Highly sensitive analysis strategies of microRNAs based on electrochemiluminescence

The ultrasensitive detection of microRNAs (miRNAs) is currently pursued for the diagnosis of diseases. Due to its outstanding sensitivity, electrochemiluminescence (ECL) is expected to be very effective toward the above goal. In this short review, bioanalytical strategies currently employed in ECL detections of miRNAs are summarized. ECL sensors based on electrochemiluminescent resonance energy transfer (ERET), hybridization chain reaction (HCR), strand displacement reaction (SDR), and other strategies, have an extremely low detection limit of 10^?18 M miRNA. In particular, the establishment of miniaturized ECL sensors has shown great potential for point-of-need testing of diseases.     

Sandwich-type electrochemiluminescence immunosensor based on Ru-silica@Au composite nanoparticles labeled anti-AFP

A simple and sensitive sandwich-type electrochemiluminescence immunosensor for α-1-fetoprotein (AFP) on a gold nanoparticles (nano-Au) modified glassy carbon electrode (GCE) was developed by using Ru-silica (Ru(bpy)₃²⁺-doped silica) doped Au (Ru-silica@Au) composite as labels. The primary antibody, anti-AFP was first immobilized on the gold nanoparticles modified electrode due to the covalent conjugation, then the antigen and the Ru-silica@Au composite nanoparticles labeled secondary antibody was conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The surfaces of Ru-silica nanoparticles were modified via the assemble of Au nanoparticles. The prepared Ru-silica@Au composite nanoparticles own the large surface area, good biocompatibility and highly effective electrochemiluminescence properties. The morphologies of the Ru-silica@Au composite nanoparticles were investigated by using transmission electronic microscope (TEM). The Ru-silica@Au composite nanoparticles labeled anti-AFP/AFP/bovine serum albumin (BSA)/anti-AFP/nano-Au modified GCE electrode was evaluated by means of cyclic voltammetry (CV) and electrogenerated chemiluminescence (ECL). The immunosensor performed high sensitivity and wide liner for detection AFP in the range of 0.05-50 ng/mL and the limit detection was 0.03 ng/mL (defined as S/N = 3).     

Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy

Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0 V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003–20 and 0.001–10 ng mL⁻¹ with the detection limits down to 1.0 and 0.8 pg mL⁻¹, respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.     

On-line galvanic cell generated electrochemiluminescence determination of acyclovir based on the flow injection sampling

A new simple, rapid and cost effective flow injection (FI) electrochemiluminescence (ECL) method was described for the determination of acyclovir (9,2-hydroxyethoxy) methyl guanine. In the presence of acyclovir, the luminol electrochemiluminescence generated by a mini on-line galvanic cell could be greatly sensitized in alkaline medium. The relative electrochemiluminescence intensity was found to increase linearly with increasing concentration of acyclovir, which was corroborated by the calculated correlation coefficient value of 0.9994 (n=7). The limit of detection was 1.6×10⁻⁷ mol l⁻¹ and the limit of quantification was 7.9×10⁻⁷ mol l⁻¹. The proposed method was applied to the determination of acyclovir in pharmaceutical formulations. The reliability of the assay method was established by parallel determination and by standard-addition method. Experiment results demonstrated the described mini analysis system, while being simple and less time consuming, was accurate, precise and reproducible (R.S.D. = 1.6%, recoveries = 99 - 103%). Further experiments indicated that there was no significant difference between the results obtained by the proposed and official methods.     

Recent advances in electrochemiluminescence imaging analysis based on nanomaterials and micro-/nanostructures

In this review, the basic principles and apparatus of ECL imaging were briefly introduced at first. Then several latest and representative applications of ECL imaging based on nanomaterials and micro-/nanostructures were overviewed. Finally, the superiorities and challenges in ECL imaging for further development were discussed.     

Recent advances in electrochemiluminescence imaging analysis based on nanomaterials and micro-/nanostructures

Electrochemiluminescence (ECL) is a kind of luminescent phenomenon caused by electrochemical reactions. Based on the advantages of ECL including low background, high sensitivity, strong spatiotemporal controllability and simple operation, ECL imaging is able to visualize the ECL process, which can additionally achieve high throughput, fast and visual analysis. With the development of optical imaging technique, ECL imaging at micro- or nanoscale has been successfully applied in immunoassay, cell imaging, biochemical analysis, single-nanoparticle detection and study of mechanisms and kinetics of reactions, which has attracted extensive attention. In this review, the basic principles and apparatus of ECL imaging were briefly introduced at first. Then several latest and representative applications of ECL imaging based on nanomaterials and micro-/nanostructures were overviewed. Finally, the superiorities and challenges in ECL imaging for further development were discussed.     

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.     

Determination of adrenaline by using inhibited Ru(bpy)3 electrochemiluminescence

Adrenaline was found to inhibit strongly the electrochemiluminescence (ECL) from the Ru(bpy)₃²⁺/tripropylamine system when a working Pt electrode was maintained at 1.05 V (versus Ag/AgCl) in pH 8.0 phosphate buffer. On this basis, a flow injection (FI) procedure with inhibited electrochemiluminescence detection has been developed for determination of adrenaline. The method exhibited a good reproducibility, sensitivity, and stability with a detection limit (signal-to-noise ratio = 3) of 7.0×10⁻⁹ mol l⁻¹ and dynamic concentration range of 2×10⁻⁸ to 1×10⁻⁴ mol l⁻¹. The relative standard deviation was 2.2% for 1.0×10⁻⁶ mol l⁻¹ adrenaline (n=11). The method was successfully applied to the determination of adrenaline in pharmaceutical samples. Moreover, ECL emission spectra, UV-Vis absorption spectra and cyclic voltammograms of Ru(bpy)₃²⁺/tripropylamine/adrenaline were studied. The inhibition mechanism has been proposed as the interaction of electrogenerated Ru(bpy)₃^2+* and the o-benzoquinone derivatives, adrenochrome and adrenalinequinone, at the electrode surface.     

分子印迹固相萃取-电化学发光检测牛奶中氯霉素

基于氯霉素(CAP)能强烈抑制Ru(bpy)32+/TPA体系的电化学发光(ECL)信号,结合分子印迹固相萃取(MISPE)样品前处理技术,建立了一种高灵敏度检测牛奶中CAP残留量的方法。在最优实验条件下,体系的ECL猝灭值ΔI与CAP浓度呈良好线性关系,线性范围为1.0×10-13～1.0×10-11g/mL,检测限为3×10-12g/mL,精密度和准确度好,可用于牛奶中CAP残留量的测定。     

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.     

Effect of Ionic Strength on the Electrochemiluminescence Generation by Tris(2,2'-bipyridyl)ruthenium(II)/Tri-n-propylamine

Electrochemiluminescence(ECL) is a powerful transduction technique used in biosensing and in vitro diagnosis, while the mechanism of ECL generation is complicated and affected by various factors. Herein the effect of ionic strength on ECL generation by the classical tris(2,2'-bipyridyl)ruthenium(II)[Ru(bpy)₃²⁺]/tri-n-propylamine(TPrA) system was investigated. It is clear that the ECL intensity decreases significantly with the increase of ionic strength, most likely arising from the reduced deprotonation rate of TPrA^+·. We further combined microtube electrode(MTE) with ECL microscopy to unravel the evolution of ECL layer with the variation of ionic strength. At a low concentration of Ru(bpy)₃²⁺, the thickness of ECL layer(TEL) nearly kept unchanged with the ionic strength, indicating the surface-confined ECL generation is dominated by the oxidative-reduction route. While at a high concentration of Ru(bpy)₃²⁺, ECL generation is dominated by the catalytic route and TEL increases remarkably with the increase of ionic strength, because of the extended diffusion length of Ru(bpy)₃³⁺ at a reduced concentration of TPrA^·.     

Application of the Ru(bipy)2(dpp)2+ / C2O4 2− electrochemiluminescence reaction to the determination of phenol

The electrochemiluminescence (ECL) of the ruthenium di(2,2′-bipyridine)- (4,7-diphenyl-1,10-phenanthroline) complex (Ru-bipy-dpp) produced on a glassy carbon electrode was studied by cyclic voltammetry. The anodic oxidation of Ru-bipy-dpp produces ECL in the presence of oxalate in oxygen-free aqueous solutions. Threefold ECL efficiencies were obtained for Ru-bipy-dpp relative to Ru(bipy)₃ as a standard. The ECL of Ru-bipy-dpp is quenched by both oxygen and phenol. The luminescence intensity was proportional to the concentration of phenol in the range of 5–100 μM. At a phenol concentration of 100 μM, the ECL of Ru-bipy-dpp peaking at 597 nm was completely quenched. Correspondence: Dan Xiao, College of Chemistry and Chemical Engineering, Sichuan University, Chengdu 610065, P.R. China     

Sensors based on galvanic cell generated electrochemiluminescence and its application

In this paper, a novel electrochemiluminescence (ECL) imaging sensor array was developed for determination of hydrogen peroxide (H₂O₂), which was based on Cu/Zn alloy galvanic cell generated ECL. In alkaline solution, Cu/Zn galvanic cell was formed because of corrosion effect, the galvanic cell could supply stable potential for ECL generation of luminol, and the weak ECL emission could be enhanced by H₂O₂. The galvanic cell sensor array was designed by putting Cu/Zn alloy in 96-well microtiter plates separately. The relative ECL intensity was proportional with the concentration of hydrogen peroxide in the range of 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol l⁻¹ and the detection limit was 3.0 × 10⁻⁷ mol l⁻¹ (3σ), the relative standard deviation (R.S.D.) for 11 parallel measurements of 1.0 × 10⁻⁵ mol l⁻¹ H₂O₂ was 4.0%.     

A novel immunoassay system and bioseparation process based on thermal phase separating polymers

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyMPAAm precipitates out of water above a critical temperature of 31°C, enabling a polymerbound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.     

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

电感耦合等离子体质谱用于多组分免疫分析研究进展

免疫分析在临床医学检测领域具有重要的地位.本课题组提出了基于电感耦合等离子体质谱(ICP-MS)的免疫分析方法,利用元素标记技术结合ICP-MS检测实现多组分免疫分析.随后,研究人员在该领域做了大量研究工作,证明该方法可用于从小分子、蛋白质、核酸到细胞等一系列生物样品的检测.本文综述了基于ICP-MS免疫分析方法的特点,对其发展方向进行了展望,希望为该领域的研究工作提供参考.     

Polyelectrolyte-based electrochemiluminescence enhancement for Ru(bpy)3 loaded by SiO2 nanoparticle carrier and its high sensitive immunoassay

In this paper the strong electrochemiluminescence (ECL) nanoparticles have been prepared based on the anionic polyelectrolyte sodium polyacrylate (PAA)-ECL enhancement for Ru(bpy)₃²⁺, which were loaded by the carrier of SiO₂ nanoparticle. There were two kinds of Ru(bpy)₃²⁺ for the as-prepared nanoparticles, the doped one and the exchanged one. The former was loaded inside the ECL nanoparticles by doping, in a form of ion-pair macromolecules PAA–Ru(bpy)₃²⁺. The corresponding ECL was enhanced about 2 times owing to the doping increase of Ru(bpy)₃²⁺. The latter was loaded on the PAA-doped Nafion membrane by ion exchange. The corresponding ECL was enhanced about 3 times owing to the ion-exchanging increase of Ru(bpy)₃²⁺. At the same time, ECL intensity of the doped-inside Ru(bpy)₃²⁺ was further enhanced 13 times because polyelectrolyte PAA in the doped membrane could obviously enhance electron transfer between the doped Ru(bpy)₃²⁺ and the working electrode. Furthermore, based on hydrophobic regions of the doped membrane antibody labeling could be easily realized by the as-prepared nanoparticles and then a high sensitive ECL immunoassay for HBsAg was developed. The linear range was between 1.0 and 100 pg mL⁻¹ (R² = 0.9912). The detection limit could be as low as 0.11 pg mL⁻¹ (signal-to-noise ratio = 3).     

光导纤维电化学发光葡萄糖传感器的研究

以碳糊为固定化载体 ,将葡萄糖氧化酶固定在碳糊电极上 ,制成了光导纤维电化学发光葡萄糖生物传感器。葡萄糖的酶催化反应、鲁米诺的电化学氧化和化学发光反应在电极表面同时发生 ,因此该传感器的信号响应在 1 0 s内达到发光强度峰值。葡萄糖浓度在 1 .0× 1 0 -5～ 2 .0× 1 0 -2 mol/L范围内与发光强度呈线性关系 ,检出限为 6.4× 1 0 -6mol/L,可应用于市售饮料中葡萄糖的测定     

Determination of epinephrine based on its enhancement for electrochemiluminescence of lucigenin

Epinephrine was found to be able to strongly enhance the electrochemiluminescence (ECL) of lucigenin system by using the anodic potential sweep. Based on which, a novel ECL method for the determination of epinephrine was developed. Under the optimum condition, the enhanced ECL intensity was linear with the epinephrine concentration in the range of 4.0 × 10⁻⁸ to 2.0 × 10⁻⁷ mol L⁻¹. The detection limit (defined as S/N = 3) was 2.4 × 10⁻⁸ mol L⁻¹, and the relative standard deviation was 2.7% for 1.0 × 10⁻⁷ mol L⁻¹ epinephrine (n = 11). The method was successfully applied to the determination of epinephrine in pharmaceutical samples with satisfactory results. In addition, the possible mechanism for the lucigenin ECL system in the presence of epinephrine has also been discussed.