[Quality control of human chorionic gonadotrophin subunit dissociation].

Human chorionic gonadotrophin (hCG), a glycohormone widely used in treatment of infertility, is a heterodimer composed of an alpha- and a beta-subunit. The heterodimer could be dissociated during production and storage with an impact on its bioactivity. A CE-SDS method for quantitative analysis of hCG subunit dissociation was established in this study by optimization of a variety of method conditions including sample preparation buffer compositions, incubation temperature, separation voltage, and capillary temperature. This method was validated for good sensitivity, linearity, precision, and accuracy for both α- and ß-subunit. CE-SDS also showed much better precision and accuracy than SDS-PAGE. The method was successfully used in both recombinant hCG (r-hCG) produced by cell culture and hCG (u-hCG) derived from urine. The CE-SDS method was used in the study of hCG development and stability. Therefore, it is an useful tool for the quality control of hCG.

A self-assemble aptamer fragment/target complex based high-throughput colorimetric aptasensor using enzyme linked aptamer assay.

Enzyme linked aptamer assay (ELAA) uses an aptamer as recognition element and enzyme as signal readout element for establishing different kinds of aptasensors. We reported herein a high-throughput colorimetric aptasensor based on ELAA only requiring a single aptamer sequence for cocaine detection. An anti-cocaine aptamer was cleaved into two fragments, one of which was immobilized on a DNA-BIND 96-well plate via 5'-labeled primary amine and the other one was biotin labeled. The presence of two aptamer fragments and the target molecule led to the formation of aptamer fragments/target complexes. Streptavidin-horseradish peroxidase (SA-HRP) was used to react with biotin in order to obtain quantitative signals. A linear response towards cocaine concentration in the range of 5-200 µM and a detection limit down to 2.8 µM (S/N=3) were achieved. The specificity and application in real sample were validated. Furthermore, a verification test of thrombin detection in the same strategy illustrated its feasibility for not only small molecule but also biomacromolecule. With the advantage of high-throughput, easy operation, high specificity, the colorimetric assay based on ELAA requiring a single aptamer sequence opens up a new approach for detecting different kinds of targets via specific affinity recognition among target and suitably cleaved aptamer fragments.

Cocaine detection by structure-switch aptamer-based capillary zone electrophoresis.

Aptamers, which are nucleic acid oligonucleotides that can bind targets with high affinity and specificity, have been widely applied as affinity probes in capillary electrophoresis (CE). Due to relative weak interaction between aptamers and small molecules, the application of aptamer-based CE is still limited in certain compounds. A new strategy that is based on the aptamer structure-switch concept was designed for small molecule detection by a novel CE method. A carboxyfluorescein (fluorescein amidite, FAM) label DNA aptamer was first incubated with partial complementary strand (CS), and then the free aptamer and the aptamer-CS duplex were well separated and determined by metal cation mediated CE/laser-induced fluorescence. When the target was introduced into the incubated sample, the hybridized form was destabilized, resulting in the changes of the fluorescence intensities of the free aptamer and the aptamer-CS duplex. The length of CS was investigated and 12 mer CS showed the best sensitivity for the detection of cocaine. The presented CE-LIF method, which combines the separation power of CE with the specificity of interactions occurring between target, aptamer, and CS, could be a universal detection strategy for other aptamer-specified small molecules.

A label-free aptasensor for the sensitive and specific detection of cocaine using supramolecular aptamer fragments/target complex by electrochemical impedance spectroscopy.

A simple and label-free aptasensor for sensitive and specific detection of cocaine was developed by measuring the change in electrochemical impedance spectra (EIS), based on the formation of a supramolecular aptamer fragments/substrate complex. An anticocaine aptamer was divided into two fragments, Cx and Cy. Three different sensing interfaces, called Au/Cx5S/MCE, Au/Cy3S/MCE and Au/Cy5S/MCE, were fabricated by immobilizing Cx or Cy on a gold electrode through modifying their 5' or 3' end with a thiolated group followed by the treatment with mercaptoethanol (MCE). The formation of the corresponding supramolecular aptamer fragments/cocaine complex was investigated via monitoring electrochemical impedance spectra in the presence of [Fe(CN)(6)](3-/4-). The interfacial electron transfer resistance (R(et)) was found to depend strongly on the cocaine concentration. Since the supramolecular aptamer fragments/cocaine complex was formed on the electrode surface, the sensing interface strongly affected the sensitivity of the aptasensor. Au/Cx5S/MCE was shown to have good sensitivity within a cocaine detection range of 0.1-20 µM. Moreover, MCE was shown to improve the sensitivity of the aptasensor greatly. Even without the help of amplification or labeling, cocaine concentrations as low as 100 nM could be easily detected by the impedimetric aptasensor developed. The specificity and regeneration of the cocaine aptasensor were also investigated and satisfactory results were obtained. The developed aptasensor was successfully applied to detect the cocaine in biological fluids.

Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis.

Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affinity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC-labeled aptamers were characterized by HPLC-MS and purified by HPLC. After desalination, the ILITC-labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE-CL) by interface of end column reaction mode, the apparatus of which was home-designed and setup. CE-CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H(2)O(2), were optimized first. The system of thrombin and its 29-mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (K(d)) and the binding site number (n), were calculated. The K(d) obtained was 124.0+/-6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE-CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE-CL apparatus and combination with aptamers for a wide range of targets.

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography.

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.

Pseudo-homogeneous immunoextraction of epitestosterone from human urine samples based on gold-coated magnetic nanoparticles.

A pseudo-homogeneous immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) for the specific extraction and quantitative analysis of epitestosterone (17alpha-hydroxy-4-androsten-3-one, abbreviated as "ET") from human urine samples by high-performance liquid chromatography (HPLC) has been developed. Half-IgG of anti-ET monoclonal antibodies were covalently immobilized onto (Fe(3)O(4))(core)-Au(shell) (Fe(3)O(4)@Au) MNPs. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC with UV detection (244nm) within 12min. The standard calibration curve for ET showed good linearity in the range of 20-200ngmL(-1) in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. Limit of detection for ET was 0.06ngmL(-1) due to an enrichment factor of 100-fold was achieved. The results obtained by the present method for spiked human urine samples were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The antibody-conjugated Fe(3)O(4)@Au MNPs are novel materials for immunoaffinity extraction. Compared with the conventional technique using immunoaffinity column, the method described here for sample pretreatment was fast, highly specific, and easy to operate.

Quantification of testosterone and epitestosterone in biological samples by capillary electrophoresis with immunoaffinity extraction.

Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL(-1), respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.