-223-Fe, MoS2/PCN-223-Fe modified electrode exhibited enhanced electrocatalytic activity to oxygen reduction. Sequentially, MoS2/PCN-223-Fe was combined with TB aptamer 2 to prepare signal probe. A sandwich-type electrochemical aptasensor was constructed for TB detection. TB aptamer 1 was fixed on electrode surface to capture TB molecules, and then TB aptamer 2 functionalized MoS2/PCN-223-Fe tracer was introduced on electrode surface. By detecting the electrochemical signal of oxygen reduction catalyzed by MoS2/PCN-223-Fe, the designed TB sensor was monitored. The proposed TB aptasensor showed a wide linear range from 0.1 pM to 100 nM and a low detection limit of 0.03 pM (S/N = 3). Meanwhile, the aptasensor showed good reproducibility, stability and selectivity, which was successfully applied for TB detection in human serum sample. This strategy broadens application of metal-organic frameworks, and could be used for other biomolecules detection.

Electrochemical thrombin aptasensor based on using magnetic nanoparticles and porous carbon prepared by carbonization of a zinc(II)-2-methylimidazole metal-organic framework.

A homogeneous electrochemical aptasensor was obtained by modifying a glassy carbon electrode (GCE) with a porous carbon nanomaterial (Z-1000, about 70nm, deteced by transmission electron microscopic) that was obtained by carbonization of a zinc(II)-2-methylimidazole metal-organic framework. Z-1000 possesses a large specific surface and outstanding electrochemical properties. A thrombin-binding aptamer (CP) was immobilized on the magnetite nanoparticles MNPs by the condensation reaction and further combined with reporter probe (RP) that is functionalized with electroactive methylene blue (MB). In the presence of thrombin, the CP was specifically recognized with it to formthe CP/MNP/Thb complex, and the RP was dissociated from MNPs. The released RP was captured by the modified GCE through π-stacking interaction between nucleobases and carbon nanostructure. The electrical signal generatedby MB can be monitored by differential pulse voltammetry (DPV). Under the optimized conditions, the DPV peak current at around -0.28V (vs.SCE) increases with thrombin concentration. The sensor has a detection limit of 0.8 fM of thrombin and a linear range that extends from 10 fM to 100nM. It was successfully applied to the analysis of spiked serum. The recoveries are 98.1-99.4% and RSDs are 3.9%-4.0%. Conceivably, this aptasensor schemecan be easily extended to other proteins and gives inspiration to manufacture sensitive aptasensor. Graphical abstract A homogeneous electrochemical aptasensor is obtained by modifying a glassy carbon electrode with the MOF-derived porous carbon. The sensor has a detection limit of 0.8 fM and a wide linear range from 10 fM to 100nM for thrombin detection.

Voltammetric aptasensor for thrombin by using a gold microelectrode modified with graphene oxide decorated with silver nanoparticles.

A sensitive electrochemical aptasensor was developed for the determination of thrombin. It is based on signal amplification by using graphene oxide (GO) decorated with silver nanoparticles (AgNP) and placed on a microelectrode. The graphene oxide decorated with silver nanoparticles (AgNP-GO) was synthesized by chemical reduction and then modified with a thrombin-binding aptamer 1 (TBA1; a 29-mer) carrying a 3'-terminal thiol group. It was immobilized on the AgNP-GO by self-assembled monolayer technique to form the signaling probe (TBA1-AgNP-GO). A capture thrombin-binding aptamer 2 (TBA-2; a 15-mer) carrying a 3'-terminal thiol group was self-assembled onto the surface of the gold electrode. After binding of thrombin and the signaling probe, a sandwich of type TBA2-thrombin-TBA1-AgNP-GO is formed. The square wave voltammetric signal of the AgNP is then recorded. The electrochemical oxidation signal (measured at 0.21V vs. Ag/AgCl) increases linearly with the concentration of thrombin in the range from 0.05nmol·L-1 to 5nmol·L-1 with a detection limit of 0.03nmol·L-1 (at S/N= 3). The aptasensor is highly specific and not affected by other proteins. It was successfully applied to the determination of thrombin in spiked human serum samples. Graphical abstract A sensitive electrochemical aptasensor for thrombin was developed based on the signal amplification of silver nanoparticles decorated graphene oxide at an ultramicroelectrode.

Aptamer based label free thrombin assay based on the use of silver nanoparticles incorporated into self-polymerized dopamine.

The authors describe an electrochemical aptasensor for thrombin that is based on the use of a glassy carbon electrode (GCE) modified with polydopamine that is loaded with silver nanoparticles (PDA/AgNPs). The use of AgNPs improves the conductivity of the film and increases the surface area of the GCE. PDA was deposited on the GCE via self-polymerization, and the thrombin binding aptamer was grafted onto the PDA-modified GCE by a single step reaction. Residual electrode surface was blocked with 6-mercapto-1-hexanol. On exposure to thrombin, the electrochemical impedance of the modified electrode increases gradually. Response is linear in the 0.1 pM to 5.0nM thrombin concentration range, and the limit of detection is as low as 36 fM. The method is selective and capable of detecting thrombin in diluted human serum. In our perception, such a GCE modified with AgNP in a PDA matrix may be applied to many other analytes for which appropriate aptamers are available. Graphical abstract Schematic of an electrochemical aptasensor for sensitive and selective thrombin detection based on the use of aself-polymerized polydopamine film loaded with silver nanoparticles.

An Electrochemical Aptasensor for Thrombin Based on a Novel Polyamidoamine Dendrimer-Streptavidin Supramolecular Architecture

This work reports the development of an electrochemical aptasensor for thrombin based on a polyamidoamine dendrimer-streptavidin supramolecular architecture prepared by the cyclic voltammetric electrodeposition of polyamidoamine dendrimer (PAMAM) followed by the drop coating of streptavidin on a glassy carbon electrode. Biotinylated aptamer probe was immobilised on the platform through biotin-streptavidin interaction. The aptasensor preparation was monitored with voltammetry and electrochemical impedance spectroscopy. The biosensor response to thrombin was evaluated with square wave voltammetry (in phosphate buffer solution and in ferro/ferric cyanide probe) and electrochemical impedance spectroscopy. Linear responses in the concentration range between 1 - 200 ng mL-1 and detection limit in the sub 0.02 ng mL-1 (defined as ( ) were calculated. The selectivity of the aptasensor was evaluated using interfering proteins such as bovine serum albumin (BSA), lysozyme and haemoglobin. The relative response obtained shows insignificant effect from the interfering proteins with BSA, lysozyme and haemoglobin having a relative response of 9.2%, 8.7% and 7.6% respectively.

Electrochemical aptasensor for thrombin using co-catalysis of hemin/G-quadruplex DNAzyme and octahedral Cu2O-Au nanocomposites for signal amplification

In this work, novel octahedral Cu2O-Au nanocomposites were synthesized and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of a DNAzyme for signal amplification. The octahedral Cu2O-Au nanocomposites have not only simultaneously served as signal amplifying molecules but have also been utilized as an ideal loading platform to immobilize a large number of electroactive substances and recognition probes. Gold nanoparticles (AuNPs) were grown directly on the surface of the octahedral Cu2O nanocrystals, and the Cu2O-Au nanocomposites obtained had the advantages of large surface areas and excellent biocompatibilities. The hemin/G-quadruplex, which was formed by intercalating hemin into the amino terminated thrombin binding aptamer (NH2-TBA), and the electroactive toluidine blue (Tb) were immobilized onto the Cu2O-Au nanocomposite surfaces through a stable Au-N bond. AuNPs, Cu2O and hemin/G-quadruplex co-catalyse the H2O2 in the working buffer to promote the electron transfer of Tb as a multiple signal amplification strategy in order to improve the performance of the electrochemical aptasensor. Under optimal conditions, the designed aptasensor exhibited sensitive detection of TB from 100 fM to 20nM with a lower detection limit of 23fM. This proposed aptasensor exhibited good sensitivity, high specificity and acceptable reproducibility and could be widely applied in bioassay analysis.

Highly sensitive electrochemical thrombin aptasensor based on peptide-enhanced electrocatalysis of hemin/G-quadruplex and nanocomposite as nanocarrier

In this work, we first conjugated a short peptide to thrombin binding aptamer (TBA) and bond hemin to the hybrid, effectively rendering hemin/G4-peptide more active over the original hemin/G4, so that a highly sensitive electrochemical thrombin (TB) aptasensor was developed based on it and PtNTs@rGO nanocomposite. It was the first report on the application of hemin/G4-peptide in electrochemical aptasensor. PtNTs@rGO with large surface area served as excellent nanocarrier for high loading of hemin/G4-peptide hybrids, resulting in the formation of hemin/G4-peptide–PtNTs@rGO bioconjugate as the secondary aptamer and further signal enhancement. The specific affinity of aptamer for target TB made the secondary aptamer go into the sensing interface, and then a noticeable current signal was obtained from hemin without additional redox mediators. Due to the collaborative electrocatalysis of hemin/G4-peptide and PtNTs toward H2O2, which was formed in situ during the process of hemin/G4-peptide-catalyzed oxidation of NADH with dissolved O2, the current intensity increased dramatically. Such an electrochemical aptasensing system could be used to detect TB with a linear range of 0.05 pM–60nM and very lower detection limit of 15fM. Notably, this method exhibited a higher sensitivity than that of many hemin/G4-based electrochemical strategies for TB detection due to the improvement of the catalytic activity of hemin/G4-peptide. The present works opened a new way for expanding the application of hemin/G4 in biological detection. With the mediator-free, proteinous enzyme-free yet high-sensitivity advantages, this electrochemical aptasensor held great promise for other biomarker detections in clinical diagnostics.

Thrombin aptasensor enabled by Pt nanoparticles-functionalized Co-based metal organic frameworks assisted electrochemical signal amplification

In this work, a Pt nanoparticles-functionalized Co-based metal organic frameworks (PtNPs@Co(II)MOFs@PtNPs) was synthesized and applied in electrochemical aptasensor for thrombin (TB) detection. First, the Co(II)MOFs@PtNPs were prepared via the mixed solvothermal method, which consists of inner Pt nanoparticles (PtNPs) encapsulated by aminofunctionalized Co(II)MOFs materials. Following that, additional PtNPs were adsorbed on the surface of Co(II)MOFs@PtNPs, resulting in the formation of PtNPs@Co(II)MOFs@PtNPs nanocomposite. The PtNPs@Co(II)MOFs@PtNPs nanocomposites with a large surface area were implimented as nanocarriers to immobilize a mass of TBA II for the formation of the TBA II bioconjugates that could be captured onto the electrode surface by sandwich-type format. Moreover, the PtNPs@Co(II)MOFs@PtNPs nanocomposites could directly use as redox tags for charge-generating and electron-transporting with the electron transfer from Co(II) to Co(III). Furthermore, in the presence of H2O2, the PtNPs@Co(II)MOF@PtNPs could effectively catalyze H2O2 oxidation with improvement electron transfer of redox probe, resulting in electrochemical signal amplification. Based on the above superior advantages, TB was determined in the concentration range from 0.1pM to 50nM with a detection limit of 0.33fM. Furthermore, the excellent sensitivity and selectivity can be easily established for quantitative analysis of other analytes.

Development of an electrochemical aptasensor for thrombin based on aptamer/Pd–AuNPs/HRP conjugates

An electrochemical aptasensor for thrombin based on aptamer/Pd–AuNPs/HRP conjugates.

A label-free and sensitive electrochemical aptasensor for thrombin based on the direct electron transfer of hemin and hemin@rGO nanosheets as the signal probe

In this work, a label-free electrochemical aptasensor for sensitive detection of thrombin was fabricated and characterized.

An electrochemical aptasensor for thrombin using synergetic catalysis of enzyme and porous Au@Pd core–shell nanostructures for signal amplification

In this work, a sensitive electrochemical aptasensor for thrombin (TB) based on synergetic catalysis of enzyme and porous Au@Pd core–shell nanostructure has been constructed. With the advantages of large surface area and outstanding catalytic performance, porous Au@Pd core–shell nanostructures were firstly employed as the nanocarrier for the immobilization of electroactive toluidine blue (Tb), hemin/G-quadruplex formed by intercalating hemin into the TB aptamer (TBA) and glucose oxidase (GOx). As a certain amount of glucose was added into the detection cell, GOx rapidly catalyzed the oxidation of glucose, coupling with the local generation of H2O2 in the presence of dissolved O2. Then, porous Au@Pd nanoparticles and hemin/G-quadruplex as the peroxidase mimics efficiently catalyzed the reduction of H2O2, amplifying the electrochemical signal and improving the sensitivity. Finally, a detection limit of 0.037pM for TB was achieved. The excellent performance of the aptasensor indicated its promising prospect as a valuable tool in simple and cost-effective TB detection in clinical application.

Porous platinum nanotubes labeled with hemin/G-quadruplex based electrochemical aptasensor for sensitive thrombin analysis via the cascade signal amplification

For the first time, a sensitive electrochemical aptasensor for thrombin (TB) was developed by using porous platinum nanotubes (PtNTs) labeled with hemin/G-quadruplex and glucose dehydrogenase (GDH) as labels. Porous PtNTs with large surface area exhibited the peroxidase-like activity. Coupling with GDH and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme, the cascade signal amplification was achieved by the following ways: in the presence of glucose and NAD+ in the working buffer, GDH electrocatalyzed the oxidation of glucose with the production of NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidation of NADH to in situ generate H2O2. Based on the corporate electrocatalysis of PtNTs and hemin/G-quadruplex toward H2O2, the electrochemical signal was significantly amplified, allowing the detection limit of TB down to 0.15pM level. Moreover, the proposed strategy was simple because the intercalated hemin offered the readout signal, avoiding the adding of additional redox mediator as signal donator. Such an electrochemical aptasensor is highly promising for sensitive detection of other proteins in clinical diagnostics.

Electrochemical Aptasensor Based on ZnO Modified Gold Electrode

AbstractWe 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.

Aptamer-linked biosensor for thrombin based on AuNPs/thionine–graphene nanocomposite

A novel protocol of electrochemical thrombin aptasensor based on a goldnanoparticles/thionine-graphene (AuNPs/Th-G) nanocomposite modified glassy carbon electrode was presented. Graphene was non-covalently functionalized by thionine viaπ-stacking interaction, and then AuNPs were electrodeposited onto the Th-G surface. The morphology and conductivity of the AuNPs/Th-G nanocomposite were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The ethanethiol-substituted oligonucleotide probe was immobilized onto the surface of the AuNPs/Th-G nanocomposite to prepare an electrochemical biosensing platform. The aptamer-thrombin reaction was monitored by differential pulse voltammetry. Under optimum conditions, the proposed biosensor exhibits high sensitivity and a low detection limit for thrombin determination. The thrombin could be quantified in a wide range of 0.5 to 40 nM and with a low detection limit of 0.093 nM (S/N = 3).

An ultrasensitive electrochemical aptasensor for thrombin based on the triplex-amplification of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme and horseradish peroxidase decorated FeTe nanorods

In the present study, we fabricated an ultrasensitive sandwich-type electrochemical aptasensor for thrombin (TB) based on a triplex signal amplification strategy. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) as well as blocking reagent-horseradish peroxidase (HRP) and iron telluride nanorods (FeTe NRs) could simultaneously amplify the electrochemical signal of thionine (Thi) in the presence of H(2)O(2). Herein, FeTe NRs, as a newly discovered HRP-mimicking enzyme, were employed to construct an aptasensor for the first time. And, the FeTe NRs decorated by gold nanoparticles (abbreviated as AuNPs@FeTe NRs), were not only used as carriers of secondary thrombin aptamer (TBA2), electron mediator thionine (Thi) and HRP, but also catalyzed the electrochemical reaction of Thi in the presence of H(2)O(2). As can be seen from experiment results, with the triplex signal amplification strategy, the reduction peak current of the fabricated aptasensor was logarithmically related to the concentration of thrombin (TB) over a wide range from 1 pM to 20 nM, and a detection limit of 0.5 pM was obtained. Hence, the proposed aptamer-based sandwich sensing approach for amplified detection of TB could provide a promising way for highly sensitive determination of other analytes.

Sensitive electrochemical thrombin aptasensor based on gold disk microelectrodearrays

The construction of a sensitive electrochemical aptamer sensor (aptasensor) for thrombin detection is described. Among the advantages of using microelectrode-based devices are the possibility to work with small sample volumes and enjoying faster mass transport rates and lower interfacial capacitance than at macroelectrodes. Therefore, gold disk microelectrode arrays are an attractive transducer option for aptasensors. The morphology of the gold disk microelectrode arrays was inspected by scanning electron microscope. The interaction between a thrombin aptamer and thrombin on gold disk microelectrode arrays was demonstrated by differential pulse voltammetry using methylene blue (MB) as an electrochemical indicator. MB adsorbed to aptamers via their guanine base. When thrombin was introduced, it displaced the MB adsorbed to the aptamers and bound to them. This resulted in a decrease of MB peak current which correlated to the concentration of thrombin over a dynamic range spanning from 10−5 to 10–12M. This method was able to linearly and selectively detect thrombin with a detection limit of 0.143pM.