A novel aptasensor based on silver nanoparticle enhanced fluorescence

In the present study, we report a novel aptasensor based on silver nanoparticle enhanced fluorescence for the detection of adenosine. First, the distance dependence nature of silver nanoparticle enhanced fluorescence was investigated through fluorescent dyes modified oligonucleotides to control the spacing distance between dyes and AgNP. The results showed that the fluorescence intensity reached the maximum value with the spacing distance of dyes about 8 nm from AgNP surface. The fluorescence intensity decreases when the spacing distance is either above or below this value. Based on this result, a fluorescence switch is constructed. In the "OFF" state, without the target molecules, there is a greater spacing distance between the Cy3 dyes and the AgNP giving comparatively lower fluorescence intensity. While in the "ON" state, in the presence of target molecules, the fluorescence signals increased for the conformation structure change of the aptamer which shorten the spacing distance between the Cy3 dyes and the AgNP to 8 nm. Using adenosine as target, the aptasensor produced a linear range from 200 nM to 200 μM with a correlation coefficient of 0.9949 and the detection limit was 48 nM estimated using 3σ. The aptasensor was also found to be specific in targeting adenosine. The presented method shows a new strategy of combining aptamer recognition and silver nanoparticle for fluorescence signal enhancement and increasing sensitivity.     

In situ enzymatic silver enhancement based on functionalized graphene oxide and layer-by-layer assembled gold nanoparticles for ultrasensitive detection of thrombin

A highly specific in situ amplification strategy was designed for ultrasensitive detection of thrombin by combining the layer-by-layer (LBL) assembled amplification with alkaline phosphatase (ALP) and gold nanoparticles (Au) mediated silver deposition. High-density carboxyl functionalized graphene oxide (FGO) was introduced as a nanocarrier for LBL assembling of alkaline phosphatase decorated gold nanoparticles (ALP-Au), which was further adopted to label thrombin aptamer II. After sandwich-type reaction, numerous ALP were captured onto the aptasensor surface and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP), which in situ generated ascorbic acid (AA), reducing Ag(+) to Ag nanoparticles (AgNPs) for electrochemical readout. Inspiringly, the in situ amplification strategy with ethanolamine as an effective blocking agent showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal, which was favorable to enhance the sensitivity of aptasensor. Our novel dramatic signal amplification strategy, with a detection limit of 2.7fM, showed about 2-3 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal enzyme-based electrochemical assay.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.     

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 6 pM.     

Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample

A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)(6)(3-/4-). The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20ng/mL and a detection limit of 0.3ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics.     

Aptamer-peptide conjugates as a new strategy to modulate human α-thrombin binding affinity

Aptamers are single-stranded RNA or DNA molecules that specifically recognize their targets and have proven valuable for functionalizing sensitive biosensors. α-thrombin is a trypsin-like serine proteinase which plays a crucial role in haemostasis and thrombosis. An abnormal activity or overexpression of this protein is associated with a variety of diseases. A great deal of attention was devoted to the construction of high-throughput biosensors for accurately detect thrombin for the early diagnosis and treatment of related diseases. Herein, we propose a new approach to modulate the interaction between α-thrombin and the aptamer TBA₁₅. To this end, TBA₁₅ was chemically conjugated to two peptide sequences (TBA-G₃FIE-Ac and TBA-G₃EIF-Ac) corresponding to a short fragment of the acidic region of the human factor V, which is known to interact directly with exosite I. Surface Plasmon Resonance (SPR) results showed enhanced analytical performances of thrombin with TBA-G₃EIF-Ac than with TBA wild-type, reaching a limit of detection as low as 44.9 pM. Electrophoresis mobility shift assay (EMSA) corroborated the SPR results. Molecular dynamics (MD) simulations support experimental evidences and provided further insight into thrombin/TBA-peptide interaction. Our findings demonstrate that the combination of TBA₁₅ with key interacting peptides offers good opportunities to produce sensitive devices for thrombin detection and potential candidates to block thrombin activity.     

A multi-amplification aptasensor for highly sensitive detection of thrombin based on high-quality hollow CoPt nanoparticles decorated graphene

In this work, we have successfully demonstrated a facile strategy to incorporate high-quality hollow CoPt bimetal alloy nanoparticles (HCoPt) onto reduced graphene oxide sheet (HCoPt-RGs). An advanced sandwich-type electrochemical aptasensor for thrombin was proposed by using the HCoPt-RGs conjugates as secondary label. The formed conjugates provided large surface area for loading plentiful redox probe thionine (Thi), horseradish peroxidase (HRP) and secondary aptamer (Apt II) with good stability and friendly biocompatibility, indicating their superior properties in electroactive mediator enrichment and biomolecule immobilization. Furthermore, activated by glutaraldehyde (GA), the chitosan-hollow CoPt alloy nanoparticle (CS-HCoPt) film can greatly facilitate the capture of primary aptamer (Apt I) and dramatically reduce the nonspecific binding. Excellent sensitivity was obtained by detecting the conspicuously enhanced electrochemical signal of Thi, which was amplified by HCoPt alloy nanoparticles and HRP toward the catalytic reduction of H₂O₂. The aptasensor displayed excellent performance for thrombin with a wide linearity in the range from 1.0 × 10⁻¹² to 5.0 × 10⁻⁸ M and a relatively low detection limit of 3.4 × 10⁻¹³ M. Moreover, the resulted aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy could pave a promising way for the wide application of graphene in clinical research.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

Enzyme-free fluorescence aptasensor for amplification detection of human thrombin via target-catalyzed hairpin assembly

Aptamers have many advantages, such as simple synthesis, good stability, high binding affinity and wide applicability, making them suitable candidates for protein detection. Since the disease-related protein may be present in very small amounts in biological samples, the development of amplification paths for aptasensors is essential. In this paper, we develop a simple and enzyme-free amplified aptasensor for protein detection via target-catalyzed hairpin assembly. This aptasensor contains two DNA hairpins termed as H1 and H2. H1, which is modified at its 5' and 3' ends with a fluorophore and a quencher respectively, consists of the aptamer sequence of human thrombin. Meanwhile, H2 is partially complementary to H1. These two hairpins H1 and H2 interact slowly with each other. Upon the addition of target protein, it can facilitate the opening of the hairpin structure of H1 and thus accelerate the hybridization between H1 and H2, resulting in the significant fluorescence enhancement of the system. By monitoring the change in fluorescence intensity, we could detect the target protein with high sensitivity. The detection limit of this aptasensor is 20 pM, which is more than two orders of magnitude lower than that of reported unamplified aptasensors. Furthermore, this amplified aptasensor shows high selectivity toward its target protein. Thus, the proposed aptasensor could be used as a simple, sensitive and selective platform for target protein detection.     

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals. Such designed aptamer/thrombin/AuNPs sensing system could not only improve the detection sensitivity compared to the reported impedimetric aptasensors but also provide a promising signal amplified model for aptamer-based protein detection. In this paper, we realize a sensitive detection limit of 0.02 nM, with a linear range of 0.05-18 nM. Meanwhile, the effect of 6-mercaptohexanol (MCH) and 2-mercaptoethanol (MCE) on the modification of the electrode is investigated.     

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.     

Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles

The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below ~12.5 μM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols.     

Nano RNA aptamer wire for analysis of vitamin B₁₂

A simple and stable RNA aptamer-based colorimetric sensor for the detection of vitamin B₁₂ using gold nanoparticles (AuNPs) has been proposed. Vitamin B₁₂ belongs to the B vitamin group and prevents pernicious anemia, which is caused by vitamin B₁₂ deficiency. A highly stable RNA aptamer that binds to vitamin B₁₂ was employed by structural modification of 2′-hydroxyl group of ribose to 2′-flouro in all pyrimidines indicated in lowercase in 35-mer aptamer (5′ GGA Acc GGu GcG cAu AAc cAc cuc AGu GcG AGc AA 3′). Aggregation of AuNPs was specifically induced by desorption of the vitamin B₁₂ binding RNA aptamer from the surface of AuNPs as a result of the aptamer–target interaction, leading to the color change from red to purple. The level of detection of vitamin B₁₂ was 0.1 μg/ml by successful optimization of the amount of the aptamer, AuNPs, salts, and stability of the aptamer. Analysis of vitamin B₁₂ was carried out, and the observed recovery was 92 to 95.3% with a relative standard deviation in the range of 2.08 to 8.27%. The results obtained were compared with those of the ultraviolet–visible (UV–vis) spectrometry method. This colorimetric aptasensor is advantageous for on-site detection with the naked eye.     

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer‐templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA–AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as‐prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA–AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd.     

适配子纤维蛋白靶向性验证及其抗凝活性测定

目的:验证适配子G81的纤维蛋白靶向性,评估适配子对凝血系统的影响。方法:以复钙法制备鼠源、人源体外纤维蛋白,将不同浓度Cy5.5标记的适配子溶液与之孵育,置于激光共聚焦显微镜下以固定的参数成像,用ImageJ软件进行相对荧光强度分析;将适配子G81溶液加入血浆中,通过倍比稀释法得到含浓度梯度适配子的血浆,采用SYSMEX CS-5100全自动血凝仪检测PT、APTT、TT,评估适配子G81对凝血功能的影响。结果:激光共聚焦显微镜显示适配子能与纤维蛋白结合,随着加入适配子量的增加其相对荧光强度逐渐增强,表明适配子可与纤维蛋白结合,统计分析提示荧光强度与适配子存在量效关系;人源、鼠源纤维蛋白结合的荧光强度无统计学差异(P0.05)。在抗凝活性检测中,血浆中适配子G81浓度达到200 pmoL/mL时,各浓度统计分析结果均显示P0.05,表明适配子对PT、APTT、TT的测量均没有统计学差异上的影响。结论:适配子G81具有纤维蛋白靶向性,且当加入的适配子剂量低于200 pmol/mL时对内、外源性凝血功能、凝血酶时间均无明显影响。     

Aptabodies - new type of artificial receptors for detection proteins

We report on a new type of artificial receptor formed by hybridization of two DNA aptamers for human thrombin (aptabody). This aptasensor based on multiwalled carbon nanotubes allowed us to detect thrombin with detection limit of 0.3 nM, which was 3 times better in comparison with conventional aptamer.     

Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.     

In-situ produced ascorbic acid as coreactant for an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) electrochemiluminescence aptasensor

Herein, an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) aptasensor using in-situ produced ascorbic acid as coreactant was successfully constructed for detection of thrombin. Firstly, the composite of Ru(bpy)(3)(2+) and platinum nanoparticles (Ru-PtNPs) were immobilized onto Nafion coated glass carbon electrode, followed by successive adsorption of streptavidin-alkaine phosphatase conjugate (SA-ALP) and biotinylated anti-thrombin aptamer to successfully construct an ECL aptasensor for thrombin determination. In our design, Pt nanoparticles in Ru(bpy)(3)(2+)-Nafion film successfully inhibited the migration of Ru(bpy)(3)(2+) into the electrochemically hydrophobic region of Nafion and facilitated the electron transfer between Ru(bpy)(3)(2+) and electrode surface. Furthermore, ALP on the electrode surface could catalyze hydrolysis of ascorbic acid 2-phosphate to in-situ produce ascorbic acid, which co-reacted with Ru(bpy)(3)(2+) to obtain quite fast, stable and greatly amplified ECL signal. The experimental results indicated that the aptasensor exhibited good response for thrombin with excellent sensitivity, selectivity and stability. A linear range of 1 × 10(-15)-1 × 10(-8) M with an ultralow detection limit of 0.33 fM (S/N=3) was obtained. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.     

Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π-π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.