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.     

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.     

量子点双标记的Fe3O4@Au磁性纳米DNA电致发光型传感器

合成了Fe3O4@Au磁性纳米粒子,并根据单链寡聚核苷酸(ss-DNA)杂交原理,利用量子点电化学发光,构建了DNA电化学传感器.在磁控玻碳电极(MCGCE)表面,将5′-SH-ssDNA捕获探针自组装在Fe3O4@Au磁性纳米粒子上,然后与目标DNA互补的一端杂交形成dsDNA,再与双标记了量子点的5′-NH2-ssDNA-NH2-3′信号探针杂交形成三明治杂交的DNA.应用循环伏安法对DNA的固定与杂交进行了表征.目标DNA浓度在1.0×10-13～1.0×10-11 mol/L范围与其响应的ECL信号呈线性关系,检出限为1.8×10-14mol/L.由于采用量子点双标记法,检测的灵敏度显著提高.     

Aptamers-based sandwich assay for silver-enhanced fluorescence multiplex detection

In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.     

利用互补核酸杂交富集金胶实现信号扩增的电化学凝血酶蛋白生物传感器研究

介绍了一种利用互补核酸杂交富集金胶实现信号扩增的蛋白质生物传感器. 以凝血酶蛋白为研究对象, 利用凝血酶蛋白相对应的两段核酸适配体, 将适配体Ⅰ固定在磁性颗粒上, 用于特异性地捕获蛋白, 将适配体Ⅱ标记金胶作为检测信标. 由凝血酶蛋白和相对应的两段核酸适配体构建三明治结构的凝血酶蛋白生物传感器. 另外, 再通过信标金胶上过剩的核酸适配体链与另一段标记有金胶的互补核酸进一步杂交, 获得金胶的选择性聚集, 实现了信号扩增. 通过信号扩增, 使此传感器的灵敏度大大提高, 对凝血酶蛋白的检测下限可达到4.52×10-15 mol/L. 平行测定浓度为7.47×10-14 mol/L的凝血酶8次, 其RSD为3.0%. 该生物传感器对凝血酶蛋白有很好的特异性, 其它蛋白如溶菌酶和牛血清白蛋白的存在对于检测没有影响.     

A novel electrochemiluminescence aptasensor for protein based on a sensitive N-(aminobutyl)-N-ethylisoluminol-functionalized gold nanoprobe

A novel electrochemiluminescence (ECL) aptasensor for platelet-derived growth factor B chain (PDGF-BB) assay was developed by assembling N-(aminobutyl)-N-ethylisoluminol functionalized gold nanoparticles (ABEI-AuNPs) with aptamers as nanoprobes. In the protocol, the biotinylated aptamer capture probes were first immobilized on a streptavidin coated gold nanoparticle (AuNPs) modified electrode, afterwards, the target PDGF-BB and the ABEI-AuNPs tagged aptamer signal probe were successively attached to the modified electrode by virtue of the dimer structure of PDGF-BB to fabricate a "sandwich" conjugate modified electrode, i.e. an aptasensor. ECL measurement was carried out with a double-step potential in carbonate buffer solution containing H(2)O(2). The aptasensor showed high sensitivity and selectivity toward PDGF-BB and specificity toward PDGF-BB aptamer. The detection limit was as low as 2.7 × 10(-14) M. In this work, the ABEI-AuNPs synthesized by a simple seed growth method have been successfully used as aptamer labels, which greatly amplified the ECL signal by binding numbers of ABEI molecules on the surface of AuNPs. The ABEI-AuNPs signal amplification is superior to other reported signal amplification strategies based on aptamer-related polymerase chain reaction or functionalized nanoparticles in simplicity, stability, labeling property and practical applicability. And the ABEI-AuNPs based nanoprobe is more sensitive than the luminol functionalized AuNPs based nanoprobe. Moreover, such an ultra-sensitive and low-cost assay can be accomplished with a simple and fast procedure by using a simple ECL instrumentation. The aptasensor was also applied for the detection of PDGF-BB in human serum samples, showing great application potential. Given these advantages, the ECL aptasensor is well suited for the direct, sensitive and rapid detection of protein in complex clinical samples.     

Sensitive Electrochemical Aptasensor for Thrombin Detection Based on Graphene Served as Platform and Graphene Oxide as Enhancer

A sensitive electrochemical aptasensor was developed with conductive graphene served as platform and inert graphene oxide (GO) as enhancer. An electrodeposited nano-Au layer was firstly formed on conductive graphene modified glass carbon electrode surface for further immobilizing of electrochemical redox probe hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was formed for immobilizing of thrombin aptamer (TBA). In the presence of thrombin, the TBA on the electrode surface could bind with thrombin, which made a barrier for electrons and inhibited the electro-transfer, resulting in the decreased electrochemical signals of NiHCFNPs. Owing to the non-conductivity property of graphene oxide, further decreased electrochemical signals of NiHCFNPs could be obtained via the sandwich reaction with GO-labeled TBA. According to the signal changes before the thrombin recognition and after sandwich reaction, trace detection of thrombin could be achieved. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range from 0.005 nM to 50 nM with a detection limit of 1 pM.     

Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design

Aptamer-based fluorescence detection of platelet-derived growth factor (PDGF) on a functionalized diamond surface was demonstrated. In this work, a sandwich design based on the ability of PDGF to bind with aptamers at its two available binding sites was employed. It was found that this sandwich design approach significantly increases the fluorescence signal intensity, and thereby a very low detection limit of 4 pM was achieved. The effect of the ionic strength of MgCl(2) buffer solution was also investigated, and the most favourable binding for PDGF-BB occurred at a Mg(2+) concentration of 5.5 mM. Since the aptamers bind to the target PDGF with high affinity, fluorescence detection exhibited high selectivity towards different biomolecules. The high reproducibility of detection was confirmed by performing three cycles of measurements over a period of three days.     

Ru(bpy)3<Superscript>2+</Superscript>/β-cyclodextrin-Au nanoparticles/nanographene functionalized nanocomposites-based thrombin electrochemiluminescence aptasensor

In this study, a functionalized nanocomposite-based electrochemiluminescence (ECL) sensor for detecting thrombin was developed. First, Ru(bpy)₃²⁺/β-cyclodextrin-Au nanoparticles (β-CD-AuNPs)/nanographene (NGP) composites were used to modify the glassy carbon electrode (GCE) surface, and then aptamers (TBA1 and TBA2 with a 1:1 M ratio) were labeled with ferrocene (Fc) acting as the probes and were attached to the composite via the host–guest recognition between β-CD and Fc. In the absence of thrombin, the quenching of Fc to [Ru(bpy)₃]²⁺ was maintained, and “signal-off” ECL was observed. However, because of the specific combination of the aptamer probes and thrombin, the configuration of aptamer probes changed and escaped from the electrode surface once thrombin appears, which results in the quenching disappearance, and the ECL signal was changed from “off” to “on.” Meanwhile, the application of nanocomposites amplified the effect of “signal-on.” By this strategy, thrombin was detected with high sensitivity and with a detection limit down to 0.23 pM. Moreover, the relatively simple ECL sensor exhibited excellent reproducibility with at least 6 cycles of recovering the original signal.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

A sensitive, non-damaging electrochemiluminescent aptasensor via a low potential approach at DNA-modified gold electrodes

Electrochemiluminescence (ECL)-based biosensors are often used in the field of DNA- and protein-assay. Although ruthenium complex-based ECL is sensitive, its high exciting potential may lead to oxidation damage to biomolecules. For the first time, a non-damaging, low potential ECL aptasensor was constructed for bioassay with lysozyme as a model. After a single-stranded anti-lysozyme aptamer was attached to a gold electrode, a double stranded (ds)-DNA formed with its complementary strand. Ru(phen)(3)(2+), as an ECL probe, was intercalated into the ds-DNA. The hybridization of lysozyme with its aptamer led to the dissociation of ds-DNA because of the high stability of the aptamer-lysozyme and therefore the Ru(phen)(3)(2+) intercalated into ds-DNA was released. A low potential ECL was observed at the ds-DNA-modified electrode because ds-DNA was able to preconcentrate tripropylamine (TPA) and acted as the acceptor of the protons released from protonated TPAH(+). While the DNA sequence (anti-lysozyme aptamer) was used as the special recognition element for lysozyme, the formed ds-DNA also provided a micro-environment for low potential ECL. The low potential ECL aptasensor achieved the determination of lysozyme with a detection limit of 0.45 pM. The day-to-day precision (RSDs, n = 5) for the determination of lysozyme was lower than 5%, showing the reliability of the aptasensor. The regeneration of the aptasensor confirmed that the low potential for ECL could decrease oxidation damage to biomolecules. Further, the proposed method was successfully used to analyze diluted egg white sample directly. The protocol exhibited a promising platform for sensitive bioassay and could be further applied for the development of other low potential ECL sensing systems.     

基于Ru(bpy)32+-SiO2纳米颗粒的核酸适配体传感器对凝血酶的电致化学发光检测

本文应用核酸适配体构建了一种新型的电致化学发光检测蛋白体系。两个核酸适配体结合凝血酶的两个不同位点,利用这两核酸适配体与凝血酶的高亲和力构建三明治传感体系检测凝血酶。一个核酸适配体固定在金电极上用来捕获凝血酶,另一个标记有包裹电致化学发光活性物Ru(bpy)₃²⁺的二氧化硅纳米颗粒,用来检测电致化学发光信号。此核酸适配体传感器对凝血酶具有特异识别性,电致化学发光信号与凝血酶的浓度直接相关,非特异性识别的牛血红蛋白、牛血清白蛋白不干扰测定。由于在检测的核酸适配体上标记的纳米颗粒包裹有多个发光活性物,因此大大提高了发光效率和灵敏度,此法对凝血酶的线性响应范围为2.0 fmol•L^-1～2.0 pmol•L^-1,检测限可达1.0 fmol•L^-1。     

Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)3(2+)-doped silica nanoparticle aptasensor via target protein-induced strand displacement

A sensitive and selective aptasensor using tri(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)₃²⁺-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (ΔI_ECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible ΔI_ECL. Under the optimal conditions, the ΔI_ECL was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)₃²⁺ molecules were labeled on the DNA probe.     

Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin

In this paper, a microchip-based sandwich-type aptasensor is developed for the detection of human thrombin. The SH-aptamer/thrombin/alkaline phosphatase-functionalized aptamer (ALP-aptamer) system was constructed in the microfluidic channels. And the substrate solution containing 4-aminophenyl phosphate (p-APP) was introduced to the microchannels for the end-column electrochemical detection. The on-chip aptasensor has a broad linear response range of 1–100 pM with a detection limit of 1 pM, which shows high sensitivity and specificity. The system was then applied to detect thrombin in human serum sample. Therefore, the on-chip aptasensor has a great promise for detecting and screening ultratrace levels of biomarkers in the complex matrices.     

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)₃]²⁺ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1 pM of PDGF-BB. A calibration curve with a linearity range from 1 pM to 10 nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.     

Development and application of an electrochemiluminescent flow-injection cell based on CdTe quantum dots modified electrode for high sensitive determination of dopamine

A nano-liter sized flow-cell is developed for constructing a flow injection analysis (FIA) system with electrochemiluminescent (ECL) detection. A sensitive ECL electrode is applied as the working electrode in this flow-cell. It is obtained by immobilizing the composite of CdTe quantum dots (QDs), carbon nanotubes (CNTs) and chitosan (Chit) on indium tin oxide (ITO) glass. The CdTe QDs were synthesized in our lab and possessed a high quantum yield. It has been demonstrated as an efficient anodic ECL material with the triethylamine (TEA) as the co-reactant. The flow-cell gives the stable ECL background under optimized conditions for parameters such as electrolytic pulse, concentration of TEA and flow rate, etc. The sensitive ECL quenching response of dopamine (DA) is realized on this FIA system within the linear range from 10 pM to 4 nM and a detection limit as low as 3.6 pM. It is practically used to determine the neurotransmitters in cerebro-spinal fluid (CSF) with DA as the index and with an average recovery of 94%.     

A signal-on electrogenerated chemiluminescent biosensor for lead ion based on DNAzyme

A highly reproducible and sensitive signal-on electrogenerated chemiluminescence (ECL) biosensor based on the DNAzyme for the determination of lead ion was developed. The ECL biosensor was fabricated by covalently coupling 5′-amino-DNAzyme-tagged with ruthenium bis (2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-ethylenediamine (Ru1-17E′) onto the surface of graphite electrode modified with 4-aminobenzoic acid, and then a DNA substrate with a ribonucleotide adenosine hybridized with Ru1-17E′ on the electrode. Upon binding of Pb²⁺ to the Ru1-17E′ to form a complex which catalyzed the cleavage of the DNA substrate, the double-stranded DNA was dissociated and thus led to a high ECL signal. The signal linearly increases with the concentration of Pb²⁺ in the range from 5.0 to 80 pM with a detection limit of 1.4 pM and a relative standard derivation of 2.3%. This work demonstrates that using DNAzyme tagged with ruthenium complex as an ECL probe and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity, good stability and significant regeneration ability is promising approach.     

A Novel Electrogenerated Chemiluminescence Reaction Scheme Using Core-Shell LuminoI-Based SiO2 Nanoparticles as a Regulator and Its Analytical Application

A novel core-shell luminol-based SiO2 nanoparticle While these nanoparticles were used as electrogenerated was synthesized by two step micro-emulsion method. chemiluminescence （ECL） reagent, the electrochemical （EC） reaction as well as the subsequent chemiluminescence （CL） reaction not only could be separated spatially, but also presented high efficiency for analytical purpose. In this case, the core-shell luminol-based SiO2 nanoparticles offered more potential to avoid the contradiction between the EC and the CL reaction conditions. A new ECL method based on the nanoparticle was developed, and isoniazid was selected as a model analyte to illustrate the characteristics of this new ECL method. Under the selected conditions, the proposed ECL response to isoniazid concentration was linear in the range of 1.0 ×10^-10 to 1.0 × 10^-6 g/mL with 2 × 10^-11g/mL detection limit.     

A Highly Sensitive Electrochemiluminescence Spermine Biosensor Based on Au−Ag Bimetallic Nanoclusters

In this study, we found that spermine (SPM) could enhance electrochemiluminescence (ECL) intensity of Au−Ag bimetallic nanoclusters (Au−Ag BNCs) with triethylamine (TEA) as a co-reactant. An ECL sensor was fabricated to detect SPM, which contained Au−Ag BNCs as ECL emitters and conductive hydrogel containing polyaniline-amino trimethylene phosphonic acid (PANI-ATMP) as an immobilizing matrix. The increased ECL intensity of SPM was linear with the logarithm of concentrations of SPM in the range of 1 pM to 10 μM with high selectivity, excellent stability, and the limit of detection is 0.11 pM (S/N=3). This sensor realized the detection of SPM in urine samples, which was fast and economic, possessing potential applications for SPM detection in clinical and bioanalysis.     

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM).