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2019 Volume 47 Issue 2
2019, 47(2): 169-180
doi: 10.19756/j.issn.0253-3820.181580
Abstract:
X-ray crystallography is the major approach in the determination of protein structure. To obtain high quality protein crystals that meet the diffraction requirements, researchers usually spend large amounts of reagents and samples for large-scale crystallization condition screening. Microfluidic technology can greatly reduce the consumption of protein samples in protein crystallization screening by manipulating ultramicro amounts of fluids. In this review, the recent progress in microfluidic protein crystallization screening methods based on the microbatch, vapor diffusion, free-interface diffusion and dialysis methods is introduced according to the difference in crystallization methods.
X-ray crystallography is the major approach in the determination of protein structure. To obtain high quality protein crystals that meet the diffraction requirements, researchers usually spend large amounts of reagents and samples for large-scale crystallization condition screening. Microfluidic technology can greatly reduce the consumption of protein samples in protein crystallization screening by manipulating ultramicro amounts of fluids. In this review, the recent progress in microfluidic protein crystallization screening methods based on the microbatch, vapor diffusion, free-interface diffusion and dialysis methods is introduced according to the difference in crystallization methods.
2019, 47(2): 181-190
doi: 10.19756/j.issn.0253-3820.181320
Abstract:
Luminescent bacteria are a kind of bacteria which emit visible light at 450 nm-490 nm. Its luminous intensity decreases with the increase of the concentration of toxic substances in test solution. The methods hased on luminescent bacteria are widely used in acute toxicity analysis of water quality because of its sensitiveness, quickness and inexpensiveness. With the rapid development of genetic modification technology, non-luminescent bacteria can also produce visible light by planting luminescent genes, which makes the application field of luminescent bacteria widen. In recent years, biosensors based on luminescent bacteria have attracted more and more attention, and the reports of bioluminescence biosensors based on microfluidic systems are increasing day by day. Based on the characteristics and mechanism of luminescent bacteria, this review introduced their applications in the environmental monitoring, and summarized several new bioluminescence sensors.
Luminescent bacteria are a kind of bacteria which emit visible light at 450 nm-490 nm. Its luminous intensity decreases with the increase of the concentration of toxic substances in test solution. The methods hased on luminescent bacteria are widely used in acute toxicity analysis of water quality because of its sensitiveness, quickness and inexpensiveness. With the rapid development of genetic modification technology, non-luminescent bacteria can also produce visible light by planting luminescent genes, which makes the application field of luminescent bacteria widen. In recent years, biosensors based on luminescent bacteria have attracted more and more attention, and the reports of bioluminescence biosensors based on microfluidic systems are increasing day by day. Based on the characteristics and mechanism of luminescent bacteria, this review introduced their applications in the environmental monitoring, and summarized several new bioluminescence sensors.
2019, 47(2): 191-197
doi: 10.19756/j.issn.0253-3820.181625
Abstract:
In this work, the cataluminescence (CTL) phenomenon of propylene oxide (PO) on the surface of nanocomposite TiO2-Y2O3 (titanium dioxide-yttrium(Ⅲ)-oxide) was studied. It was found that the nanocomposite had high sensitivity and good selectivity for the detection of PO. The common volatile organic compounds such as acetone, acetaldehyde and benzene showed no response to the catalysis of TiO2-Y2O3. Based on this phenomenon, a PO CTL sensor was designed. The ratio of TiO2 and Y2O3, and the sintering temperatures of the composites were optimized. It was found that when the mass ratio of TiO2 and Y2O3 was 1:3 and the sintering temperature was 500℃, the catalytic materials showed the best performance. The temperature, wavelength and carrier gas flow rate of the CTL system were also optimized, and 197℃, 490 nm and 0.3 L/min were selected as the optimal conditions. The quantitative analysis was performed under the optimized conditions and CTL intensity was linear with PO concentration in the range from 4.5 mg/L to 1375 mg/L with a detection limit (3σ) of 1.25 mg/L. The sensor was used for quantitative analysis and real-time monitoring of PO residues in fumigation cereals. The result obtained by this CTL sensor was consistent with that by gas chromatography. The CTL sensor proposed here had many merits such as high sensitivity, rapidity and simple operation, and had potential application prospects in the rapid detection of PO in food. In addition, the mechanism of catalytic oxidation of PO was discussed as well.
In this work, the cataluminescence (CTL) phenomenon of propylene oxide (PO) on the surface of nanocomposite TiO2-Y2O3 (titanium dioxide-yttrium(Ⅲ)-oxide) was studied. It was found that the nanocomposite had high sensitivity and good selectivity for the detection of PO. The common volatile organic compounds such as acetone, acetaldehyde and benzene showed no response to the catalysis of TiO2-Y2O3. Based on this phenomenon, a PO CTL sensor was designed. The ratio of TiO2 and Y2O3, and the sintering temperatures of the composites were optimized. It was found that when the mass ratio of TiO2 and Y2O3 was 1:3 and the sintering temperature was 500℃, the catalytic materials showed the best performance. The temperature, wavelength and carrier gas flow rate of the CTL system were also optimized, and 197℃, 490 nm and 0.3 L/min were selected as the optimal conditions. The quantitative analysis was performed under the optimized conditions and CTL intensity was linear with PO concentration in the range from 4.5 mg/L to 1375 mg/L with a detection limit (3σ) of 1.25 mg/L. The sensor was used for quantitative analysis and real-time monitoring of PO residues in fumigation cereals. The result obtained by this CTL sensor was consistent with that by gas chromatography. The CTL sensor proposed here had many merits such as high sensitivity, rapidity and simple operation, and had potential application prospects in the rapid detection of PO in food. In addition, the mechanism of catalytic oxidation of PO was discussed as well.
2019, 47(2): 198-206
doi: 10.19756/j.issn.0253-3820.181563
Abstract:
Fluorescent sensing methods, because of their simple, cheap, fast, high selectivity and sensitivity, are widely used in the detection of cationic, anionic and other chemicals. Anthraquinone derivatives have many pharmacological activities, rigid planar structure and large ring conjugate system. They have moderate fluorescence emission wavelengths, good optical stabilities and high luminous efficiencies. In this work, anthraquinone derivatives were employed as fluorophore to prepare new fluorescent probes. Based on the mechanism of intramolecular charge transfer (ICT), a series of symmetric bis-1,2,3-triazole hydrazine derivatives K1 and K2, which were confirmed by 1H NMR and 13C NMR, were designed and synthesized. The spectral properties were also investigated. The structure and theoretical spectrum of K1 were calculated using GaussView 9.0 quantization program and time-dependent density functional theory. The results showed that exhibited good selectivity to Ag+ in a mixed solution of DMSO/HEPES (1:7, V/V). Binding analysis using the method of continuous variations (Job's plot) established that the stoichiometry of K1-Ag+ complex was estimated to be 1:1. The detection limit of K2 for Ag+ was 21.2 μmol/L (S/N=3). A complexation mechanism of K1-Ag+ was proposed, which was consistent with the calculated results. The fluorescence of K1 was increased dramatically in the presence of Ag+, and it remained nearly constant in the range of pH 6.0-8.0. Other metal ions, such as Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, Fe3+, K+, Mn2+, Na+, NH4+, Ni2+, Pb2+ and Zn2+, induced negligible fluorescence increasing for under the same conditions. The synthesized fluorescent probe K1 could be applied to image intracelluar Ag+ in living HepG2 cells, indicating that it was a useful tool in bioanalytical applications.
Fluorescent sensing methods, because of their simple, cheap, fast, high selectivity and sensitivity, are widely used in the detection of cationic, anionic and other chemicals. Anthraquinone derivatives have many pharmacological activities, rigid planar structure and large ring conjugate system. They have moderate fluorescence emission wavelengths, good optical stabilities and high luminous efficiencies. In this work, anthraquinone derivatives were employed as fluorophore to prepare new fluorescent probes. Based on the mechanism of intramolecular charge transfer (ICT), a series of symmetric bis-1,2,3-triazole hydrazine derivatives K1 and K2, which were confirmed by 1H NMR and 13C NMR, were designed and synthesized. The spectral properties were also investigated. The structure and theoretical spectrum of K1 were calculated using GaussView 9.0 quantization program and time-dependent density functional theory. The results showed that exhibited good selectivity to Ag+ in a mixed solution of DMSO/HEPES (1:7, V/V). Binding analysis using the method of continuous variations (Job's plot) established that the stoichiometry of K1-Ag+ complex was estimated to be 1:1. The detection limit of K2 for Ag+ was 21.2 μmol/L (S/N=3). A complexation mechanism of K1-Ag+ was proposed, which was consistent with the calculated results. The fluorescence of K1 was increased dramatically in the presence of Ag+, and it remained nearly constant in the range of pH 6.0-8.0. Other metal ions, such as Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, Fe3+, K+, Mn2+, Na+, NH4+, Ni2+, Pb2+ and Zn2+, induced negligible fluorescence increasing for under the same conditions. The synthesized fluorescent probe K1 could be applied to image intracelluar Ag+ in living HepG2 cells, indicating that it was a useful tool in bioanalytical applications.
2019, 47(2): 207-213
doi: 10.19756/j.issn.0253-3820.181417
Abstract:
The masD and bamA are key genes for anaerobic degradation of alkanes and aromatic hydrocarbons, respectively. In this study, the SYBR Green I real-time quantitative polymerase chain reaction (Real Time-qPCR) method was established with the two genes as targets. With reference to the GenBank sequence of related degraded oil strains, the anaerobic degradation gene amplification primers masD-f, masD-r and bamA-f, bamA-r for alkanes and aromatic hydrocarbons were designed and synthesized. Two pairs of primers were amplified by conventional PCR to obtain 389 bp and 354 bp fragments, respectively. The amplified products were confirmed as fragments of masD and bamA by being sequenced and queried in the NCBI database. The positive clone plasmid was extracted and serially diluted to construct a real-time qPCR standard curve. The best reaction conditions for the 25-μL amplification system were as follows:0.2 μmol/L pre-primer and post-primer, 12.5 μL of 2×Trans Start Top Green qPCR Super Mix, 52℃ of annealing temperatures for masD and 56℃ for bamA genes. Real Time-qPCR technology showed high sensitivity and repeatability, which was 100 times higher than traditional PCR technology. The quantitative detection of anaerobic genes in the anaerobic degradation system of petroleum hydrocarbons promoted by graphene oxide showed that the addition of different concentrations of graphene oxide increased the copy numbers of bamA gene, but had no significant effect on the copy numbers of masD gene.
The masD and bamA are key genes for anaerobic degradation of alkanes and aromatic hydrocarbons, respectively. In this study, the SYBR Green I real-time quantitative polymerase chain reaction (Real Time-qPCR) method was established with the two genes as targets. With reference to the GenBank sequence of related degraded oil strains, the anaerobic degradation gene amplification primers masD-f, masD-r and bamA-f, bamA-r for alkanes and aromatic hydrocarbons were designed and synthesized. Two pairs of primers were amplified by conventional PCR to obtain 389 bp and 354 bp fragments, respectively. The amplified products were confirmed as fragments of masD and bamA by being sequenced and queried in the NCBI database. The positive clone plasmid was extracted and serially diluted to construct a real-time qPCR standard curve. The best reaction conditions for the 25-μL amplification system were as follows:0.2 μmol/L pre-primer and post-primer, 12.5 μL of 2×Trans Start Top Green qPCR Super Mix, 52℃ of annealing temperatures for masD and 56℃ for bamA genes. Real Time-qPCR technology showed high sensitivity and repeatability, which was 100 times higher than traditional PCR technology. The quantitative detection of anaerobic genes in the anaerobic degradation system of petroleum hydrocarbons promoted by graphene oxide showed that the addition of different concentrations of graphene oxide increased the copy numbers of bamA gene, but had no significant effect on the copy numbers of masD gene.
2019, 47(2): 214-220
doi: 10.19756/j.issn.0253-3820.181387
Abstract:
Poly(2,2-bithiophene-co-5-methoxyindole)-oxide graphene composite coating was prepared by voltammetry for headspace solid-phase microextraction and gas chromatography determination of antidepressants (i.e., citalopram, fluoxetine and nortriptyline). The coating was uniform and showed cauliflower-like microstructure. It had high thermal stability (up to 400℃) and could be used for at least 180 times of solid-phase microextraction (SPME) without decrease in extraction performance. Furthermore, it presented high extraction capacity for the antidepressants due to the hydrogen bond and π-π interaction between the analytes and the coating. Under optimized conditions, good linearity (R>0.9958), wide linear range (0.01-50 μg/L) and low limits of detection (4.25-6.25 ng/L) were achieved for these analytes. The relative standard deviation was less than 5.3% for five successive measurements with one fiber, and the relative standard deviations for fiber-to-fiber were 4.8%-6.5% (n=5). The SPME-GC method was applied to the determination of lake water, with recoveries of 93.6%-109%.
Poly(2,2-bithiophene-co-5-methoxyindole)-oxide graphene composite coating was prepared by voltammetry for headspace solid-phase microextraction and gas chromatography determination of antidepressants (i.e., citalopram, fluoxetine and nortriptyline). The coating was uniform and showed cauliflower-like microstructure. It had high thermal stability (up to 400℃) and could be used for at least 180 times of solid-phase microextraction (SPME) without decrease in extraction performance. Furthermore, it presented high extraction capacity for the antidepressants due to the hydrogen bond and π-π interaction between the analytes and the coating. Under optimized conditions, good linearity (R>0.9958), wide linear range (0.01-50 μg/L) and low limits of detection (4.25-6.25 ng/L) were achieved for these analytes. The relative standard deviation was less than 5.3% for five successive measurements with one fiber, and the relative standard deviations for fiber-to-fiber were 4.8%-6.5% (n=5). The SPME-GC method was applied to the determination of lake water, with recoveries of 93.6%-109%.
2019, 47(2): 221-228
doi: 10.19756/j.issn.0253-3820.181433
Abstract:
The motion characteristics of different cells in the multi-electrode array microchip were studied under the dielectrophoretic force, and the two cells with the same external morphology but different internal histone protein were separated. The multi-electrode array microfluidic chip was embedded in the 5 square cross section of the channel, and 8 electrodes were embedded on each side of the cross section. The structure enlarged the size of the microfluidic channel, which was beneficial for the high flow separation of the cells under the action of the dielectrophoretic force. To study the characteristics of cell motion in the microfluidic device, the electric field distribution in a cross-section was calculated, and the optimal electrode combination pattern was obtained, which generated a uniform electric field distribution and a greatest the dielectrophoretic force. After that, the motion characteristics of MRC-5 in human lung fibroblasts in different frequencies and complex electric fields at different frequencies and complex electric fields were analyzed. By analyzing the spectrum of the dielectrophoretic force, the separation frequency of the two kinds of cells of wild type (WT) and histone-GFP type was f=30 kHz, and the separation rate of cell was calculated by adding different proportions of sucrose solution and two cell mixture at two inlets. That was, when the flow-rate ratio of the 2 inlets was 12:1, the separation rate of the two types of cells was 93.5%. The method for cell separation using microfluidic chips provided a basis for the rapid separation of cells in the future.
The motion characteristics of different cells in the multi-electrode array microchip were studied under the dielectrophoretic force, and the two cells with the same external morphology but different internal histone protein were separated. The multi-electrode array microfluidic chip was embedded in the 5 square cross section of the channel, and 8 electrodes were embedded on each side of the cross section. The structure enlarged the size of the microfluidic channel, which was beneficial for the high flow separation of the cells under the action of the dielectrophoretic force. To study the characteristics of cell motion in the microfluidic device, the electric field distribution in a cross-section was calculated, and the optimal electrode combination pattern was obtained, which generated a uniform electric field distribution and a greatest the dielectrophoretic force. After that, the motion characteristics of MRC-5 in human lung fibroblasts in different frequencies and complex electric fields at different frequencies and complex electric fields were analyzed. By analyzing the spectrum of the dielectrophoretic force, the separation frequency of the two kinds of cells of wild type (WT) and histone-GFP type was f=30 kHz, and the separation rate of cell was calculated by adding different proportions of sucrose solution and two cell mixture at two inlets. That was, when the flow-rate ratio of the 2 inlets was 12:1, the separation rate of the two types of cells was 93.5%. The method for cell separation using microfluidic chips provided a basis for the rapid separation of cells in the future.
2019, 47(2): 229-236
doi: 10.19756/j.issn.0253-3820.181684
Abstract:
A small embedded wireless monitoring system for potentiometric sensors was designed and fabricated. The system employed a dual precision op amp of TLC4502 as the preamplifier and a bluetooth low energy (BLE) chip of CC2541 as the main controller, and the main controller integrated 14 bits analog to digital convertor (ADC). The system used the BLE development application program (App) to establish a Bluetooth connection with the CC2541 for wireless real-time reading of sensing potentials. The monitoring system software was developed on the basis of a BLE-CC254X-1.41 protocol stack by the embedded development software of IAR Embedded Workbench. To verify the reliability and accuracy of the system, a precision regulated power supply was used for analog potential acquisition test, and a pH composite electrode was used for monitoring the pH change. The test results showed that the system could be applied to real time measurement of potential signals for three times amplification with an accuracy of 0.4 mV, and a consistent response to pH change was able to quickly output with a linear correlation coefficient of R2=0.9994. Furthermore, the concentration change of L-cysteine (L-cys) was monitored in real time by the BLE system with a self-made L-cys sensor, and the dynamic response change was consistent with that of commercial multimeter, indicating that the rapid transmission and real time monitoring of potentiometric signals could be well realized through the BLE wireless sensing system.
A small embedded wireless monitoring system for potentiometric sensors was designed and fabricated. The system employed a dual precision op amp of TLC4502 as the preamplifier and a bluetooth low energy (BLE) chip of CC2541 as the main controller, and the main controller integrated 14 bits analog to digital convertor (ADC). The system used the BLE development application program (App) to establish a Bluetooth connection with the CC2541 for wireless real-time reading of sensing potentials. The monitoring system software was developed on the basis of a BLE-CC254X-1.41 protocol stack by the embedded development software of IAR Embedded Workbench. To verify the reliability and accuracy of the system, a precision regulated power supply was used for analog potential acquisition test, and a pH composite electrode was used for monitoring the pH change. The test results showed that the system could be applied to real time measurement of potential signals for three times amplification with an accuracy of 0.4 mV, and a consistent response to pH change was able to quickly output with a linear correlation coefficient of R2=0.9994. Furthermore, the concentration change of L-cysteine (L-cys) was monitored in real time by the BLE system with a self-made L-cys sensor, and the dynamic response change was consistent with that of commercial multimeter, indicating that the rapid transmission and real time monitoring of potentiometric signals could be well realized through the BLE wireless sensing system.
2019, 47(2): 237-243
doi: 10.19756/j.issn.0253-3820.181544
Abstract:
Reaction mass spectrometry can provide specific information through the introduction of reactive gas into ionization source. A new compound-specific stable chlorine isotope analysis (CSIA-Cl) method was developed to overcome drawbacks of existing methods. Contemporary methods often require laborious off-line procedures prior to isotope analysis to obtain the chlorine isotope ratios from the mass spectra. In contrast to previous methods, our on-line setup was based on the use of chemical ionisation mass spectrometry through the introduction of methane. The cleanest ion traces of 35Cl-(m/z 35) and 37Cl-(m/z 37) were recorded to directly determine the isotopic ratios of chlorine and the method completely avoided isobaric interferences. The stable chlorine fragments were obtained from each specific chlorinated compound using the appropriate energy and an optimized acquisition mode, and the precision was maintained by the control of reactive gas flow and filament current. The principle and the mechanism of this method were presented and explained using trichloroethylene (TCE) and perchloroethylene(PCE). Method validation was investigated using chlorinated alkenes, chlorinated benzenes, and organochlorine pesticides. The sensitivity of the method was significantly improved, and standard deviations (1σ) was almost below 2.0‰. Further reaction mass spectrometry scheme was proposed in order to improve the precision, which offered an innovative approach to compound-specific chlorine isotope analysis, and it could facilitate the application of CSIA-Cl in many fields.
Reaction mass spectrometry can provide specific information through the introduction of reactive gas into ionization source. A new compound-specific stable chlorine isotope analysis (CSIA-Cl) method was developed to overcome drawbacks of existing methods. Contemporary methods often require laborious off-line procedures prior to isotope analysis to obtain the chlorine isotope ratios from the mass spectra. In contrast to previous methods, our on-line setup was based on the use of chemical ionisation mass spectrometry through the introduction of methane. The cleanest ion traces of 35Cl-(m/z 35) and 37Cl-(m/z 37) were recorded to directly determine the isotopic ratios of chlorine and the method completely avoided isobaric interferences. The stable chlorine fragments were obtained from each specific chlorinated compound using the appropriate energy and an optimized acquisition mode, and the precision was maintained by the control of reactive gas flow and filament current. The principle and the mechanism of this method were presented and explained using trichloroethylene (TCE) and perchloroethylene(PCE). Method validation was investigated using chlorinated alkenes, chlorinated benzenes, and organochlorine pesticides. The sensitivity of the method was significantly improved, and standard deviations (1σ) was almost below 2.0‰. Further reaction mass spectrometry scheme was proposed in order to improve the precision, which offered an innovative approach to compound-specific chlorine isotope analysis, and it could facilitate the application of CSIA-Cl in many fields.
2019, 47(2): 244-248
doi: 10.19756/j.issn.0253-3820.181097
Abstract:
A method of headspace gas chromatography-mass spectrometry (HS-GC/MS) for simultaneous qualitative and quantitative analysis of off-flavour in well and rock salt was developed. For qualitative analysis, the sample were analyzed by headspace injection, then separated by a HP-innoWax column (60 m×0.25 mm×0.25 μm), and detected under electron ionization using scan mode, based on the NIST database retrieval and double column confirmed using standards, 13 and 5 kinds of short-chain fatty acids(SCFAs) were identified in the raw brine samples and finished smelled salt respectively. For the purpose of quantitative analysis short-chain fatty acids in finished salt, a selected ion monitor mode and external standard with matrix matched method was used. The result demonstrated that good linear relationships in the range of 0.13-5 mg/kg were obtained and the correlation coefficients (R2) of the 5 SCFAs were greater than 0.995. The limits of detection (LODs, S/N=3) ranged from 0.05 to 0.20 mg/kg. The mean recoveries varied from 82% to 104% at spiked levels of 0.25, 0.5 and 2.5 mg/kg, and the relative standard deviations (RSDs) ranged from 5.1% to 9.8% (n=6). The proposed method is simple and sensitive for simultaneous determination of SCFAs in well and rock salt.
A method of headspace gas chromatography-mass spectrometry (HS-GC/MS) for simultaneous qualitative and quantitative analysis of off-flavour in well and rock salt was developed. For qualitative analysis, the sample were analyzed by headspace injection, then separated by a HP-innoWax column (60 m×0.25 mm×0.25 μm), and detected under electron ionization using scan mode, based on the NIST database retrieval and double column confirmed using standards, 13 and 5 kinds of short-chain fatty acids(SCFAs) were identified in the raw brine samples and finished smelled salt respectively. For the purpose of quantitative analysis short-chain fatty acids in finished salt, a selected ion monitor mode and external standard with matrix matched method was used. The result demonstrated that good linear relationships in the range of 0.13-5 mg/kg were obtained and the correlation coefficients (R2) of the 5 SCFAs were greater than 0.995. The limits of detection (LODs, S/N=3) ranged from 0.05 to 0.20 mg/kg. The mean recoveries varied from 82% to 104% at spiked levels of 0.25, 0.5 and 2.5 mg/kg, and the relative standard deviations (RSDs) ranged from 5.1% to 9.8% (n=6). The proposed method is simple and sensitive for simultaneous determination of SCFAs in well and rock salt.
2019, 47(2): 249-255
doi: 10.19756/j.issn.0253-3820.181614
Abstract:
A methylation-based approach for analysis of phosphatidic acids (PAs) using ultra-high performance liquid chromatography-Orbitrap mass spectrometry with incorporation of in-house database was established. This method allowed high throughput analysis with confident molecular identification and quantitation. Phosphatidic acids were extracted using the mixture of chloroform and methanol with subsequent derivatization using (trimethylsilyl) diazomethane. Derivatized PAs (Me-PA) were analyzed in data dependent acquisition (Full MS/dd-MS2) mode of high resolution mass spectrometer. In-house database compatible with Tracefinder was derived allowing lipid identification based on MS/MS spectra. In this work, to achieve confident molecular identification and quantitation, combinatorial analysis of positive and negative ion modes was performed. The method was evaluated by the linearity, lower limit of detection (LLOQ), inter-and intra-day precision using standard molecule PA(14:0/14:0), and R2>0.99, LLOD of 0.125 μg/mL, inter-and intra-day precision<8.47% were achieved. PAs in aged mice brains were analyzed using this high throughput approach. A total of 14 phosphatidic acid molecules were identified and quantified in C57BL/6 mice brain. Moreover, decreased level of phosphatidic acid containing polyunsaturated fatty acid chain was observed in aged brains. Conclusively, this orbitrap-based platform with methylation provides a sensitive and accurate approach to analyze phosphatidic acid quantitatively.
A methylation-based approach for analysis of phosphatidic acids (PAs) using ultra-high performance liquid chromatography-Orbitrap mass spectrometry with incorporation of in-house database was established. This method allowed high throughput analysis with confident molecular identification and quantitation. Phosphatidic acids were extracted using the mixture of chloroform and methanol with subsequent derivatization using (trimethylsilyl) diazomethane. Derivatized PAs (Me-PA) were analyzed in data dependent acquisition (Full MS/dd-MS2) mode of high resolution mass spectrometer. In-house database compatible with Tracefinder was derived allowing lipid identification based on MS/MS spectra. In this work, to achieve confident molecular identification and quantitation, combinatorial analysis of positive and negative ion modes was performed. The method was evaluated by the linearity, lower limit of detection (LLOQ), inter-and intra-day precision using standard molecule PA(14:0/14:0), and R2>0.99, LLOD of 0.125 μg/mL, inter-and intra-day precision<8.47% were achieved. PAs in aged mice brains were analyzed using this high throughput approach. A total of 14 phosphatidic acid molecules were identified and quantified in C57BL/6 mice brain. Moreover, decreased level of phosphatidic acid containing polyunsaturated fatty acid chain was observed in aged brains. Conclusively, this orbitrap-based platform with methylation provides a sensitive and accurate approach to analyze phosphatidic acid quantitatively.
2019, 47(2): 256-261
doi: 10.19756/j.issn.0253-3820.181403
Abstract:
The effect of taurine (Tau) on inhibiting the proliferation of myofibroblasts (myoFbs) of neonatal rats were studied, and the inhibiting mechanism was explored with the combination of different analytical techniques. Angiotensin Ⅱ (AngⅡ) was used to induce the proliferation of myoFbs in cultured neonatal rats, and myofibrosis mode was established in the experiment. The proliferation of myoFbs was detected based on methyl thiazolyl tetrazolium (MTT) assay, and the results showed that different concentrations of Tau (0.03, 0.06, 0.12 mol/L) could significantly inhibit myoFbs proliferation in a dose-dependent way. Furthermore, the expression and contents of reactive oxygen species (ROS), cell cycle protein E (CyclinE), nuclear factor-kappa Bp65(NF-κBp65) and transforming growth factor-β1(TGF-β1) were determined based on the immunocytochemistry, laser confocal scanning microscopy and ELISA kit method. It was found that Tau could decrease the content of ROS and the expression of TGF-β1, and inhibit the translocation and expression of NF-κBp65 and CyclinE in nucleus, which resulted in the inhibition of proliferation of myoFbs and the effect of anti-fibrosis.
The effect of taurine (Tau) on inhibiting the proliferation of myofibroblasts (myoFbs) of neonatal rats were studied, and the inhibiting mechanism was explored with the combination of different analytical techniques. Angiotensin Ⅱ (AngⅡ) was used to induce the proliferation of myoFbs in cultured neonatal rats, and myofibrosis mode was established in the experiment. The proliferation of myoFbs was detected based on methyl thiazolyl tetrazolium (MTT) assay, and the results showed that different concentrations of Tau (0.03, 0.06, 0.12 mol/L) could significantly inhibit myoFbs proliferation in a dose-dependent way. Furthermore, the expression and contents of reactive oxygen species (ROS), cell cycle protein E (CyclinE), nuclear factor-kappa Bp65(NF-κBp65) and transforming growth factor-β1(TGF-β1) were determined based on the immunocytochemistry, laser confocal scanning microscopy and ELISA kit method. It was found that Tau could decrease the content of ROS and the expression of TGF-β1, and inhibit the translocation and expression of NF-κBp65 and CyclinE in nucleus, which resulted in the inhibition of proliferation of myoFbs and the effect of anti-fibrosis.
2019, 47(2): 262-270
doi: 10.19756/j.issn.0253-3820.181447
Abstract:
A simple and rapid sample pretreatment method was developed based on magnetic nanoparticles for multi-pesticides residue analysis in soil samples. The target analytes were extracted by acetonitrile under the assistance of ultra-sonication, followed by the purification of dispersive solid phase extraction. The magnetite (Fe3O4) nanoparticles modified with 3-(N,N-diethylamino) propyltrimethoxysilane (Fe3O4-PSA) and commercial C18 were selected as the cleanup adsorbents to remove the matrix interferences. The target analytes were further analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The factors influencing the recoveries of analytes, such as extraction time, amount of Fe3O4-PSA and C18, were optimized. The optimal extraction time was 10 min, and the amount of Fe3O4-PSA and C18 were 40 mg and 30 mg, respectively. The method showed good linearity in the concentration range from 2 μg/L to 250 μg/L except for 3-hydroxy-carbofuran and isocarbophos (5 μg/L to 250 μg/L). The limits of detection (LODs) ranged from 0.1 μg/L to 1.0 μg/L. The recoveries ranged from 74.1% to 120.0% at three spiked concentration levels with relative standard deviations of lower than 18.1%. The proposed method was rapid, simple, low-cost and effective for detection of trace multi-pesticides residue in soil.
A simple and rapid sample pretreatment method was developed based on magnetic nanoparticles for multi-pesticides residue analysis in soil samples. The target analytes were extracted by acetonitrile under the assistance of ultra-sonication, followed by the purification of dispersive solid phase extraction. The magnetite (Fe3O4) nanoparticles modified with 3-(N,N-diethylamino) propyltrimethoxysilane (Fe3O4-PSA) and commercial C18 were selected as the cleanup adsorbents to remove the matrix interferences. The target analytes were further analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The factors influencing the recoveries of analytes, such as extraction time, amount of Fe3O4-PSA and C18, were optimized. The optimal extraction time was 10 min, and the amount of Fe3O4-PSA and C18 were 40 mg and 30 mg, respectively. The method showed good linearity in the concentration range from 2 μg/L to 250 μg/L except for 3-hydroxy-carbofuran and isocarbophos (5 μg/L to 250 μg/L). The limits of detection (LODs) ranged from 0.1 μg/L to 1.0 μg/L. The recoveries ranged from 74.1% to 120.0% at three spiked concentration levels with relative standard deviations of lower than 18.1%. The proposed method was rapid, simple, low-cost and effective for detection of trace multi-pesticides residue in soil.
2019, 47(2): 271-280
doi: 10.19756/j.issn.0253-3820.181574
Abstract:
The porous graphene (PG) with excellent structure was successfully prepared by a simple pyrolysis process, which was then applied to the construction of electrochemical sensor (PG@GCE) for detection of calycosin. The porous graphene was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. Also, the electrochemical properties of the proposed sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), indicating that the sensor had relatively large specific surface area and higher electron transport rate. Based on all those characteristics, the PC@GCE sensor displayed a great response to calycosin, a traditional Chinese medicine (TCM) active ingredient. Under the optimized conditions, the sensors displayed a good linear relationship between the peak current and the calycosin concentration in the range of 1.8×10-7-4.4×10-5 mol/L with a detection limit of 5.8×10-8 mol/L (S/N=3). This study provided a novel analytical method for trace detection of calycosin, and rapid identification of Radix Hedysari and Radix Astragali in biological samples. At the same time, it would also deepen the application of porous graphene in the field of pharmaceutical analysis.
The porous graphene (PG) with excellent structure was successfully prepared by a simple pyrolysis process, which was then applied to the construction of electrochemical sensor (PG@GCE) for detection of calycosin. The porous graphene was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. Also, the electrochemical properties of the proposed sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), indicating that the sensor had relatively large specific surface area and higher electron transport rate. Based on all those characteristics, the PC@GCE sensor displayed a great response to calycosin, a traditional Chinese medicine (TCM) active ingredient. Under the optimized conditions, the sensors displayed a good linear relationship between the peak current and the calycosin concentration in the range of 1.8×10-7-4.4×10-5 mol/L with a detection limit of 5.8×10-8 mol/L (S/N=3). This study provided a novel analytical method for trace detection of calycosin, and rapid identification of Radix Hedysari and Radix Astragali in biological samples. At the same time, it would also deepen the application of porous graphene in the field of pharmaceutical analysis.
2019, 47(2): 281-287
doi: 10.19756/j.issn.0253-3820.181695
Abstract:
Fingerprint identification is one of the most reliable methods to identify cognizance in the field of forensic science. It is particular important to investigate the development and enhancement of latent fingerprints on complex backgrounds. In this study, diatomaceous earth-based colored powders were used to develop latent fingerprints on complex backgrounds and image enhancement analyses were performed after development process. Diatomaceous earth after pickling and heat treatment was used as the matrix material. The particle morphology exhibited disc shape with a radius of about 15 μm. Nitrogen adsorption-desorption measurement showed that diatomaceous earth had a BET surface area of 38.7 m2/g, a narrow pore-size distribution centered at 4.5 nm, and a cumulative volume of 0.067 cm3/g. Organic dyes were incorporated into diatomaceous earth based on physical adsorption. Malachite green and crystal violet were chosen because of the similarity in molecular structure but the difference in color. According to the UV-Vis spectra, the standard curves of malachite green and crystal violet were y=0.1336x-0.01304 (R2=0.99945) and y=0.1136x-0.00400 (R2=0.99939) respectively. The adsorption capacity of diatomaceous earth to malachite green and crystal violet was 0.60 and 0.62 mg/g, respectively, and on the basis of this, the diatomaceous earth-based colored powders were prepared. Latent fingerprints on complex backgrounds including food packaging and beverage label were developed by the as-prepared powders. To qualitatively and quantitatively analyze the effect of image enhancement, R, G and B channels in RGB color images of fingerprints captured by a digital camera were extracted by Photoshop, and gray analysis was performed by Image J. The results indicated that the combination of diatomaceous earth-based colored powders and image processing software could effectively avoid background interference and eventually achieve clear fingerprint images.
Fingerprint identification is one of the most reliable methods to identify cognizance in the field of forensic science. It is particular important to investigate the development and enhancement of latent fingerprints on complex backgrounds. In this study, diatomaceous earth-based colored powders were used to develop latent fingerprints on complex backgrounds and image enhancement analyses were performed after development process. Diatomaceous earth after pickling and heat treatment was used as the matrix material. The particle morphology exhibited disc shape with a radius of about 15 μm. Nitrogen adsorption-desorption measurement showed that diatomaceous earth had a BET surface area of 38.7 m2/g, a narrow pore-size distribution centered at 4.5 nm, and a cumulative volume of 0.067 cm3/g. Organic dyes were incorporated into diatomaceous earth based on physical adsorption. Malachite green and crystal violet were chosen because of the similarity in molecular structure but the difference in color. According to the UV-Vis spectra, the standard curves of malachite green and crystal violet were y=0.1336x-0.01304 (R2=0.99945) and y=0.1136x-0.00400 (R2=0.99939) respectively. The adsorption capacity of diatomaceous earth to malachite green and crystal violet was 0.60 and 0.62 mg/g, respectively, and on the basis of this, the diatomaceous earth-based colored powders were prepared. Latent fingerprints on complex backgrounds including food packaging and beverage label were developed by the as-prepared powders. To qualitatively and quantitatively analyze the effect of image enhancement, R, G and B channels in RGB color images of fingerprints captured by a digital camera were extracted by Photoshop, and gray analysis was performed by Image J. The results indicated that the combination of diatomaceous earth-based colored powders and image processing software could effectively avoid background interference and eventually achieve clear fingerprint images.
2019, 47(2): 288-296
doi: 10.19756/j.issn.0253-3820.181450
Abstract:
A method for determination of 16 kinds of N-nitrosamines in drinking water was established using solid phase extraction-gas chromatography-orbitrap mass spectrometry. The water sample was extracted by solid phase extraction, concentrated by nitrogen blowing, separated by DB-35MS column, detected by high-resolution mass spectrometry, and quantified by internal standard method. The chromatographic separation conditions, such as ion source temp, carrier gas flow, injection mode and surge pressure of the method were optimized, and the optimal conditions were obtained including 280℃ of ion source temp, 2.0 mL/min of carrier gas flow, and 200 kPa of splitless and surge pressure. Based on four-factor three-level orthogonal experiment, the solid phase extraction conditions were optimized as Chromabond HR-P and 10 mL of ethyl acetate. The results showed that the limits of detection (LOD) of the analytes were 0.05-0.50 ng/L, and the limits of quantitation (LOQ) were 0.2-2.0 ng/L, which were far lower than the literature value. The linear range was 0.2-500 μg/L with correlation coefficients (R2) of 0.9943-0.9997. The recoveries at four different spiking concentrations ranged from 72.4% to 114.8%, and the relative standard deviations (RSD, n=6) ranged from 0.8% to 9.5%. Finally, 12 actual water samples collected in Beijing were measured. Among them, five kinds of nitrosamines within 0.9 ng/L to 20.4 ng/L were detected. The method exhibited high sensitivity, selectivity and accuracy, and was suitable for the determination of trace nitrosamines in water.
A method for determination of 16 kinds of N-nitrosamines in drinking water was established using solid phase extraction-gas chromatography-orbitrap mass spectrometry. The water sample was extracted by solid phase extraction, concentrated by nitrogen blowing, separated by DB-35MS column, detected by high-resolution mass spectrometry, and quantified by internal standard method. The chromatographic separation conditions, such as ion source temp, carrier gas flow, injection mode and surge pressure of the method were optimized, and the optimal conditions were obtained including 280℃ of ion source temp, 2.0 mL/min of carrier gas flow, and 200 kPa of splitless and surge pressure. Based on four-factor three-level orthogonal experiment, the solid phase extraction conditions were optimized as Chromabond HR-P and 10 mL of ethyl acetate. The results showed that the limits of detection (LOD) of the analytes were 0.05-0.50 ng/L, and the limits of quantitation (LOQ) were 0.2-2.0 ng/L, which were far lower than the literature value. The linear range was 0.2-500 μg/L with correlation coefficients (R2) of 0.9943-0.9997. The recoveries at four different spiking concentrations ranged from 72.4% to 114.8%, and the relative standard deviations (RSD, n=6) ranged from 0.8% to 9.5%. Finally, 12 actual water samples collected in Beijing were measured. Among them, five kinds of nitrosamines within 0.9 ng/L to 20.4 ng/L were detected. The method exhibited high sensitivity, selectivity and accuracy, and was suitable for the determination of trace nitrosamines in water.
2019, 47(2): 297-305
doi: 10.19756/j.issn.0253-3820.181118
Abstract:
A ultra performance liquid chromatography-tandem mass spectrometry method for determination of triazole fungicides in pork, pork liver and pork fat tissue was established. Samples were extracted with acetonitrile, concentrated, and well-mixed with water. The mixed extract was then purified by a PRiME HLB column, and the elution was collected and detected by ultra performance liquid chromatography-tandem mass spectrometry. The matrix-matched standard curve was applied for quantification. The curve was linear in the range of 0.5-100 μg/L with the correlation coefficients of higher than 0.99. The recoveries ranged from 69.5% to 109.5% at the spiked levels of 2.0 μg/kg, 5.0 μg/kg and 10.0 μg/kg, with relative standard deviations of 0.4%-10.8%. The method has many advantages such as simple operation, high sensitivity and good precision, and can largely eliminate the interference in the matrix and is suitable for the residue detection of 21 triazolecompounds in the muscle, liver and fat tissues of animal derived food.
A ultra performance liquid chromatography-tandem mass spectrometry method for determination of triazole fungicides in pork, pork liver and pork fat tissue was established. Samples were extracted with acetonitrile, concentrated, and well-mixed with water. The mixed extract was then purified by a PRiME HLB column, and the elution was collected and detected by ultra performance liquid chromatography-tandem mass spectrometry. The matrix-matched standard curve was applied for quantification. The curve was linear in the range of 0.5-100 μg/L with the correlation coefficients of higher than 0.99. The recoveries ranged from 69.5% to 109.5% at the spiked levels of 2.0 μg/kg, 5.0 μg/kg and 10.0 μg/kg, with relative standard deviations of 0.4%-10.8%. The method has many advantages such as simple operation, high sensitivity and good precision, and can largely eliminate the interference in the matrix and is suitable for the residue detection of 21 triazolecompounds in the muscle, liver and fat tissues of animal derived food.
2019, 47(2): 306-314
doi: 10.19756/j.issn.0253-3820.171266
Abstract:
A sensitive method was developed for simultaneous determination of chlorpyrifos (CPF) and its main metabolite 3,5,6-trichloro-2-pyridinol (TCP) in tea samples by QuEChERS and ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Tea samples were extracted with acidic acetonitrile (containing 5% acetic acid), and cleaned up by the mixture of adsorbents containing 100 mg of graphitized carbon black (GCB) and 200 mg of C18. The extracts were diluted with deionized water equally in volume prior to instrumental analysis. Scheduled multiple reaction monitoring in positive ion mode was applied to detection of chlorpyrifos and selected ion recording in negative mode was applied to detection of TCP. The matrix-matched internal standard method was used for quantification. The results showed that the method provided linear ranges from 0.4 to 400 μg/kg and 0.4 to 80 μg/kg for CPF and TCP respectively with the correlation coefficients of larger than 0.99. The limits of detection (S/N=3) were 0.06 μg/kg for CPF and 0.04-0.08 μg/kg for TCP, while the limits of quantification (S/N=10) were 0.20 μg/kg for CPF and 0.14-0.26 μg/kg for TCP. The recoveries were ranged from 93.9% to 111.4% for CPF and 92.4% to 118.4% for TCP respectively with the relative standard deviations (RSD, n=6) of 3.2%-11.1%. This method was sensitive, rapid, simple and had good reproducibility, indicating that it was suitable for simultaneous determination of CPF and TCP in tea samples.
A sensitive method was developed for simultaneous determination of chlorpyrifos (CPF) and its main metabolite 3,5,6-trichloro-2-pyridinol (TCP) in tea samples by QuEChERS and ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Tea samples were extracted with acidic acetonitrile (containing 5% acetic acid), and cleaned up by the mixture of adsorbents containing 100 mg of graphitized carbon black (GCB) and 200 mg of C18. The extracts were diluted with deionized water equally in volume prior to instrumental analysis. Scheduled multiple reaction monitoring in positive ion mode was applied to detection of chlorpyrifos and selected ion recording in negative mode was applied to detection of TCP. The matrix-matched internal standard method was used for quantification. The results showed that the method provided linear ranges from 0.4 to 400 μg/kg and 0.4 to 80 μg/kg for CPF and TCP respectively with the correlation coefficients of larger than 0.99. The limits of detection (S/N=3) were 0.06 μg/kg for CPF and 0.04-0.08 μg/kg for TCP, while the limits of quantification (S/N=10) were 0.20 μg/kg for CPF and 0.14-0.26 μg/kg for TCP. The recoveries were ranged from 93.9% to 111.4% for CPF and 92.4% to 118.4% for TCP respectively with the relative standard deviations (RSD, n=6) of 3.2%-11.1%. This method was sensitive, rapid, simple and had good reproducibility, indicating that it was suitable for simultaneous determination of CPF and TCP in tea samples.
2019, 47(2): 315-321
doi: 10.19756/j.issn.0253-3820.181658
Abstract:
The analytical near infrared (NIR) spectroscopy model of 1,3-dichloro-2-propanol in the production process of 1,3-dichloro-2-propanol was established with sampling error profile analysis (SEPA) method. In SEPA, with a number of randomly sampling (1000 sampling in this work), 1000 sub-models were built and a series of cross validation errors under several PLS factors were obtained. With the errors, an error profile was plotted and some statistic parameters, including median, standard deviation, skewness and kurtosis were calculated. Condition optimization, modeling and evaluation of the model were carried out by comprehensively investigating the statistic performances. It was found that four spectral pretreatment methods showed good performance, and they were used to the following process of wavenumber selection to further improve the model with SEPA. The optimization results showed that the method was 1st derivative coupled with standard normal variate, the wavenumber region selected here was 6931-6017 cm-1, and five PLS factors were employed. Under the optimal conditions, the root mean squared error of calibration, the cross validation and the prediction were 0.881%, 1.282% and 1.167%, respectively. The selected wavenumber region was explainable, and the built model was reasonable and believable in view of statistics, which indicated that SEPA could supply a useful and robust NIR model.
The analytical near infrared (NIR) spectroscopy model of 1,3-dichloro-2-propanol in the production process of 1,3-dichloro-2-propanol was established with sampling error profile analysis (SEPA) method. In SEPA, with a number of randomly sampling (1000 sampling in this work), 1000 sub-models were built and a series of cross validation errors under several PLS factors were obtained. With the errors, an error profile was plotted and some statistic parameters, including median, standard deviation, skewness and kurtosis were calculated. Condition optimization, modeling and evaluation of the model were carried out by comprehensively investigating the statistic performances. It was found that four spectral pretreatment methods showed good performance, and they were used to the following process of wavenumber selection to further improve the model with SEPA. The optimization results showed that the method was 1st derivative coupled with standard normal variate, the wavenumber region selected here was 6931-6017 cm-1, and five PLS factors were employed. Under the optimal conditions, the root mean squared error of calibration, the cross validation and the prediction were 0.881%, 1.282% and 1.167%, respectively. The selected wavenumber region was explainable, and the built model was reasonable and believable in view of statistics, which indicated that SEPA could supply a useful and robust NIR model.
