Human erythropoietin （hEPO）, an endogenous glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant human erythropoietin （rhEPO） has made it ...Human erythropoietin （hEPO）, an endogenous glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant human erythropoietin （rhEPO） has made it possible for its abuse in competitive sports. In this work, pre-capillary and on-capillary derivatization by 5-furoylquinoline-3-carboxaldehyde （FQ） and fluorescein isothiocyanate （FITC） for the detection of rhEPO by capillary electrophoresis with laser-induced fluorescence detection （CE-LIF） were compared. FQ pre-capillary labeling improves sensitivity but degrades the glycoforms separation due to the inhomogeneity of the reaction products from multiple labeling. Compared with FITC pre-capillary derivatization with the excess fluorescent background, the on-capillary FQ derivatization method can provide shorter analysis time, lower background, and better selectivity. It is demonstrated that, through optimizing reaction conditions of FQ on-capillary derivatization, both high sensitivity and satisfactory resolution for the analysis of the be used for the glycoforms profiling and quality control of rhEPO doping control analysis. glycoforms of rhEPO could be obtained. This method can It may be used as a candidate method for fast screening in展开更多
This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary ele...This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis（CE） coupled with laser-induced fluorescence（LIF） detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range（2.0-500 ng/mL） of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation（RSD） less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.展开更多
N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals. With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine ...N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals. With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine the free morphine in urine of abusers. The detection limit was calculated to be 40 ng/mL. Simulated urine samples were analyzed with good recoveries, which showed the feasibility of its application in specific morphine determination in urine of morphine abusers.展开更多
A novel approach for analysis of amino acids in individual erythrocytes was established. In this method, the derivatization reagent was introduced into the living cells by electroporation. After derivatization, the am...A novel approach for analysis of amino acids in individual erythrocytes was established. In this method, the derivatization reagent was introduced into the living cells by electroporation. After derivatization, the amino acids in a single cell were determined by capillary electrophoresis with laser-induced fluorescence detection.展开更多
The main goal of this work was to quantify the detection of colistin at low levels in urine samples through the practical application of mixed surfactant micellar electrokinetic chromatography–laser-induced fluoresce...The main goal of this work was to quantify the detection of colistin at low levels in urine samples through the practical application of mixed surfactant micellar electrokinetic chromatography–laser-induced fluorescence (MEKC-LIF) analysis method using its advantage of sensitivity and to examine direct injection of biological samples. Colistin (po- lymyxin E) has neither strong UV chromophore nor fluorophore. So, its assay for metabolism, pharmacokinetics studies for bioavailability and bioequivalence are difficult because of poor detectability. Therefore an enhanced UV or fluores-cence detection by chemical derivatization is required. MEKC-LIF method was proposed for colistin with a 488/520 nm argon-ion laser using a pre-CE derivatization with fluorescein isothiocyanate (FITC). Borate buffer was used as background buffer (BGB). The different parameters affecting the proposed derivatization reaction including concentration of the derivatizing reagent, reaction time and temperature were studied and optimized. The derivative was stable for up to 3 days. Different micelles (TX-100 and SDS) were examined as BGB additives separately but negative-charged mixed micelles (SDS/TX-100) were shown to be the best additive to BGB for the analysis of colistin particularly in human urine as they enhance both selectivity and sensitivity of the proposed method. BGB was used with pH 9.5, 10 kV, 8 s inj time, capillary length 75 cm × 75 μm ID (66 cm effective length), detection was LIF Ex 488 nm;Em 520 nm. The method was applied to colistin analysis in human urine and the recovery was > 98% (n = 5). LOD and LOQ in urine after pre-column derivatization using FITC were 100 and 250 ng/ml, respectively. Urine samples were analysed by direct injection without sample pre-treatment. The mechanism of enhancement of fluorescence of the derivative by surfactant was proposed.展开更多
FITC labeled amino acids have been separated using a home-built capillary electrophoresis with a laser-induced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino ...FITC labeled amino acids have been separated using a home-built capillary electrophoresis with a laser-induced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.展开更多
A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino a...A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.展开更多
Proteomics is becoming more and more mature, but the detection of low abundance proteins is still a difficult task. Laser-induced fluorescence（LIF） detection is one of the most sensitive detection methods in a capil...Proteomics is becoming more and more mature, but the detection of low abundance proteins is still a difficult task. Laser-induced fluorescence（LIF） detection is one of the most sensitive detection methods in a capillary electrophoresis（CE） system. However, most proteins do not exhibit favourable native fluorescence, a derivatization procedure is necessary for LIF detection of proteins. Since the derivatization reaction between protein and fluorescent reagent takes place after the separation of protein, the separation cannot be compromised by multiple derivatization products, the post-column derivatization becomes an attractive method for derivatization in CE-LIF system.展开更多
Caspases play important roles in cell apoptosis.Meas-urement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the...Caspases play important roles in cell apoptosis.Meas-urement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development,screening,and evaluation of anticancer drugs that target apoptotic pathways.The fluorescence resonance energy transfer(FRET)technique provides a valuable approach for defining the dynamics of apop-tosis with high spatio-temporal resolution.However,FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation.In the cur-rent study,a FRET sensor was combined with capillary electrophoresis(CE)to achieve a high throughput method for cellular caspase detection.The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs,such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide,as well as their combination with tumor necrosis factor(TNF).A posi-tive correlation between the caspase-3 activation ve-locity and drug concentration was observed when the cells were treated with cisplatin,but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations.Moreover,differ-ent types of cells presented distinct sensitivities under the same drug treatment,and the combination treat-ment of TNF and anticancer drugs significantly accel-erated the caspase-3 activation process.Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investi-gating the mechanisms of anticancer drugs and anti-cancer drug screening.展开更多
基金National Natural Science Foundation of China(Grant No.20635001 and 21175005)
文摘Human erythropoietin （hEPO）, an endogenous glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant human erythropoietin （rhEPO） has made it possible for its abuse in competitive sports. In this work, pre-capillary and on-capillary derivatization by 5-furoylquinoline-3-carboxaldehyde （FQ） and fluorescein isothiocyanate （FITC） for the detection of rhEPO by capillary electrophoresis with laser-induced fluorescence detection （CE-LIF） were compared. FQ pre-capillary labeling improves sensitivity but degrades the glycoforms separation due to the inhomogeneity of the reaction products from multiple labeling. Compared with FITC pre-capillary derivatization with the excess fluorescent background, the on-capillary FQ derivatization method can provide shorter analysis time, lower background, and better selectivity. It is demonstrated that, through optimizing reaction conditions of FQ on-capillary derivatization, both high sensitivity and satisfactory resolution for the analysis of the be used for the glycoforms profiling and quality control of rhEPO doping control analysis. glycoforms of rhEPO could be obtained. This method can It may be used as a candidate method for fast screening in
基金supported by the National Natural Science Foundation of China(No30470886)
文摘This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis（CE） coupled with laser-induced fluorescence（LIF） detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range（2.0-500 ng/mL） of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation（RSD） less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.
文摘N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals. With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine the free morphine in urine of abusers. The detection limit was calculated to be 40 ng/mL. Simulated urine samples were analyzed with good recoveries, which showed the feasibility of its application in specific morphine determination in urine of morphine abusers.
基金supported by the National Natural Science Foundation of China,the State Key Laboratory of Microbial Technology,Shandong University.
文摘A novel approach for analysis of amino acids in individual erythrocytes was established. In this method, the derivatization reagent was introduced into the living cells by electroporation. After derivatization, the amino acids in a single cell were determined by capillary electrophoresis with laser-induced fluorescence detection.
文摘The main goal of this work was to quantify the detection of colistin at low levels in urine samples through the practical application of mixed surfactant micellar electrokinetic chromatography–laser-induced fluorescence (MEKC-LIF) analysis method using its advantage of sensitivity and to examine direct injection of biological samples. Colistin (po- lymyxin E) has neither strong UV chromophore nor fluorophore. So, its assay for metabolism, pharmacokinetics studies for bioavailability and bioequivalence are difficult because of poor detectability. Therefore an enhanced UV or fluores-cence detection by chemical derivatization is required. MEKC-LIF method was proposed for colistin with a 488/520 nm argon-ion laser using a pre-CE derivatization with fluorescein isothiocyanate (FITC). Borate buffer was used as background buffer (BGB). The different parameters affecting the proposed derivatization reaction including concentration of the derivatizing reagent, reaction time and temperature were studied and optimized. The derivative was stable for up to 3 days. Different micelles (TX-100 and SDS) were examined as BGB additives separately but negative-charged mixed micelles (SDS/TX-100) were shown to be the best additive to BGB for the analysis of colistin particularly in human urine as they enhance both selectivity and sensitivity of the proposed method. BGB was used with pH 9.5, 10 kV, 8 s inj time, capillary length 75 cm × 75 μm ID (66 cm effective length), detection was LIF Ex 488 nm;Em 520 nm. The method was applied to colistin analysis in human urine and the recovery was > 98% (n = 5). LOD and LOQ in urine after pre-column derivatization using FITC were 100 and 250 ng/ml, respectively. Urine samples were analysed by direct injection without sample pre-treatment. The mechanism of enhancement of fluorescence of the derivative by surfactant was proposed.
基金Project supported by The National Natural Science Foundation of China (Grant No. 29825112) and CAS (No. KJ951-A1-507).
文摘FITC labeled amino acids have been separated using a home-built capillary electrophoresis with a laser-induced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.
文摘A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.
基金Supported by the Surface Project of the National Natural Science Foundation of China（No.21075039/B050106） and the Spe- cialized Research Fund for the Doctoral Program of Higher Education of China（No.20100074110016）.
文摘Proteomics is becoming more and more mature, but the detection of low abundance proteins is still a difficult task. Laser-induced fluorescence（LIF） detection is one of the most sensitive detection methods in a capillary electrophoresis（CE） system. However, most proteins do not exhibit favourable native fluorescence, a derivatization procedure is necessary for LIF detection of proteins. Since the derivatization reaction between protein and fluorescent reagent takes place after the separation of protein, the separation cannot be compromised by multiple derivatization products, the post-column derivatization becomes an attractive method for derivatization in CE-LIF system.
基金supported by the National Natural Science Foundation of China(Grant Nos.30800339 and 30800208).
文摘Caspases play important roles in cell apoptosis.Meas-urement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development,screening,and evaluation of anticancer drugs that target apoptotic pathways.The fluorescence resonance energy transfer(FRET)technique provides a valuable approach for defining the dynamics of apop-tosis with high spatio-temporal resolution.However,FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation.In the cur-rent study,a FRET sensor was combined with capillary electrophoresis(CE)to achieve a high throughput method for cellular caspase detection.The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs,such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide,as well as their combination with tumor necrosis factor(TNF).A posi-tive correlation between the caspase-3 activation ve-locity and drug concentration was observed when the cells were treated with cisplatin,but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations.Moreover,differ-ent types of cells presented distinct sensitivities under the same drug treatment,and the combination treat-ment of TNF and anticancer drugs significantly accel-erated the caspase-3 activation process.Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investi-gating the mechanisms of anticancer drugs and anti-cancer drug screening.
