A sensitive and rapid immunoassay for quantification of CA125 in human sera by capillary electrophoresis with enhanced chemiluminescence detection

In this paper we have presented a sensitive and rapid immunoassay (IA) method by capillary electrophoresis with an enhanced chemiluminescence detection system (CE-CL) based on the catalytic effects of horseradish peroxidase (HRP) on the luminol-hydrogen peroxide reaction. The conditions for the CL reaction and electrophoresis were systematically investigated using HRP as a model sample. The linear range from 2.5 x 10(-11) to 1.0 x 10(-9) mol/L (R = 0.999), and the detection limit of 1.0 x 10(-12) mol/L (signal-to-noise ratio = 3) for HRP were achieved using para-iodophenol as CL enhancer. The relative standard deviations of the migration time and peak area for 5.0 x 10(-10) mol/L HRP (n = 7) were 0.26 and 4.8%, respectively, using a CE system with a home-built CL detector. Under the optimal condition, the HRP-labeled CA125 antibody (Ab) and the Ab-antigen complex were well separated within 4 min by CE using a high-pH buffer (pH 10.20). The assay was successfully used for quantification of CA125 in human sera from health controls and patients associated with ovarian cancer, and the recoveries of the standard addition experiments were 93-109%. Our primary results demonstrated that IA based on CE-CL detection is a powerful tool for clinical diagnosis combined with these commercial IA kits.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

癌胚抗原毛细管电泳-化学发光均相免疫分析

设计了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测均相免疫分析新方法。采用四苯硼酸钠增强luminol-H2O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物。测定癌胚抗原的线性范围2.0~80.0μg/L(R=0.9921),检出限为0.1μg/L(绝对检出限为0.75 fg)。     

癌胚抗原毛细管电泳-化学发光均相免疫分析

建立了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测的均相免疫分析新方法.采用四苯硼钠增强luminol-H2 O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物.测定癌胚抗原的线性范围2.0～80.0 μg/L(R=0.9921),检出限为0.1 μg/L(绝对检出限为0.75 fg).     

毛细管电泳-化学发光法测定人血清中的异烟肼

基于碱性介质中异烟肼对laminol-K3Fe(CN)6化学发光体系的增敏作用,设计了一个经毛细管电泳(CE)分离,在线化学发光检测异烟肼的新方法.研究并优化了毛细管电泳分离及化学发光检测的条件.在优化的实验条件下,该方法测定异烟肼的线性范围为4.0×10-7～1.0×10-5g/mL(R2=0.9984),检出限(3σ)为1×10-8g/mL,对6.0×10-6g/mL的异烟肼进行6次平行测定,其相对标准偏差为4.0%.方法已用于血清中异烟肼的测定.     

流动注射化学发光法灵敏检测人血清白蛋白

基于人血清白蛋白(HSA)在碱性介质中对luminol-H2O2化学发光体系有很强的增敏作用,提出了一种流动注射化学发光测定HSA的新方法。在优化条件下,HSA的线性范围为7.5×10-10～2.8×10-7mol/L,检出限为9.1×10-11mol/L,样品检测频率达102个/h。对2.0×10-8mol/L HSA平行测定11次,RSD为0.9%。方法可应用于实际样品人血清中HSA含量的测定。结合化学发光光谱和紫外可见吸收光谱,对该反应机理进行了探讨。     

Utilization of magnetic nanobeads for analyzing haptoglobin in human plasma as a marker of Alzheimer’s disease by capillary electrophoretic immunoassay with laser-induced fluorescence detection

Alzheimer’s disease (AD) is a neurodegenerative disorder resulting from an impaired cholinergic function with loss of cognitive activity in the brain. Haptoglobin is a useful biomarker for AD analysis. Compared to the conventional enzyme-linked immunosorbent assay for haptoglobin analysis, the proposed immunoassay procedure reduces sample analysis time by approximately 55 min. Therefore, immunoassay was coupled with capillary electrophoresis (CE) to determine haptoglobin concentrations indirectly by using magnetic nanobeads (MBs) as a support and laser-induced fluorescence detection. In human plasma sample, the haptoglobin was immobilized on the MBs and reacted with the purified anti-haptoglobin antibody. The optimum separation time for the analyte was shorter than 6 min at 25 °C with a fused-silica capillary column of 40.2 cm × 50 μm ID (effective length 30 cm) and a run buffer containing 25 mM phosphate (pH 8.0) with 0.01% poly(ethylene oxide) (PEO). When using Atto 495 NHS ester as an internal standard (IS) (250.0 ng mL⁻¹), the linear range of the proposed method for indirect determination of haptoglobin was 0.2–3.0 mg mL⁻¹. The method was further used to monitor the course of AD in patients with behavioral and psychological symptoms of dementia (BPSD).     

Cation-exchange antibody labeling for simultaneous electrochemical detection of tumor markers CA15-3 and CA19-9

We report on a new kind of non-covalent multi-label electrochemical immunoassay that was applied to simultaneously quantify the tumor markers CA15-3 and CA19-9. The method employs a nanohybrid composed of an ionomer and conductive titanium dioxide nanoparticles that act as a matrix support for the antibodies. The two antibodies (anti-CA153 and anti-CA199) were labeled (a) with a cobaltous dipyridine complex, and (b) with methylene blue. Labeling is based on cation-exchange interaction rather than on covalent conjugation. The redox potentials of the two labels are separated by an interval of 0.3 V. The resulting sandwich-type immunosensor was read out by differential pulse voltammetry. The potential sites and currents of the two redox probes reflect the concentration of the two analytes. The two analytes were determined with a detection limit of 1.6 U?mL^?1 for CA19-9, and of 0.3 U?mL^?1 for CA15-3.

Application of capillary electrophoretic immunoassay to analysis of estradiol in women's serum

Summary A rapid and sensitive capillary electrophoretic immunoassay is described for determination of estradiol in women's serum. Addition of thermally reversible hydrogel in the buffer, serving as a replaceable packing material, improved the reproducibility of the method. Using a laser-induced fluorescence detector this method can be applied to the determination of estradiol at concentrations as low as 30.6 pg mL¹. Estradiol levels in 16 normal women's serum were measured at the range 115≈370 pg mL¹. The results of this method have been found to correlate well with those of chemiluminescent immunoassay.     

Direct capillary electrophoretic detection of carbohydrate-deficient transferrin in neat serum

Transferrin, an iron transport protein found in serum and cerebrospinal fluid, is known to be microheterogeneous with respect to its carbohydrate and sialic acid content. The forms of transferrin deficient in sialic acid and/or carbohydrate, termed carbohydrate-deficient transferrin (CDT), have been of clinical interest for almost two decades as a result of the initial finding that elevated CDT concentrations are associated with chronic, excessive alcohol abuse. We demonstrate the utility of capillary electrophoresis for examining the CDT sialoform profile via the direct electrophoresis of serum. The need for negligible preelectrophoresis sample preparation and absence of postelectrophoresis processing dramatically decreases analysis time compared to slab gel-based separations. Using a fluorocarbon-coated capillary containing a hydroxyethyl cellulose/borate buffer, the high resolution separation of serum components is effected in less than 30 min. Under these conditions, the beta region proteins (including transferrin) are well resolved from the alpha-2 and gamma zone proteins in a window where the individual transferrin sialoforms can be detected. The usefulness of this method is demonstrated with the electrophoresis of serum from subjects known to be either non-alcoholic and alcoholic.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

Sensitive Laser induced fluorescence detection of Polyamine-Fluoresceinisothiocyanate-derivatives after capillary zone electrophoretic separation

Capillary zone electrophoresis (CZE) has been applied for the separation and quantification of the polyamines putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), after their derivatization with the fluoresceinisothiocyanate isomer I (FITC).The derivatization conditions (character of the derivatization medium and concentration, organic modifier, pH and temperature) and CZE operation conditions have been optimized with respect to a sensitive Laser induced fluorescence detection (_excitation=488 nm/_detection=520 nm). A complete separation of CAD from PUT was impossible under the conditions chosen. The detection limits and relative standard deviations for SPM, SPD, CAD, PUT were determined to be 2.9 (12%), 3.2 (7.6%), 0.7 (4.6%), 1.2 (7.6%) nmol/L, respectively. The method has been applied to a pine needle extract.List of abbreviations a intercept - b slope - b_norm normalized slope of the calibration plot - CAD cadaverine - CZE capillary zone electrophoresis - DL detection limit - EOF electroosmotic flow - FITC fluoresceinisothiocyanate - FMOC 9-fluorenylmethyl chloroformate - HEC hydroxyethyl-cellulose - LIF Laser induced fluorescence - MW molecular weight - OPA o-phthalaldehyde - P/ACE programmable/automatical capillary electrophoresis - PUT putrescine - q charge - RSD relative standard deviation - SDS sodium dodecylsulfate - SPD spermidine - SPM spermine - t_migr migration time - w basic peak width - _eff effective mobility     

Determination of myoglobin in human serum by high-performance liquid chromatography with chemiluminescence detection

A highly sensitive method for the determination of myoglobin in serum is described, based on high-performance size-exclusion chromatography with chemiluminescence detection. Serum proteins are separated according to their molecular masses on columns packed with TSK-SW gel and those containing haem are detected selectively by post-column chemiluminescence reaction with luminol using a conventional fluorimetric detector. The method is rapid (30 min) and sufficiently sensitive for the diagnosis of myocardial infarction. The minimum detectable myoglobin concentration is 10 ng/ml.     

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E₂) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H₂O₂ chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E₂, without cross-reaction for the major steroids, including estrone (E₁), estriol (E₃), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E₂ in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E₂-6-HRP conjugate and anti-E₂ polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51 pg mL⁻¹ with a larger working range of 15-1000 pg mL⁻¹. The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E₂ in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E₂ in human serum.     

Sensitive and efficient determination of gabapentin in human serum based on capillary electrophoresis with laser-induced fluorescence detection

A sensitive and efficient analytical method for gabapentin (GBP) in human serum based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection has been established. 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new synthesized fluorescent reagent, was used for precolumn derivatization of the non-fluorescent drug in serum. γ-Aminobutyric acid (GABA) was used as an internal standard (I.S.). The best derivative condition was obtained in phosphate buffer (pH 8) at room temperature for 10 min. Optimal separation and detection were obtained with a background electrolyte (BGE) of 3.5 × 10^?2 M phosphate buffer (pH 5.5) and laser-induced fluorescence detection excited at 473 nm. The method developed for GBP was linear over the concentration range of 4.0 × 10^?9 to 4.0 × 10-7 M. The concentration limit of detection was 2.0 × 10^?10 M (signal-to-noise ratio = 3). The sensitive method was used for the determination of GBP in serum samples.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Determination of medroxyprogesterone acetate residues by CE immunoassay with chemiluminescence detection

A rapid and simple method is developed for the determination of medroxyprogesterone acetate (MPA) by CE immunoassay with chemiluminescence (CL). This method is based on the competitive reactions between horseradish peroxidase (HRP)-labeled MPA (MPA-HRP) and free MPA with anti-MPA antiserum. The influencing factors on the electrophoresis and CL detection were studied completely and the optimal conditions of separation and determination were obtained. The linear range was 2.0-50 nmol/L and the LOD for MPA was 0.9 nmol/L. The present method was applied to the analysis of pork tissues.     

Sensitive and rapid determination of nitric oxide in human serum using microchip capillary electrophoresis with laser-induced fluorescence detection

Microfluidic chip capillary electrophoresis with laser-induced fluorescence detection is employed for direct determination of trace nitric oxide in human blood using diaminorhodamines as the fluorescence probe. Factors influencing the separation and detection processes were systematically studied. Complete and fast separation of the highly fluorescent triazole formed was achieved within 45 s, and the relative standard deviations values of migration time and peak area were less than 3%. The detection limit of NO was 3.0 nmol.L^-1 (at a signal-to-noise ratio of 3) and the liner range was from 1.0?×?10^-8?mol.L^-1 to 3.0?×?10^-6?mol.L^-1. The method has been applied to the determination of NO in serum of healthy persons and patients suffering from diseases, with recoveries varying from 92.65 to 98.43%.