Label-free Electrochemical Aptasensor Research Articles

A highly sensitive label-free electrochemical aptasensor for interferon-gamma detection based on graphene controlled assembly and nuclease cleavage-assisted target recycling amplification

We report here a highly sensitive and label-free electrochemical aptasensing technology for detection of interferon-gamma (IFN-γ) based on graphene controlled assembly and enzyme cleavage-assisted target recycling amplification strategy. In this work, in the absence of IFN-γ, the graphene could not be assembled onto the 16-mercaptohexadecanoic acid (MHA) modified gold electrode because the IFN-γ binding aptamer was strongly adsorbed on the graphene due to the strong π–π interaction. Thus the electronic transmission was blocked (eT OFF). However, the presence of target IFN-γ and DNase I led to desorption of aptamer from the graphene surface and further cleavage of the aptamer, thereby releasing the IFN-γ. The released IFN-γ could then re-attack other aptamers on the graphene, resulting in the successive release of the aptamers from the graphene. At the same time, the “naked” graphene could be assembled onto the MHA modified gold electrode with hydrophobic interaction and π-conjunction, mediating the electron transfer between the electrode and the electroactive indicator. Then, measurable electrochemical signals were generated (eT ON), which was related to the concentration of the IFN-γ. By taking advantages of graphene and enzyme cleavage-assisted target recycling amplification, the developed label-free electrochemical aptasensing technology showed a linear response to concentration of IFN-γ range from 0.1 to 0.7pM. The detection limit of IFN-γ was determined to be 0.065pM. Moreover, this aptasensor shows good selectivity toward the target in the presence of other relevant proteins. Our strategy thus opens new opportunities for label-free and amplified detection of other kinds of proteins.

An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification

In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.

Label-free electrochemical IgE aptasensor based on covalent attachment of aptamer onto multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite modified electrode

A simple, sensitive and label-free aptamer-based biosensor for the detection of human immunoglobulin E (IgE) is developed using the electrochemical transduction method. A special immobilization interface consisting of multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite (MWCNTs/IL/Chit) is utilized to improve the conductivity and performance characteristics of the biosensor as well as to increase the loading amount of aptamer DNA sequence. A 5'-amino-terminated aptamer is covalently attached onto MWCNTs/IL/Chit modified glassy carbon (GC) electrode via a linker of glutaraldehyde (GA). Methylene blue (MB) is used as an electrochemical indicator which is intercalated into the aptamer through the specific interaction with its guanine bases. In the absence of IgE, MB bound to the aptamer produces a strong differential pulse voltammetric (DPV) signal. But when IgE exists, the intercalated MB releases from the aptamer, resulting an obviously decreased DPV signal. This phenomenon can be applied for human IgE detection. The peak current of MB linearly decreases with the concentration of IgE over a range of 0.5–30nM with a detection limit of 37pM. By using Bovine serum albumin (BSA) and lysozyme, the excellent specificity of this sensing system for the detection of IgE is also demonstrated. Finally, the proposed aptasensor is successfully used to IgE analysis in human serum sample. The obtained result is well agreed with the value obtained by the standard ELISA method. The herein described approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.

Label-free electrochemical aptasensor for sensitive thrombin detection using layer-by-layer self-assembled multilayers with toluidine blue–graphene composites and gold nanoparticles

In the present study, toluidine blue–graphene (Tb–Gra) nanocomposites were prepared to design a Lable-free electrochemical aptasensor for highly sensitive detection of thrombin based on layer-by-layer (LBL) technology. The nanocomposites with excellent redox electrochemical activities were first immobilized on the gold nanoparticles (nano-Au) modified glassy carbon electrodes (GCE). Then, the LBL structure was performed by electrostatic adsorption between the positively charged Tb–Gra and negatively charged nano-Au, which formed {Tb–Gra/nano-Au}n multilayer films for electroactive species enrichment and biomolecule immobilization. Subsequently, the thiolated thrombin binding aptamer (TBA) was assembled on the nano-Au surface through Au–S bond. In the presence of target thrombin (TB), 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 Tb–Gra nanocomposites. Under the optimal conditions, a wide detection range from 0.001nM to 80nM and a low detection limit of 0.33 pM (defined as S/N=3) for thrombin were obtained. In addition, the sensor exhibited excellent selectivity against other proteins.

A novel label-free electrochemical aptasensor based on graphene–polyaniline composite film for dopamine determination

A novel label-free electrochemical aptasensor based on graphene–polyaniline (GR–PANI) nanocomposites film for dopamine (DA) determination was reported. The resulting GR–PANI layer exhibited good current response for DA determination. The good electron transfer activity might be attributed to the effect of GR and PANI. The highly conductive and biocompatible nanostructure of GR–PANI nanocomposites was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). To quantify the amount of DA, the peaks of square-wave voltammetry (SWV) were monitored using the redox couple of an [Fe(CN)6]4−/3− probe. The electrochemical aptasensor showed a linear response to DA in the range 0.007–90nmol/L and a limit of detection of 0.00198nmol/L (S/N=3). The electrochemical aptasensor was successfully tested on human serum samples.

A Reusable Impedimetric Aptasensor for Detection of Thrombin Employing a Graphite-Epoxy Composite Electrode

Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]4− using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

Electrochemical Sensing of Aptamer-Facilitated Virus Immunoshielding

Oncolytic viruses (OVs) are promising therapeutics that selectively replicate in and kill tumor cells. However, repetitive administration of OVs provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effects. In this work, we selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus (VSV), to protect it from nAbs. A label-free electrochemical aptasensor was used to evaluate the degree of protection (DoP). The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VSV-specific aptamer. Electrochemical impedance spectroscopy was employed to quantitate VSV in the range of 800-2200 PFU and a detection limit of 600 PFU. The aptasensor was also utilized for evaluating binding affinities between VSV and aptamer pools/clones. An electrochemical displacement assay was performed in the presence of nAbs and DoP values were calculated for several VSV-aptamer pools/clones. A parallel flow cytometric analysis confirmed the electrochemical results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22, ZMYK-23, and ZMYK-28, showed the highest protective properties with dissociation constants of 17, 8, 20, and 13 nM, respectively. Another four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities to VSV without protecting it from nAbs and can be further utilized in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential to allow efficient delivery of VSV through the bloodstream without compromising the patient's immune system.

Design and construction of a label free aptasensor for electrochemical detection of sodium diclofenac

The present manuscript describes a label free electrochemical aptasensor for the detection of sodium diclofenac (DCF). In order to construct the biosensor, the amino-functionalized diclofenac binding aptamer (DBA) was covalently immobilized on the surface of the glassy carbon electrode (GCE). The conformation of the DBAs on the surface of the electrode is changed when this is exposed to different concentrations of DCF. The introduction of DCF induces an alteration in the conformation of the surface immobilized DBA and causes a decrease in the charge transfer resistance of the aptasensor. However, the charge transfer resistance is increased by incubation of GCE/DBA/DCF in the secondary DBA. The changes in the charge transfer resistance have been monitored using the voltammetric and electrochemical impedance spectroscopic (EIS) techniques. The aptasensor shows two different linear dynamic ranges over 0–5.0μM and 10μM to 1mM, and the sensitivity of 15.7kΩμM−1 and detection limit of 2.7×10−7M were obtained. The validity of the method and applicability of the aptasensor were successfully evaluated by detection of DCF in a blood serum sample without interference from the sample matrix. Furthermore, the aptasensor has shown good stability.

Porphyrin functionalized graphene nanosheets-based electrochemical aptasensor for label-free ATP detection

A facile strategy for the preparation of meso-terakis(4-methoxyl-3-sulfonatophenyl) porphyrin (T(4-Mop)PS4)-graphene hybrid nanosheets (TGHNs) was demonstrated for the first time, which combines the features of both graphene (high conductivity and large specific surface area) and porphyrins (photochemical electron-transfer ability and excellent electrochemical activity). The as-obtained TGHNs were characterized by UV-vis spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, atomic force microscopy, transmission electron microscopy and cyclic voltammetry, which confirmed that the porphyrin had been effectively functionalized on the surface of graphene. Combined with the high affinity and specificity of an aptamer, a simple, rapid, sensitive and label-free electrochemical aptasensor was successfully fabricated for adenosine triphosphate (ATP) detection. The proposed TGHN-based aptasensor achieved the direct electron transfer of porphyrin and showed good affinity and specificity towards ATP, which avoided interference such as the introduction of labeled-substances and the [Fe(CN)6]3−/4− probe. The preferable linear range for ATP was from 2.2 nM to 1.3 μM (R = 0.9982) with a detection limit of 0.7 nM.

Impedimetric thrombin aptasensor based on chemically modified graphenes

Highly sensitive biosensors are of high importance to the biomedical field. Graphene represents a promising transducing platform for construction of biosensors. Here for the first time we compare the biosensing performance of a wide set of graphenes prepared by different methods. In this work, we present a simple and label-free electrochemical impedimetric aptasensor for thrombin based on chemically modified graphene (CMG) platforms such as graphite oxide (GPO), graphene oxide (GO), thermally reduced graphene oxide (TR-GO) and electrochemically reduced graphene oxide (ER-GO). Disposable screen-printed electrodes were first modified with chemically modified graphene (CMG) materials and used to immobilize a DNA aptamer which is specific to thrombin. The basis of detection relies on the changes in impedance spectra of redox probe after the binding of thrombin to the aptamer. It was discovered that graphene oxide (GO) is the most suitable material to be used as compared to the other three CMG materials. Furthermore, the optimum concentration of aptamer to be immobilized onto the modified electrode surface was determined to be 10 μM and the linear detection range of thrombin was 10-50 nM. Lastly, the aptasensor was found to demonstrate selectivity for thrombin. Such simply fabricated graphene oxide aptasensor shows high promise for clinical diagnosis of biomarkers and point-of-care analysis.

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).

An electrochemical aptasensor for chiral peptide detection using layer-by-layer assembly of polyelectrolyte-methylene blue/polyelectrolyte-graphene multilayer

Here we demonstrate for the first time that by physically adsorbing aptamer onto conductive film assembled via alternate adsorption of graphene/polyelectrolyte and methylene blue/polyelectrolyte, a label-free electrochemical aptasensor with high sensitivity and selectivity for peptide detection is constructed. Graphene multilayer derived from layer-by-layer assembly has played significant roles in this sensing strategy: allowing accumulation of methylene blue, facilitating electron transfer and providing much more adsorption site. As compared to previous electrochemical aptasensors, the current sensor based on graphene multilayer alternated with electroactive molecule layer offers extremely high capability for sensitive detection of target without interference of environmental surrounding. This electroactive probe-confined graphene multilayer confers great flexibility to combine with differential pulse voltammetry (DPV) together. In the presence of target d entiomer of arginine vasopressin ( d-VP), the binding of peptide to aptamer block the electron transfer process of MB, leading to decreased current peak of DPV. By this way, this electrochemical aptasensor based on electroactive molecule-intercalated graphene multilayer provide highly sensitive and specific detection of d-VP with the lowest detectable concentration of 1 ng mL −1 and a wide detection range from 1 to 265 ng mL −1.

Graphene-promoted 3,4,9,10-perylenetetracarboxylic acid nanocomposite as redox probe in label-free electrochemical aptasensor

Graphene/3,4,9,10-perylenetetracarboxylic acid (GPD) with three-dimensional porous structure has been successfully synthesized and served as redox probe to construct ultrasensitive electrochemical aptasensor. The GPD nanocomposite shows promoted electrochemical redox-activity of 3,4,9,10-perylenetetracarboxylic acid (PTCA) with an obvious well-defined cathodic peak from −0.7 to 0 V that never been seen from graphene or PTCA, which avoids miscellaneous redox peaks of PTCA in electrochemical characterization, offering a novel redox probe for electrochemical sensors with highly electrochemical active area and conductivity. To the best of our knowledge, this is the first study that utilizes PTCA self-derived redox-activity as redox probe in electrochemical sensors. Moreover, the interesting GPD possesses the advantages of membrane-forming property, providing a direct immobilization of redox probes on electrode surface. This simple process not only diminishes the conventional fussy immobilization of redox probes on the electrode surface, but also reduces the participation of the membrane materials that acted as a barrier of the electron propagation in redox probe immobilization. With thrombin as a model target, the redox probe-GPD based label-free electrochemical aptasensor shows a much higher sensitivity (a detection range from 0.001 nM to 40 nM with a detection limit of 200 fM) to that of analogous aptasensors produced from other redox probes.

Label‐free electrochemical aptasensor for thrombin detection with platinum nanoparticles and blocking reagent horseradish peroxidase as enhancers

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with platinum nanoparticles (Pt) and blocking reagent horseradish peroxidase (HRP) as enhancers. A Nafion®-graphene-coated electrode was first modified with an electrochemical probe of methylene blue (MB) through electrostatic interaction. Then Pt was electrodeposited onto an electrode for immobilization of the thrombin aptamer (TBA). Subsequently, HRP served as blocking reagent instead of bovine serum albumin (BSA). With the synergistic effect between Pt and HRP, the prepared aptasensor showed a superior catalytic efficiency toward H(2) O(2) in the presence of MB. After the combination of target thrombin on electrode surface, the TBA-thrombin complex made a barrier for electrocatalysis of Pt and HRP and inhibited the electrotransfer, resulting in a greater decrease in MB signals. As a result, the proposed approach showed a high sensitivity and a much wider linearity to thrombin in the range from 0.005 to 50 nM with a detection limit of 1 pM.

Label-free electrochemical aptasensor for thrombin detection based on the nafion@graphene as platform

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with nafion@graphene as platform. With electrostatic interaction between nafion and methylene blue (MB), positive charged MB was successfully assembled on nafion@graphene modified electrode surface, which provided amounts of redox probes for electrochemical aptasensor. In the presence of thrombin, the thrombin aptamer (TBA) on the electrode surface would catch the target on the electrode interface, which made a barrier for electrons and inhibits the electro-transfer, resulting in the decreased differential pulse voltammetry signals of MB. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range 0.01-50 nM with a detection limit of 6pM.

Label-free, regenerative and sensitive surface plasmon resonance and electrochemical aptasensors based on graphene

Label-free, regenerative and sensitive surface plasmon resonance (SPR) and electrochemical aptasensors based on graphene for the detection of α-thrombin have been reported, which propose a new, simple way for protein recognition with high sensitivity and selectivity.

Label-free Electrochemical Cocaine Aptasensor Based on a Target-inducing Aptamer Switching Conformation

A novel label-free electrochemical nucleic acid aptasensor for the determination of cocaine by the immobilization of thiolated self-assembled DNA sequences on a gold nanoparticles-modified electrode is presented. When cocaine was complexed specifically to the aptamer, the configuration of the nucleic acid aptamer switched to a locked structure and the interface of the biosensor changed, resulting in a variation of the corresponding peak current of an electrochemical probe ([Fe(CN)(6)](3-/4-)). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to characterize modifications of the electrode surface. The peak current was detected by differential pulse voltammetry (DPV). Under the optimized experimental conditions, the presented sensor exhibits a nice specificity towards cocaine. The decrease of the peak current response of the aptasensor has a linear relationship with the concentration of cocaine ranging from 1.0 × 10(-6) to 1.5 × 10(-4) mol L(-1) with a detection limit of 3 × 10(-7) mol L(-1) at 3σ. The proposed aptasensor can be easily regenerated by the denaturalization of aptamer-target complexes in a heated water bath at 80-90°C. Besides, this biosensor has a high reproducibility and selectivity, which can be a promising method to detect cocaine in real samples.

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.

Solid-State Probe Based Electrochemical Aptasensor for Cocaine: A Potentially Convenient, Sensitive, Repeatable, and Integrated Sensing Platform for Drugs

Aptamers, which are artificial oligonucleotides selected in vitro, have been employed to design novel biosensors (i.e., aptasensors). In this work, we first constructed a label-free electrochemical aptasensor introducing a probe immobilization technique by the use of a layer-by-layer (LBL) self-assembled multilayer with ferrocene-appended poly(ethyleneimine) (Fc-PEI) on an indium tin oxide (ITO) array electrode for detection of cocaine. The Fc-PEI and gold nanoparticles (AuNPs) were LBL assembled on the electrode surface via electrostatic interaction. Then, cocaine aptamer fragments, SH-C2, were covalently labeled onto the outermost AuNP layer. When the target cocaine and cocaine aptamer C1 were present simultaneously, the SH-C2 layer hybridized partly with C1 to bind the cocaine, which led to a decreased differential pulse voltammetry (DPV) signal of Fc-PEI. This DPV signal change could be used to sensitively detect cocaine with the lowest detectable concentration down to 0.1 microM and the detection range up to 38.8 microM, which falls in the the expected range for medical use of detecting drug abuse involving cocaine. Meanwhile, the sensor was specific to cocaine in complex biologic fluids such as human plasma, human saliva, etc. The sensing strategy had general applicability, and the detection of thrombin could also be realized, displayed a low detection limit, and exhibited worthiness to other analytes. The aptasensor based on the array electrode held promising potential for integration of the sensing ability in multianalysis for simultaneous detection.

Layer-by-layer electrochemical biosensor with aptamer-appended active polyelectrolyte multilayer for sensitive protein determination

Herein, we report two simple label-free electrochemical aptasensors for protein detection using layer-by-layer (LBL) self-assembled multilayers with ferrocene-appended poly(ethyleneimine) (Fc-PEI), carbon nanotubes (CNTs) and aptamer. In one sensing strategy, the Fc-PEI, CNTs and DNA aptamer are LBL assembled on the electrode surface via electrostatic interaction. In the presence of target, the aptamer on the outermost layer of the LBL self-assembled multilayer would catch the target on the electrode interface, which makes a barrier for electrons and inhibits the electro-transfer, resulting in the decreased DPV signals of Fc-PEI. Using this strategy, a wide detection range (0.3–165 ng ml −1) for model target thrombin is obtained, with a low detection limit of 0.14 ng ml −1. In the similar sensing strategy for detection of lysozyme, a wide detection range (0.2 ng ml −1 to 1.66 μg ml −1) and a low detection limit (0.17 ng ml −1) are obtained. These results prove that the LBL sensing strategies developed possess sensitivity, selectivity, stability and generality.