Integration of a flow-type chemiluminescence detector on a glass electrophoresis chip

A glass electrophoresis microchip integrated a flow-type chemiluminescence (CL) detection cell has been developed and evaluated. The chip pattern is a double-T-type electrophoretic sample injection and separation combining with a Y-type chemiluminecent detector. The double-T geometry allows for high-efficiency sample injection and geometric definition of sample plug size. The branch of Y was used as CL reagent channel, and the CL reagent was delivered by a lab-made micropump. Bis[(2,4,6-trichlorophenyl)]oxalate-H₂O₂ CL system was employed to detect dansyl amino acids. On this microchip, dansyl-phenylalanine and -sarcosine were successfully separated by electrophoresis and detected within 250 s. The detection limits (S/N=3) of dansyl-phenylalanine and -sarcosine could reach to 2.8 and 3.2 μM, respectively, due to the vigorous dilution of sample with CL reagent and timely removal of the waste solution from reaction area.     

On-line chemiluminescence detection for isoelectric focusing of heme proteins on microchips

In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol-H2O2 enhanced by para-iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c, myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c, myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N=3) were 1.2x10(-7), 1.6x10(-7), and 1.0x10(-10) M, respectively.     

Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection

We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol-microperoxidase-hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm x 40 mm x 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor.     

微流控芯片直接化学发光检测方法研究

基于Ru(bipy)₃²⁺的化学发光反应,建立了一种新型微流控芯片化学发光检测方法.以草酸为研究对象,探讨了表面活性剂CTAB的浓度、化学发光反应酸度以及Ru(bipy)₃²⁺浓度等因素对峰形以及检测灵敏度和重现性的影响.草酸的线性范围为1.0×10^-4~5.0×10^-6mol/L,检测限达到2.0×10^-6mol/L.该方法的建立为微流控芯片分离检测某些有机酸、药物、环境物质及核酸等提供了新方法.     

A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection

A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H(2)O(2) system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30 s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0 nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.     

采用甲基丙烯酸丁酯整体微柱芯片系统测定血样中痕量异烟肼

将合成的甲基丙烯酸丁酯(BMA)整体柱与微流控芯片技术结合,在PMMA芯片上以K3Fe(CN)6-NaOH-异烟肼化学发光体系为样品对象,在优化混合发光试剂比例和流速以及选择适合的洗脱液基础之上,实现了BMA整体微柱对异烟肼样品的富集作用,平均富集倍数和回收率分别达到16.8和84.2%,由此建立了流动注射化学发光(FIA-CL)芯片系统测定血液中痕量异烟肼的浓度的方法,可有效地实现异烟肼血药浓度分析的片上预处理和快速测定,检出限低于0.2 mg/L。     

异硫氰酸荧光素-人血清白蛋白标记物化学发光反应的研究

采用反相流动注射分析方法,研究了异硫氰酸荧光素（FITC）－人血清白蛋白（HSA）标记物的化学发光性能和反应的最佳条件,建立了化学发光测定人血清白蛋白的新方法。体系的化学发光强度与人血清白蛋白的含量在0．08－16μg/mL内呈线性关系,方法的检出限为0．04μg/mL。讨论了蛋白质标记物化学发光性增强的原因。     

Microchip-based multiplex electro-immunosensing system for the detection of cancer biomarkers

Microfluidic-based microchips have become the focus of research interest for immunoassays and biomarker diagnostics. This is due to their aptitude for high-throughput processing, small sample volume, and short analysis times. In this paper, we describe the development of a microchip-based multiplex electro-immunosensing system for simultaneous detection of cancer biomarkers using gold nanoparticles and silver enhancer. Our microchip is composed of biocompatible poly(PDMS) and glass substrates. To fix the antibody-immobilized microbeads, we used pillar-type microfilters within a reaction chamber. An immunogold silver staining (IGSS) method was used to amplify the electrical signal that corresponded to the immune complex. To demonstrate this approach, we simultaneously assayed three cancer biomarkers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) on the microchip. The electrical signal generated from the result of the immunoreaction was measured and monitored by a PC-based system. The overall assay time was reduced from 3-8 h to about 55 min when compared to conventional immunoassays. The working range of the proposed microchip was from 10(-3) to 10(-1) microg/mL of the target antigen.     

A study on the system of nonaqueous microchip electrophoresis with on‐line peroxyoxalate chemiluminescence detection

This article describes a further development of our previously reported miniaturized analysis system of microchip electrophoresis with on‐line chemiluminescence detection. The system, developed first time for nonaqueous microchip electrophoresis with peroxyoxalate chemiluminescence detection, consists of a suction pressure device for sample or reagent introduction, a constant voltage supplied for electrophoretic separation, an either hydrophilic or hydrophobic porous polymer plug for preventing chemiluminescence reagent flowing upstream and a spiral detection channel for enhancement of both detection sensitivity and reproducibility. Especially, by using organic solvent as BGE medium, the developed system avoided the interface problem between aqueous running buffer and low‐water‐content chemiluminescence solvent in previous reports. The influencing factors on chemiluminescence signal were optimized using rhodamine 6G as model molecule. The system performance was further investigated in the experiment of separation of hydrophilic rhodamine dyes and analysis of hydrophobic polycyclic aromatic hydrocarbon, providing the detection limit (S/N = 3) of 3.5 nmol/L for rhodamine 123, 6.8 nmol/L for rhodamine 6G, and 60 nmol/L for 1‐aminopyrene, respectively. The experimental results showed the system offered a number of benefits, including compact structure, high sensitivity, good reproducibility, and a wide range of application prospect.     

连续进样的重力驱动微流控芯片流动分析系统

建立了一种可连续进样的集成化重力驱动微流控芯片流动分析系统.该系统可实现连续高通量引入试样,换样时间仅为15s;还采用了水平贮液池和出口引流管等重力驱动流体控制技术,显著降低了试剂和样品的消耗,提高分析速度,增加系统连续工作时间.利用Luminol-K₃[Fe(CN)₆]-H₂O₂化学发光体系考察了该系统的分析性能,系统对不同试样的分析速度达80～100样/h,对H₂O₂检测的线性范围为1×10^-6～1×10^-4mol/L,检出限为2.0×10^-7mol/L,RSD为0.3%(n=5).     

Microchip electrophoresis coupled with on-line magnetic separation and chemiluminescence detection for multiplexed immunoassay

A facile and universal strategy for multiplexed immunoassay is proposed. The strategy is based on microchip electrophoresis (MCE) coupled with on-line magnetic separation and chemiluminescence (CL) detection. The system consisted of a microchip, an electromagnet, and a photomultiplier. The realization of multiplexed immunoassay protocol involves sampling magnetic nanoparticles (MNPs) labeled antibodies, N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) labeled antigens and free antigens in the precolumn reactor, on-line immunoreaction, capturing the MNPs-immunocomplexes, and the separation of unconjugated ABEI-labeled antigens. After on-line magnetic separation, the free ABEI-labeled antigens were transported into the separation channel, and mixed with hydrogen peroxide (H(2) O(2) ) in the presence of horseradish peroxidase in the postcolumn reactor, and producing CL emission. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. As a proof-of-concept, the multiplexed immunoassay was evaluated for the simultaneous determination of five model analytes (i.e. hydrocortisone, corticosterone, digoxin, testosterone, and estriol). The results exhibited excellent precision and sensitivity, the relative standard deviations for nine times detection were lower than 4.7% for all the five components, and the detection limits of five analytes were in the range of 3.6-4.9 nM. The MCE system was validated using two human serum-based control samples containing five analytes.     

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL⁻¹ in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL⁻¹ was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80–110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level).     

Label-free, non-derivatization CRET detection platform for 6-mercaptopurine based on the distance-dependent optical properties of gold nanoparticles

A label-free, non-derivatization chemiluminescence resonance energy transfer (CRET) detection platform has been developed for the detection of the non-fluorescent small molecule 6-mercaptopurine. This CRET process arose from a chemiluminescent (CL) donor-acceptor system in which the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2)-fluorescein (maximum emission at 521.6 nm) served as the donor and gold nanoparticles (AuNPs, maximum absorption at 520.0 nm) served as the acceptor. This process caused a significant decrease in the CL signal of the TCPO-H(2)O(2)-fluorescein reaction. The presence of 6-mercaptopurine induced an aggregation of AuNPs with the assistance of Cu(2+) ions through cooperative metal-ligand interactions that was accompanied by a distinct change in color and optical properties. The maximum absorption band of the AuNPs was red-shifted to 721.0 nm and no longer overlapped with the CL spectrum of the reaction; as a result, the CL signal was restored. This CRET system exhibited a wide linear range, from 9.0 nmol L(-1) to 18.0 μmol L(-1), and a low detection limit (0.62 nmol L(-1)) for 6-mercaptopurine. The applicability of the proposed CRET system was evaluated by analysis of 6-mercaptopurine in spiked human plasma samples.     

Capillary electrophoretic system incorporating an UV/CL dual detector

We developed a capillary electrophoretic system incorporating an ultra-violet absorption (UV)/chemiluminescence (CL) dual detector, taking advantage of the CL reaction of luminol-hydrogen peroxide and the batch-type CL detection cell. UV detection was carried out using the on-capillary method while CL detection was performed using the end-capillary method. Examination of isoluminol isothiocyanate (ILITC) as a model sample revealed two main peaks with UV detection and one main peak with CL detection. The first peak in the UV detection data corresponded to the main peak in the CL detection data. We then determined that the ILITC sample included natural ILITC as well as an impurity that had absorption behavior but did not have CL properties and labeling ability. Furthermore, the components of a mixture containing glycine, glycylglycine and glycylglycylglycine, all labeled with ILITC, were well separated and detected using the present system. The present system easily, rapidly, and simultaneously produces useful information due to the presence of both UV and CL detectors.     

流动注射-鲁米诺-高碘酸钾-过氧化氢体系的化学发光行为及其应用

详细研究了流动注射－鲁米诺－高碘酸钾－过氧化氢体系化学发光行为，给出了反应的最佳条件。拟定了一种化学发光测定过氧化氢的新方法。方法的检测限为３．０Ｘ１０－８ｍｏｌ／ＬＨ２Ｏ２；线性范围为２．０×１０－７～６．０×１０－４ｍｏｌ／Ｌ。评价了该体系的应用前景。     

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3σ).     

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.     

A novel chemiluminescence technique for quantitative measurement of low concentration human serum albumin.

An efficient and highly sensitive chemiluminescence (CL) technique is proposed in the current study for detection of low levels of human serum albumin (HSA). Chemiluminescence (CL) produced during interaction between fluoresceinyl cypridina luciferin analog (FCLA)-1O2 can be modified with the presence of HSA. The conventional CL technique uses a quenching effect of HSA for its quantitative measurement. We are reporting here that the CL intensity can be enhanced, rather than quenched, by the addition of HSA. The CL signal can be linearly correlated with the HSA concentration over a clinically interesting range of 5 x 10(-9) - 8 x 10(-8) mol L(-1), with a detection limit of 2.5 x 10(-9) mol L(-1). The determination result was consistent with that obtained from conventional methods. One possible mechanism of HSA detection technique using CL enhancement approach is discussed. Intermolecular energy transfer in chemiluminescence systems and changes of microenvironment are likely to be contributors of the CL enhancement with HSA.     

Solubilisation and binding characteristics of a recombinant beta2-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals

The on-line incorporation of cloud point extraction (CPE) to flow injection analysis (FIA) was previously based on the use of a cotton-packed column to entrap the analyte-containing surfactant aggregates after salt-induced CPE, and then the preconcentrated analyte was eluted into a separate detection cell for subsequent chemiluminescence (CL) detection (via the peroxyoxalate CL reaction). In the work, the on-line CPE/FIA technique was improved by the following: (1) sample preconcentration and CL detection were both carried out directly inside the collection column, thus avoiding the decrease in detection sensitivity due to sample dispersion and dilution, and (2) CL detection was performed through the reaction between nitrite and hydrogen peroxide, which is compatible with aqueous samples and should allow for chemical excitation to occur more efficiently inside the collection column. In addition to more effective sample preconcentration, the CL detection of the entrapped analytes directly inside the collection column, i.e., a unique heterogeneous microenvironment in which analyte-containing surfactant aggregates were embedded within the densely packed filtering material, may also contribute to the overall increase in CL intensity (e.g., a CL enhancement factor of ca. 1000). Under optimum experimental conditions, the calibration curve was found to be linear for the CL detection of bilirubin (5 to 120 μg L⁻¹), the limit of detection (S/N = 3) was 1.8 μg L⁻¹, and the R.S.D. was ca. 2.6% (n = 30) for 20 μg L⁻¹ bilirubin. Good agreements were obtained for the determination of total bilirubin in certified reference human serum samples between the present approach and an established clinical method.     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.