基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.     

Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.     

Iridium Oxide Film-Enhanced Impedance Immunosensor for Rapid Detection of Carcinoembyronic Antigen

A simple, rapid and sensitive impedance immunosensor based on iridium oxide （IrOx） thin film for the detection of carcinoembyronic antigen （CEA） in human sera has been proposed. Gold electrode was electrochemically modified with IrOx thin film and simultaneously functionalized with protein A （PA） to bind anti-CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentration ranging from 36.2 to 460.0 ng/mL, with a detection limit of 28.0 ng/mL. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay （CLIA）, indicating the feasibility of using the proposed method for CEA immunoassay in clinical laboratory.     

Use of 2-(tert-butoxy)-N-(3-carbamothioylphenyl)acetamide and graphene oxide for separation and preconcentration of Fe(Ⅲ),Ni(Ⅱ),Cu(Ⅱ) and Zn(Ⅱ) ions in different samples

A simple and selective method using a column packed with graphene oxide(GO) as a solid phase extractant has been developed for the multi-element preconcentration of Fe(Ⅲ),Ni(Ⅱ),Cu(Ⅱ) and Zn(Ⅱ)ions prior to flame atomic absorption spectrometric determinations.The method is based on the sorption of mentioned ions on synthesized GO using 2-(tert-butoxy)-N-(3-carbamothioylphenyl)acetamide as a chelating agent.Several parameters on the extraction and complex formation were optimized.Under the optimized conditions(pH 6,flow rate 9 mL/min),metal ions were retained on the column,then quantitatively eluted by HNO3solution(5 mL,3.0 mol/L).The preconcentration factor was calculated as250.The detection limits for the analyte ions of interest were found in the range of 0.11 ng/mL(Ni2+) to0.63 ng/mL(Cu2+).The column packed with GO was adequate for metal ions separation in matrixes containing alkali,alkaline earth,transition and heavy metal ions.     

Flow—injection Chemiluminescence Determination of Reserpine in Medicine and Biological Fluids with Controlled—Reagent—Release Technology

A sensitive and rapid chemiluminescence(CL) flow injection with controlled-reagent-release technology for the determination of reserpine was proposed.The Cl reagents,luminol and dichromate,uses in this sensor,were all immobilized on anion-exchange resin.Through injection of 100μl of water,the reagents on the anion-ex-change resin column were eluted and in the presence of reserpine ,the CL intensity was decreased,by which reserpine could be sensed.Reserpine was quantified by measuring the decrement of CL intensity,which was observed linear with the logrithm of reserpine concentration in the rage of 1.0-500.0ng/mL,and the limit of detection was 0.4ng/mL(3σ)with a relative standard deviation of less than 3.0?The proposed procedure was applied in the assay of reserpine in pharmaceutical preparation and biological fhuids without any pre-treatment process and with sampling frequencies of 72 times per hour.     

高效液相色谱法荧光检测测定鱼中痕量硒含量

,5-benzopiazselenol (NSD)formed by reaction of Se(IV) and 2,3-diaminonaphthalene(DAN) was ex-tracted with cyclohexane and then injected into an HPLC ODS column( 150mm × 5mm)with a moblie phaseof acetonitrile at a flow rate of 1.0mL/min and detected fluorimetrically. The selenium content in fish hasbeen determined by this method with good results and recoveries. The limit of detection was 0.1ng.     

Determination of catecholamines by ion chromatography coupled to acidic potassium permanganate chemiluminescence detection

A simple,fast,sensitive,highly selective and eco-friendly analytical method for the determination of catecholamines in human urine by ion chromatography（IC） with chemiluminescence（CL） detection was described in this paper.Using 12 mmol/L H₂SO₄ without any organic additive as eluent,three catecholamines including epinephrine（EP）,norepinephrine（NE） and dopamine（DA） were well separated on a cation-exchange column.The CL detection was based on the reaction of analytes with acidic potassium permanganate in the presence of formaldehyde as an enhancer.The absence of methanol and acetonitrile in eluent made the proposed method more sensitive and eco-friendly.Under the optimal conditions,the linear range of the proposed method was in the range of 0.02-0.5μg/mL.The limit of detection（LOD） was in the range of 0.6 and 5.1μg/L.The relative standard deviations （RSD） for 0.1μg/mL mixed standard solution were in the range of 0.8-1.9%（n = 11）.The method has been applied to the determination of catecholamines in human urine successfully.Excellent spiked recoveries were achieved for catecholamines ranged from 91.2%to 112.7%.     

HOMOGENEOUS ENZYME IMMUNOASSAY OF SERUM AFP BY FLUORIMETRIC KINETIC METHOD

The activity of the Ab-bound enzyme(HRP)changed afterimmunochemical reaction,and can be indicated by a sensitive fluorimetrickinetic method.The finding set the stage for developing sensitivehomogeneous immunoassay.AFP was measured as an example.     

Detection of complement C4 with a piezoelectric immunosensor

A piezoelectric immunosensor has been developed for the detection of complement C4. Anti-C4 antibody was immobilized onto the gold electrodes of a 9 MHz AT-cut piezoelectric crystal. The coated crystal with the physical adsorption method to immobilize antibody showed the better results than the polyethyleneimine adhesion, glutaraldehyde cross-linking method with respect to sensitivity and reproducibility. The antibody-bound crystal with the physical adsorption method was successfully used for the detection of human complement C4 in the concentration range of 0.1-10 μg/mL for 40 min incubation time. The immunosensor system had good selectivity, and other materials in human serum did not interfere the detection remarkably. The crystal could be regenerated nearly 15 times when the bound materials on the crystal surface were eluted by strong acid and strong alkali solutions and subsequently cleaned in a ultrasonic cleaner.     

A novel indirect inhibitive immunoassay for sulfamethoxazole

<正>A new indirect inhibitive immunoassay using surface plasmon resonance(SPR) coupled with molecularly imprinted polymer(MIP) was developed.A sulfamethoxazole(SMX) MIP coating capillary was produced and used as an in-tube solid phase extraction(SPE) device.The MIP coating formed a nanometer film on the inner wall of the capillary.The anti-SMX mono-antibody was inhibited by SMX extracted by the MIP coating in a dose-dependent manner.The calibration curve was generated by linear fit over the range of 0.04-10.00 ng/mL.The limit of detection was 0.01 ng/mL.This method has high sensitivity and can be performed automatically.     

Development of a competitive format sorbent assay for the determination of parathion in water using molecular imprinted polymer as specific sorbent carrier

<正>A rapid,simple,and reliable competitive molecular imprinted polymer sorbent assay(MIPSA) was developed and validated for measurement of parathion in water samples.This assay employed a molecular imprinted polymer(MIP) that was synthesized with non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase(para-HRP) as an enzyme label.The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding sites of the MIP.The optimized analysis conditions of 10 ng mL~(-1) para-HRP and 10 mg mL~(-1) polymer were found.The assay was acceptable to detect parathion in water samples under the optimized conditions,with a limit of detection of 50 ng mL~(-1).Mean analytical recoveries of added parathion in water samples ranged from 101.2%to 105%.The precision of the assay was satisfactory; relative standard deviation ranged from 4.3%to 6%.     

Kinetic spectrophotometric method for the determination of selenium(IV) by its catalytic effect on the reduction of spadns by sulphide in micellar media

A simple kinetic spectrophotometric method was developed for the determination of ultratrace amounts of Se(IV). The method is based on the reduction of spadns by sulphide in micellar media. The reaction was monitored spectrophotometrically by measuring the decrease in the absorbance of spadns at 515 nm with a fixed-time method. The decrease in the absorbance of spadns is proportional to the concentration of Se(IV) in the range 0.5–100 ng/mL with a fixed time of 2.5–7.0 min from the initiation of the reaction. The limit of detection is 0.3 ng/mL Se(IV). The relative standard deviation for the determination of 0.02 and 0.10 μg/mL Se(IV) was 2.10 and 1.95%, respectively. The method was applied to the determination of Se(IV) in water. The text was submitted by the authors in English.     

A novel method for the determination of trace copper in cereals by dispersive liquid-liquid microextraction based on solidification of floating organic drop coupled with flame atomic absorption spectrometry

A novel,simple,rapid,efficient and environment-friendly method for the determination of trace copper in cereal samples was developed by using dispersive liquid-liquid microextraction based on solidification of floating organic drop(DLLME-SFO) followed by flame atomic absorption spectrometry.In the DLLME-SFO,copper was complexed with 8-hydroxy quinoline and extracted into a small volume of 1-dodecanol,which is of low density,low toxicity and proper melting point near room temperature. The experimental parameters affecting the extraction efficiency were investigated and optimized.Under the optimum conditions, the calibration graph exhibited linearity over the range of 0.5—500 ng/mL with the correlation coefficient(r) of 0.9996.The enrichment factor was 122 and the limit of detection was 0.1 ng/mL.The method was applied to the determination of copper in the complex matrix samples such as rice and millet with the recoveries for the spiked samples at 5.0 and 10.0 u,g/g falling in the range of 92.0-98.0%and the relative standard deviation of 3.9-5.7%.     

FLAME ATOMIC ABSORPTION DETERMINATION OF COPPER IN CEREALS FOOD SAMPLES WITH THE PRECONCENTRATION OF POTASSIUM TETRATITANATE WHISKER

A simple and reliable method has been developed for separation and preconcentration of trace amounts of copper ions in cereals food for subsequent measurement by flame atomic absorption spectrometry （FAAS）. The Cu^2＋ ions are adsorbed selectively and quantitatively during the passage. The retained copper ions were desorbed from the potassium tetratitanate whisker with 10.0mL of 2mol/L sulphuric acid solutions as eluent and were determined by FAAS. The linear range was 0.05μg/mL-0.20μg/mL in the original solution with a correlation coefficient of 0.9998. The detection limit of the proposed method is 2. lng/mL in the original solution （3σ, n=9）. Determination of copper in standard ions showed that the proposed method has good accuracy （recovery was more than 95%）. The method was successfully applied for recovery and determination of copper in cereals food samples     

Development and Validation of a Reverse-phase High Performance Liquid Chromatography Method for Determination of Exenatide in Poly（lactic-co-glycolic acid） Microspheres

Exenatide(synthetic exendin-4), which has been approved by the Food and Drug Administration(FDA) for the adjunctive treatment of patients with type 2 diabetes, is an incretin mimetic agent. The development and validation of a RP-HPLC method for the quantification of the exenatide in poly(lactic-co-glycolic acid)(PLGA) microspheres is described. Separation was performed on a C4 column via a mobile phase consisting of ACN:KH2PO4(0.02 mol/L, pH=2.5) gradient elution from 30:70 to 45:55(volume ratio) in 30 min. Multi-diode array detection(DAD) appears to be most appropriate to evaluate the spectral purity of exenatide. The limits of detection and quantification of exenatide were 0.4 and 1.2 μg/mL, respectively. The calibration curve of exenatide was linear in a range of 0.025―0.2 mg/mL with a correlation coefficient of 0.9995. The results of validation study show that this method is specific, accurate(recovery95%), precise(RSD2.0%) and robust.     

Electrochemical Enzyme Immunoassay of Tumor Marker CA15—3 with Capilary Electrophoresis

Tumor marker CA15-3 was determined by using capilary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) ,The method can be used to detect CA15-3 with a limit of 0.024 U/mL.     

高效液相色谱法测定福丽生发液中柚皮甙的含量

 This paper describes a method for determining naringin in Fully Hair Regenerator by reversed-phaseHPLC with a 150mm×4.6mm i.d.YWGCis column.It is eluted with methanol-acetic acid-water(20∶ 4∶76)mixture at a flowrate of 1.0mL/min and detected at 283 nm.The coefficient of variation was less than1.0%,and the recoverv was more than 97.4%.The method is simple,rapid,sensitive and of excellent pre-cision.     

A Study on the Interaction of the DAS-K with Bovine Serum Albumin by On-line Ultrafiltration and Chemiluminescence

Potassium dehydroandrographolide succinate （DAS-K） has antibacterial and antiviral effects. It has been used widely for the treatment of virus pneumonia, malaria and respiratory infections. In this work, a novel flow-injection chemiluminescence （CL） method for the determination of DAS-K was proposed. The method is based on the reaction between DAS-K and hexacyanoferrate（III） in alkaline solution to give weak CL signal, which is enhanced by rhodamine B. The experimental conditions for the CL reaction were optimized and the possible reaction mechanism was discussed. Under the optimum conditions, the concentration of DAS-K is proportional to the CL intensity in the range of 0.1-80 μmol·L^-1 with a detection limit of 0.05 μmol·L^-1. The interaction of the DAS-K with bovine serum albumin by on-line ultrafiltration and flow-injection chemiluminescence was studied. The concentrations of unbound DAS-K from ultra filter tube were determined by the flow-injection CL method. The binding parameters were estimated by the Scatchard plot and Klotz plot. The proposed system proved that FIA-CL coupled with on-line ultrafiltration sampling was a fast and simple technique for the study of drug-protein interaction.     

新型液相色谱-荧光检测法测定药物剂型和人血浆中的他达拉非和盐酸达泊西汀(英文)

Tadalafil(TAD)and dapoxetine HCl(DAP)are recently co-formulated and both show native fluorescence.Therefore,a novel,accurate,specific and sensitive reversed-phase high performance liquid chromatographic method with fluorescence detection was developed and validated for their separation and quantitation in dosage form and human plasma using avanafil as an internal standard(IS).Separation was achieved using isocratic elution within 7.0 min on C18column and acetonitrile-0.15%triethylamine(40∶60,v/v;pH 4)as a mobile phase.The flow rate was 1.0 mL/min and the detection was time-programmed at 330,410 and 370 nm for TAD,DAP and IS,respectively,after excitation at 236 nm.The linear ranges from 0.01 to 30.00μg/mL for each drug with the limits of detection of 4.20 and 7.20 ng/mL for TAD and DAP,respectively.The method was validated in accordance to the International Conference on Harmonization(ICH)guidelines and was successfully applied to spiked human plasma with mean recoveries of 98.17%and 98.83%for TAD and DAP respectively.     

Determination of Tetracyclines in Honey Using Liquid Chromatography with Ultraviolet Absorbance Detection and Residue Confirmation by Mass Spectrometry

A determination method has been optimized and validated for the simultaneous analysis of tetracycline （TC）, oxytetracycline （OTC）, chlortetracycline （CTC） and doxycycline （DC） in honey. Tetracyclines （TCs） were removed from honey samples by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns and eluted with Na2EDTA-Mcllvaine pH 4.0 buffers. Extracts were further cleaned up by Oasis HLB solid-phase extraction （SPE）, while other solid-phase extraction cartridges were compared. Chromatographic separation was achieved using a polar end-capped C 18 column with an isocratic mobile phase consisting of oxalic acid, acetonitrile and methanol. LC with ultraviolet absorbance at 355 nm resulted in the quantitation of all four tetracycline residues from honey samples fortified at 15, 50, and 100 ng/g, with liner ranges for tetracyclines of 0.05 to 2 μg/mL. Mean recoveries for tetracyclines were greater than 50% with R.S.D. values less than 10% （n= 18）. Detection limits of 5, 5, 10, 10 ng/g for oxytetracycline, tetracycline, chlortetracycline and doxycycline, respectively and quantitation limits of 15 ng/g for all the four tetracyclines were determined. Direct confirmation of the four residues in honey （2-50 ng/g） was realized by liquid chromatography-tandem mass spectrometry （LC/MS/MS）. The linear ranges of tetracyclines determined by LC/MS/MS were between 5 to 300 ng/mL, with the linear correlation coefficient r〉 0.995. The limits of detection of 1 to 2 ng/g were obtained for the analysis of the TCs in honey.