Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay

An innovatory ECL immunoassay strategy was proposed to detect the newly developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)(3)(2+). The cocaine-encapsulated liposomes were successfully characterized by TEM. The quantificational calculation proved the ～5.3×10(3) cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng mL(-1) range, and a detection limit down to 0.77 pg mL(-1).     

Highly selective trapping of enteropathogenic E. coli on Fabry-Pérot sensor mirrors

A novel strategy for the enhancement of electrochemiluminescence (ECL) was developed by combining CdSe quantum dots (QDs) with graphene oxide-chitosan (GO-CHIT). The ECL sensor fabricated with CdSe QDs/GO-CHIT composite exhibited high ECL intensity, good biocompatibility and long-term stability, and was used to detect of cytochrome C (Cyt C). The results show that the ECL sensor has high sensitivity for Cyt C with the linear range from 4.0 to 324 μM and the detection limit of 1.5 μM. Furthermore, the ECL sensor can selectively sense Cyt C from glucose and bovine serum albumin (BSA).     

Cathodic electrochemiluminescent behavior of luminol at nafion-nano-TiO2 modified glassy carbon electrode

The electrochemiluminescence (ECL) behavior of luminol on a nafion-nano-TiO(2) modified glassy carbon electrode (nafion-nano-TiO(2)--GCE) was studied. Two ECL peaks (ECL-1 and ECL-2) were found during cathodic potential scanning. ECL-1 at ca -0.4 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and active oxygen anion produced at the GCE surface directly, while ECL-2 at ca -0.9 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and the active oxygen anion catalyzed by TiO(2.) The possible mechanism for the generation of both ECL peaks has been proposed. The reproducibility of the ECL intensities on nafion-nano-TiO(2)--GCE at ECL-1 and ECL-2 was good, with relative standard deviations (n = 10) of 4.3 and 1.3%, respectively. The ECL-2 generated at the nafion-nano-TiO(2)--GCE surface was further developed to detect the dissolved oxygen, and a detection limit of 0.02 mg/L was achieved. The proposed method was applied to detect dissolved oxygen in water with satisfactory result.     

Highly amplified electrochemiluminescence of peroxydisulfate using bienzyme functionalized palladium nanoparticles as labels for ultrasensitive immunoassay

An immunosensor based on the electrochemiluminescence (ECL) of peroxydisulfate was firstly proposed by coupling the cooperation of two enzymes to in situ generate coreactant with palladium nanoparticles (PdNPs) as catalyst for the ECL reaction. PdNPs were previously synthesized, which successfully attached to functional carbon nanotubes (FCNTs), to bind the secondary antibody and bienzyme (horseradish peroxidase and glucose oxidase). Then the prepared bioconjugates were introduced to the electrode via sandwich immunoreactions. Accordingly, a dramatically amplified ECL signal was obtained for that GOD catalyzed glucose to produce H(2)O(2) which was subsequently reduced by HRP to in situ generate O(2), then PdNPs as catalyst for the ECL reaction of peroxydisulfate/O(2). The present immunosensor was used to detect α-1-fetoprotein (AFP) and showed a wide linear range of 1×10(-5)-100ngmL(-1), with a low detection limit of 3.3fgmL(-1)(S/N=3). This new signal amplification strategy for preparation of the ECL immunosensor could be easily realized and has a potential application in ultrasensitive bioassays.     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

Electrogenerated chemiluminescence detection of adenosine based on triplex DNA biosensor

A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2'-bipyridine) ruthenium (Ru(bpy)(3)(2+)) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy)(3)(2+) derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer-adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7×10(-10) mol L(-1) and 2.3×10(-9) mol L(-1) for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.     

TiO(2) nanocrystals electrochemiluminescence quenching by biological enlarged nanogold particles and its application for biosensing

Electrochemiluminescence (ECL) of TiO(2) nanocrystals with different crystal styles modified fluorine-doped tin oxide (FTO) electrode was investigated in H(2)O(2) solution. The amorphous TiO(2) nanospheres were facilely synthesized by the hydrothermal and condensation method. Crystal TiO(2), namely anatase and rutile, were prepared by calcination of the amorphous TiO(2) nanospheres at 450 and 800°C, respectively. The transmission electron microscope (TEM) and electron diffraction pattern were used to characterize the obtained TiO(2) nanoparticles morphology and the corresponding crystal styles. The electrochemical and ECL behaviors were investigated by cyclic voltammetry. The ECL quenching was observed by introduction of gold nanoparticles. Based on the quenching effect, a sensitive glucose ECL biosensor as a model was fabricated by in-situ growing-up gold seeds in AuCl(4)(-) solution induced by biologically generated H(2)O(2). The linear range to detect glucose is from 5.0×10(-7)M to 4.0×10(-3)M with the limit of detection of 2.5×10(-7)M.     

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.     

Fast and Sensitive Detection of Bacillus anthracis Spores by Immunoassay

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.     

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

高灵敏度电化学发光PCR方法检测基因点突变研究

近年来发展了一种用于定量检测基因点突变的电化学发光PCR方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对待测基因进行PCR扩增;随后,采用特定的限制性内切酶对扩增产物进行酶切,由于野生型样品和突变型样品间存在酶切位点的变化,其中只有一种基因型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到样品池中;进行电化学发光检测,通过所得信号的有无可以判断其基因型。我们分别将该法用于Presenilin-1基因和H-ras癌基因的点突变检测,结果均可明显区分突变型样品和野生型样品。该法具有灵敏、快速、简便、安全等优点,是一种实用的基因点突变检测方法。     

Quantitative electrochemiluminescence detection of proteins: Avidin-based sensor and tris(2,2'-bipyridine) ruthenium(II) label

Quantitative electrochemiluminescence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin–avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2′-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2′-bipyridine) ruthenium(II) label was attached to the prepared avidin surface. After binding of BSA labeled with tris(2,2′-bipyridine) ruthenium(II) derivative to the surface-immobilized avidin through biotin, ECL response was generated when the self-assembled modified electrode was immersed in a TPA-containing electrolyte solution. Such double protein labeling protocol with a biotin label for biorecognition and ruthenium label for ECL detection facilitated the detection of protein compared to the classical double antibody sandwich format. The ECL intensity was linearly proportional to the feed concentration of BSA over two orders of magnitude in the range of 15 nM to 7.5 μM. The detection limit was estimated to be 1.5 nM. Further application to the lysozyme analysis was carried out to validate the present approach for an effective and favorable protocol for the quantitative detection of proteins. The dynamic range of lysozyme was from 0.001 g L⁻¹ to 0.1 g L⁻¹ and the detection limit was 0.1 mg L⁻¹. Electrochemical impedance and cyclic voltammetric measurements along with some necessary control experiments were conducted to characterize the successful formation of self-assembled modified electrodes and to grant the whole detection process.     

Detection of Prorocentrum minimum (Pavillard) Schiller with an Electrochemiluminescence–Molecular Probe Assay

The occurrence of harmful algal blooms (HABs) caused by Prorocentrum minimum (Pavillard) Schiller is a crucial subject in the study of HABs. An electrochemiluminescence-molecular probe assay (ECL-MP) was developed to qualitatively and quantitatively detect P. minimum. It was based on the sandwich hybridization integrated with a nuclease protection assay (NPA-SH) and improved by electrochemiluminescence (ECL). An ECL analyzer was established in this study, and it was shown that this analyzer was stable and highly sensitive, with a detection range of 0.4?pmol to 4?nmol Ru(bpy)(3)Cl(2)·6H(2)O under optimal reaction conditions of 1.0?V, 1.0?mA, 1.5?mol·L(-1) TPrA, and pH?7.4. The optimal amount of magnetic beads for separation of labeled NPA probes in a 20-μL hybridization mixture was 4?μg. The ECL counts per second was linear with the number of P. minimum cells in a range of 6.25?×?10(2) to 4?×?10(4), and there was no significant difference between ECL-MP and microscopy, with a 95% confidence level (t test) when individual, mixed cultures and field samples were treated. This study provides a convenient method for fast and accurate detection of P. minimum in the marine environment.     

Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.     

Plasticity Response in the Contralesional Hemisphere after Subtle Neurotrauma: Gene Expression Profiling after Partial Deafferentation of the Hippocampus

Neurotrauma or focal brain ischemia are known to trigger molecular and structural responses in the uninjured hemisphere. These responses may have implications for tissue repair processes as well as for the recovery of function. To determine whether the plasticity response in the uninjured hemisphere occurs even after a subtle trauma, we subjected mice to a partial unilateral deafferentation of the hippocampus induced by stereotactically performed entorhinal cortex lesion (ECL). The expression of selected genes was assessed by quantitative real-time PCR in the hippocampal tissue at the injured side and the contralesional side at day 4 and 14 after injury. We observed that expression of genes coding for synaptotagmin 1, ezrin, thrombospondin 4, and C1q proteins, that have all been implicated in the synapse formation, re-arrangement and plasticity, were upregulated both in the injured and the contralesional hippocampus, implying a plasticity response in the uninjured hemisphere. Several of the genes, the expression of which was altered in response to ECL, are known to be expressed in astrocytes. To test whether astrocyte activation plays a role in the observed plasticity response to ECL, we took advantage of mice deficient in two intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP^−/−Vim^−/−) and exhibiting attenuated astrocyte activation and reactive gliosis. The absence of GFAP and vimentin reduced the ECL-induced upregulation of thrombospondin 4, indicating that this response to ECL depends on astrocyte activation and reactive gliosis. We conclude that even a very limited focal neurotrauma triggers a distinct response at the contralesional side, which at least to some extent depends on astrocyte activation.     

Target-cycling synchronized rolling circle amplification strategy for biosensing Helicobacter pylori DNA

Helicobacter pylori is closely linked to many gastric diseases such as gastric ulcers and duodenal ulcers. Therefore, biosensing H. pylori has attracted wide attention from both scientists and clinicians. Here, we proposed an electrochemiluminescence (ECL)-based platform that could sensitively detect H. pylori DNA. In this platform, a novel target-cycling synchronized rolling circle amplification was used for signal amplification. Silver nanoclusters (Ag NCs) were synthesized on the circle DNA products, embedding them with the ability to catalyze the electrochemical reduction of K₂S₂O₈, in turn resulting in rapid consumption of the ECL co-reactant near the working electrode, and leading to a decrease in the ECL emission intensity. In addition to its excellent stability and selectivity, the proposed strategy had a low detection limit of 10 pM, an indication that it can be beneficially applied to test biosamples. Furthermore, a biosensing chip was designed to improve the throughput and shed new light on large-scale clinical biosensing applications.     

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.     

An ultrasensitive electrochemiluminescent sensor based on a pencil graphite electrode modified with CdS nanorods for detection of chlorogenic acid in honeysuckle

In this paper, a novel and ultrasensitive electrochemiluminescent sensor employing a solvothermal‐synthesized CdS nanorod‐modified pencil graphite electrode (CdS/PGE) for the determination of chlorogenic acid (CA) is fabricated. In the first step, the PGE surface is modified using CdS nanorods. In the next step, the developed electrode is used to detect CA using a electrochemiluminescent (ECL) technique, in which potassium persulfate (K₂S₂O₈) served as a co‐reactant. The possible ECL mechanism is investigated, and the influences of pH and cyclic voltammetric scanning rate on the signal response are studied. The ECL intensity decreases quantitatively in relation to the concentration of the target molecule. Under optimized conditions, the linear correlation between the quenched ECL intensity and the logarithm of CA concentration is observed in the range from 2 × 10^?9 to 8 × 10^?7 mol L^?1 with a limit of detection of 1 × 10^?9 mol L^?1. This proposed method is applied to the analysis of CA in honeysuckle flower, giving recoveries of 99‐107%. The experimental results demonstrate that this ECL sensor shows good stability and reproducibility.