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.     

A electrochemiluminescence aptasensor for detection of thrombin incorporating the capture aptamer labeled with gold nanoparticles immobilized onto the thio-silanized ITO electrode

A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as β- and γ-thrombins had negligible response, which indicated a high specificity of α-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analysis.     

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.     

A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification

In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS₂), we prepared poly(diallyldimethylammonium chloride)–graphene/molybdenum disulfide (PDDA–G–MoS₂) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA–G–MoS₂ (PdNPs/PDDA–G–MoS₂) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H₂O₂. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H₂O₂, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis.     

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.     

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.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

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.     

Aptamer-based Biosensor for Detection of Phenylalanine at Physiological pH

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

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.     

Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

Label-free aptasensor for platelet-derived growth factor (PDGF) protein

A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN)₆]^3−/4−, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R_ct was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.     

An electronic channel switching-based aptasensor for ultrasensitive protein detection

Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 × 10⁻⁶ μg mL⁻¹ (22.7 fM, 220 zmol in 10-μL sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 × 10⁻⁶ to 4.44 × 10⁻¹ μg mL⁻¹. We believe that the described sensing concept here might open a new avenue for the detection of proteins and other biomacromolecules.     

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.     

An aptamer-based biosensor for sensitive thrombin detection

A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using quantum dots as electrochemical label. CdSe quantum dots were labeled to the secondary aptamer, which were determined by the square wave stripping voltammetric analysis after dissolution with nitric acid. The aptasensor has a lower detection limit at 1 pM, while the sample consumption is reduced to 5 μl. The proposed approach shows high selectivity and minimizes the nonspecific adsorption, so that it was used for the detection of target protein in the human serum sample. Such an aptamer-based biosensor provides a promising strategy for screening biomarkers at ultratrace levels in the complex matrices.     

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.     

A novel low-cost and easy to develop functionalization platform. Case study: Aptamer-based detection of thrombin by surface plasmon resonance

A novel low-cost platform to assess biomolecular interactions was investigated using surface plasmon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalized with a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dextran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The high end commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor (TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1-150 nM both in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on data derived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stability and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.     

Polymerized ionic liquid-wrapped carbon nanotubes: The promising composites for direct electrochemistry and biosensing of redox protein

Polymerized ionic liquid-wrapped carbon nanotubes (PIL-CNTs) were firstly designed for direct electrochemistry and biosensing of redox proteins. The CNTs were coated successfully with polymerized ionic liquid (PIL) layer, as verified by transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and Fourier transform infrared (FT-IR) spectroscopy. The PIL-CNTs were dispersed better in water and showed superior electrocatalysis toward O₂ and H₂O₂ comparing to pristine CNTs and the mixture of IL monomer and CNTs. With glucose oxidase (GOD) as a protein model, the direct electrochemistry of the redox protein was investigated on the PIL-CNTs modified glassy carbon (GC) electrode and excellent direct electrochemical performance of GOD molecules was observed. The proposed biosensor (GOD/PIL-CNTs/GC electrode) displayed good analytical performance for glucose with linear response up to 6 mM, response sensitivity of 0.853 μA mM⁻¹, good stability and selectivity.     

Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection

A novel and ultrasensitive sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Core/shell Fe₃O₄/Au magnetic nanoparticles (AuMNPs) loading aptamer1 (Apt1) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5–100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in protein detection and disease diagnosis.     

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H₂O₂ (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.