Aptamer-based highly sensitive electrochemiluminescent detection of thrombin via nanoparticle layer-by-layer assembled amplification labels

The preparation and use of a new class of signal amplification label, the CdTe quantum dot layer-by-layer assembled polystyrene microbead composite, for amplified ultrasensitive electrochemiluminescent detection of thrombin is described.     

Aptamer-based electrochemical approach to the detection of thrombin by modification of gold nanoparticles

This paper presents a simple electrochemical approach for the detection of thrombin, using aptamer-modified electrodes. The use of gold nanoparticles results in significant signal enhancement for subsequent detection. 1,6-Hexanedithiol was used as the medium to link Au nanoparticles to a bare gold electrode. Anti-thrombin aptamers were immobilized on the gold nanoparticles’ surfaces by self-assembly. The packing density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH₃)₆]³⁺) which were electrostatically bound to the DNA phosphate backbones. The results indicate that the total amount of aptamer probes immobilized on the gold nanoparticle surface is sixfold higher than that on the bare electrode, leading to increased sensitivity of the aptasensor and a detection limit of 1 pmol L⁻¹. Based on the Langmuir model, the sensor signal displayed an almost perfect linear relationship over the range of 1 pmol L⁻¹ to 30 nmol L⁻¹. Moreover, the proposed aptasensor is highly selective and stable. In summary, this biosensor is simple, highly sensitive, and selective, which is beneficial to the ever-growing interest in fabricating portable bio-analytical devices with simple electrical readout procedures.     

Background current reduction and biobarcode amplification for label-free, highly sensitive electrochemical detection of pathogenic DNA

By using exonuclease I and biobarcode nanoparticles, we describe a novel background current reduction strategy for amplified electrochemical detection of uropathogen specific sequences at ultralow concentrations.     

Dual amplification strategy of highly sensitive thrombin amperometric aptasensor based on chitosan-Au nanocomposites

A highly sensitive and selective electrochemical aptasensor for thrombin was developed. By introducing chitosan-gold nanoparticles and horseradish peroxidase (CS-AuNPs-HRP) conjugates to the sensitive union, the thrombin detection signal was dual amplified. The capture probe was prepared by immobilizing an anti-thrombin aptamer on core-shell Fe(3)O(4)-Au magnetic nanoparticles (AuMNPs) and which was served as magnetic separation material as well. The detection probe was prepared from another anti-thrombin aptamer, horseradish peroxidase (HRP), thiolated CS nanoparticle and gold nanoparticle (CS-AuNPs-HRP-Apt2). In the presence of thrombin, the sandwich structure of AuMNPs-Apt1/thrombin/Apt2-CS-AuNPs-HRP was formed and abundant HRP was captured in it. The resultant conjugates are of magnetic characters and were captured onto the surface of a screen printed carbon electrode (SPCE) to prepare the modified electrode by a magnet located on the outer flank of the SPCE. It was demonstrated that the oxidation of hydroquinone (HQ) with H(2)O(2) was dramatically accelerated by the captured HRP. The electrochemical signal, which correlated to the reduction of BQ (the oxidation product of HQ), was amplified by the catalysis of HRP toward the reaction and the enrichment of HRP on the electrode surface. Under optimized conditions, ultrasensitive and high specific detection for thrombin was realized with the proposed assay strategy. The signal current was linearly correlated to the thrombin concentration in the range of 0.01-10 pM with a detection limit of 5.5 fM (S/N = 3). These results promise extensive applications of this newly proposed signal amplification strategy in protein detection and disease diagnosis.     

Highly sensitive lable-free electrochemical aptasensor for thrombin detection with cobalt hexacyanoferrate as the electrochemical probe

A highly sensitive label-free electrochemical aptasensor has been constructed for the electrochemical detection of thrombin (TB), where two layers of cobalt hexacyanoferrate (CoHCF) redox probes sandwiched with carbon nanotubes–Nafion were directly immobilized on the electrode surface by electrodeposition. Through the strong interaction between CN^? (CoHCF) and gold nanoparticles (GNPs), GNPs were assembled on the CoHCF-modified electrode for the immobilization of thiolated thrombin aptamers (TBA). In the presence of target TB, TBA on the electrode surface could catch TB to form TBA–TB complex, which made a barrier for the electron transfer, resulting in a greater decrease in CoHCF redox probe signals. Thus, the proposed aptasensor showed a high sensitivity and a much wider linearity to TB in the range of 1.0 pg/mL?～?1.0 μg/mL with a detection limit of 0.28 pg/mL.     

Carbon nanospheres for highly sensitive electrochemical detection of sequence-specific protein-DNA interactions

A carbon nanosphere-based electrochemical detection method is developed for the highly sensitive detection of Sp1-DNA interactions.     

Aptamer-based origami paper analytical device for electrochemical detection of adenosine

Paper biosensors: an origami sensor is printed on a single piece of paper, folded into a three-dimensional fluidic device, and encapsulated by thermal lamination. Aptamer is trapped in the fluidic channel, where it binds to the target and releases an enzyme to generate a signal. The device is read out using a digital multimeter.     

A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H₂O₂. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.     

Aptamer-based turn-on fluorescent four-branched quaternary ammonium pyrazine probe for selective thrombin detection

In this thrombin detection system, the bright fluorescence of TASPI is almost eliminated by the DNA aptamer TBA (turn-off); however, in the presence of thrombin, it specifically binds to TBA by folding unrestricted TBA into an anti-parallel G-quadruplex structure and then releasing TASPI molecules, resulting in vivid and facile fluorescence recovery (turn-on).     

Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy

A label-free sensing assay for ethanolamine (EA) detection based on G-quadruplex-EA binding interaction is presented by using G-rich aptamer DNA (Ap-DNA) and electrochemical impedance spectroscopy (EIS). The presence of K⁺ induces the Ap-DNA to form a K⁺-stabilized G-quadruplex structure which provides binding sites for EA. The sensing mechanism was further confirmed by circular dichroism (CD) spectroscopy and EIS measurement. As a result, the charge transfer resistance (R_CT) is strongly increased as demonstrated by using the ferro/ferricyanide ([Fe(CN)₆]^3−/4−) as a redox probe. Under the optimized conditions, a linear relationship between ΔR_CT and EA concentration was obtained over the range of 0.16 nM and 16 nM EA, with a detection limit of 0.08 nM. Interference by other selected chemicals with similar structure was negligible. Analytical results of EA spiked into tap water and serum by the sensor suggested the assay could be successfully applied to real sample analysis. With the advantages of high sensitivity, selectivity and simple sensor construction, this method is potentially suitable for the on-site monitoring of EA contamination.     

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with β-galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c₅₀ was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.     

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with -galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c₅₀ was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.     

G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection

Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.     

Proximity-based electrochemical biosensor for highly sensitive determination of methyltransferase activity using gold nanoparticle-based cooperative signal amplification

We describe a turn-on electrochemical biosensor for the detection of methyltransferases (MTases) causing DNA adenine methylation. This biosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles (AuNPs), and a signal probe-dragging strategy. A double-stranded DNA (dsDNA) containing identical MTase and methylation-resistant endonuclease (Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding. The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene (Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags (at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to the log of the MTase activity in the 0.1 to 40 U·mL⁻¹ range. The dynamic range extends from 0.05 to 50 U·mL⁻¹, and the limit of detection is 0.024 U·mL⁻¹ (at an S/N ratio of 3). The assay is well reproducible and highly selective. In our perception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other MTase by exchanging the corresponding DNA.

Proximity-based electrochemical biosensor for highly sensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity using methylation-resistant cleavage coupled with gold nanoparticle based cooperative signal amplification.

     

Fabrication of highly catalytic silver nanoclusters/graphene oxide nanocomposite as nanotag for sensitive electrochemical immunoassay

Silver nanoclusters and graphene oxide nanocomposite (AgNCs/GRO) is synthesized and functionalized with detection antibody for highly sensitive electrochemical sensing of carcinoembryonic antigen (CEA), a model tumor marker involved in many cancers. AgNCs with large surface area and abundant amount of low-coordinated sites are synthesized with DNA as template and exhibit high catalytic activity towards the electrochemical reduction of H₂O₂. GRO is employed to assemble with AgNCs because it has large specific surface area, super electronic conductivity and strong π-π stacking interaction with the hydrophobic bases of DNA, which can further improve the catalytic ability of the AgNCs. Using AgNCs/GRO as signal amplification tag, an enzyme-free electrochemical immunosensing protocol is designed for the highly sensitive detection of CEA on the capture antibody functionalized immunosensing interface. Under optimal conditions, the designed immunosensor exhibits a wide linear range from 0.1 pg mL⁻¹ to 100 ng mL⁻¹ and a low limit of detection of 0.037 pg mL⁻¹. Practical sample analysis reveals the sensor has good accuracy and reproducibility, indicating the great application prospective of the AgNCs/GRO in fabricating highly sensitive immunosensors, which can be extended to the detection of various kinds of low abundance disease related proteins.     

Aptamer-based detection of thrombin by acoustic method using DNA tetrahedrons as immobilisation platform

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (K_D) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by K_D = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH₃)₆]³⁺ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.     

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 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.