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2020 Volume 48 Issue 1
2020, 48(1): 1-12
doi: 10.19756/j.issn.0253-3820.191322
Abstract:
Genome editing has become a vital tool in medical biology research. The critical mission of facilitating the progress of genome editing is to enrich positive edited cells quickly and effectively. In recent years, researchers have established various reporter systems for selection and enrichment of editing-induced positive cells based on genome repair mechanisms, such as non-homologous end joining, homology directed repair, single strand annealing and inversion, and the principle of the expression of fluorescent protein or resistance tag after genome repair. The T7E1 assay or sequencing method can analyze the mutation of enriched cells with lower background signals and higher mutation ratio. Therefore, these reporter systems can profit the characterization of genome editing effectiveness. Besides, positive cells can be cultured continuously, so this technology possesses a promising prospect of mutated cell line construction and the research of mutated cell functions. This article summarized the design principles and applications of these reporter systems and provided a reference to construct a more perfect evaluating system for genome editing.
Genome editing has become a vital tool in medical biology research. The critical mission of facilitating the progress of genome editing is to enrich positive edited cells quickly and effectively. In recent years, researchers have established various reporter systems for selection and enrichment of editing-induced positive cells based on genome repair mechanisms, such as non-homologous end joining, homology directed repair, single strand annealing and inversion, and the principle of the expression of fluorescent protein or resistance tag after genome repair. The T7E1 assay or sequencing method can analyze the mutation of enriched cells with lower background signals and higher mutation ratio. Therefore, these reporter systems can profit the characterization of genome editing effectiveness. Besides, positive cells can be cultured continuously, so this technology possesses a promising prospect of mutated cell line construction and the research of mutated cell functions. This article summarized the design principles and applications of these reporter systems and provided a reference to construct a more perfect evaluating system for genome editing.
2020, 48(1): 13-21
doi: 10.19756/j.issn.0253-3820.191566
Abstract:
As a post-translational modification, protein glycosylation not only reflects on body health, but also serves as the target of drug treatment. Therefore, it is of great significance to study protein glycosylation for the diagnosis and treatment of diseases. However, many glycoproteins of biological importance exhibit relatively low abundance, high complexity, and wide dynamic range in biological samples. It is indispensable to explore highly efficient sample pretreatment methods. In recent years, monolithic columns have attracted extensive attention in the field of separation and enrichment of glycoproteins due to their advantages of rapid separation, high efficiency, and good biological compatibility. In this paper, recent progress of monolithic columns in the field of separation and enrichment of glycoproteins is surveyed, and the future development is briefed.
As a post-translational modification, protein glycosylation not only reflects on body health, but also serves as the target of drug treatment. Therefore, it is of great significance to study protein glycosylation for the diagnosis and treatment of diseases. However, many glycoproteins of biological importance exhibit relatively low abundance, high complexity, and wide dynamic range in biological samples. It is indispensable to explore highly efficient sample pretreatment methods. In recent years, monolithic columns have attracted extensive attention in the field of separation and enrichment of glycoproteins due to their advantages of rapid separation, high efficiency, and good biological compatibility. In this paper, recent progress of monolithic columns in the field of separation and enrichment of glycoproteins is surveyed, and the future development is briefed.
2020, 48(1): 22-27
doi: 10.19756/j.issn.0253-3820.191550
Abstract:
A miniaturized dielectric barrier discharge ionization (DBDI) source assisted with a sample rapid evaporation platform was developed. With the DBDI source, the sample solution could be evaporated rapidly into aerosol state and then transferred to plasma ionization region, which improved the efficiency of sample transmission and ionization. The miniaturized DBDI source was coupled to a homemade miniaturized linear ion trap mass spectrometer which had a total volume of 31.5 cm×27.3 cm×27.3 cm and a weight of about 18 kg. In the experiment, the flow rate of carrier gas (He) and the temperature of sample rapid evaporation platform were optimized, and the limit of detection (LOD) and stability of the instrument were evaluated. Experimental results indicated that a LOD of 0.2 ng/mL (caffeine solution, mass selected storage, m/z 195) was achieved. The relative standard deviation (RSD) of the mass spectrum intensity was calculated to be 11.1%. Furthermore, various samples including propofol, arginine, SIPI5286 and reserpine were analyzed using the instrument. The combination of rapid evaporation assisted DBDI source and the homemade miniaturized linear ion trap mass spectrometer had the characteristics of strong qualitative ability, fast analytical speed, low limit of detection and high stability, and showed a great potential in the field of real-time and in-situ analysis.
A miniaturized dielectric barrier discharge ionization (DBDI) source assisted with a sample rapid evaporation platform was developed. With the DBDI source, the sample solution could be evaporated rapidly into aerosol state and then transferred to plasma ionization region, which improved the efficiency of sample transmission and ionization. The miniaturized DBDI source was coupled to a homemade miniaturized linear ion trap mass spectrometer which had a total volume of 31.5 cm×27.3 cm×27.3 cm and a weight of about 18 kg. In the experiment, the flow rate of carrier gas (He) and the temperature of sample rapid evaporation platform were optimized, and the limit of detection (LOD) and stability of the instrument were evaluated. Experimental results indicated that a LOD of 0.2 ng/mL (caffeine solution, mass selected storage, m/z 195) was achieved. The relative standard deviation (RSD) of the mass spectrum intensity was calculated to be 11.1%. Furthermore, various samples including propofol, arginine, SIPI5286 and reserpine were analyzed using the instrument. The combination of rapid evaporation assisted DBDI source and the homemade miniaturized linear ion trap mass spectrometer had the characteristics of strong qualitative ability, fast analytical speed, low limit of detection and high stability, and showed a great potential in the field of real-time and in-situ analysis.
2020, 48(1): 28-33
doi: 10.19756/j.issn.0253-3820.191607
Abstract:
A home-made vacuum ultraviolet (VUV) photoionization time-of-flight mass spectrometer (VUVPI-TOFMS) was assembled to study atmospheric radical reactions. In the work, a commercial VUV discharge lamp with photon energy at 10.6 eV was installed as light source and a cage-shaped photoionization source was employed to efficiently extract and focus ions. With this spectrometer, a limit of detection (LOD) of 0.03 μg/L (S/N=3) for benzene detection was obtained. Then a microwave discharge flow tube was employed as reactor to produce free radicals and model their atmospheric reactions. The concentration of the methyl radical was measured with its self-reaction kinetics within the flow tube. As a representative example, the reaction of CH3 and NO was investigated by VUVPI-TOFMS. Both the stable species and radicals involved in the reaction were probed and the reaction rate constant at room temperature and 300 Pa pressure was measured to be k(CH3+NO)=1.2×10-12 cm3/(molecule·s).
A home-made vacuum ultraviolet (VUV) photoionization time-of-flight mass spectrometer (VUVPI-TOFMS) was assembled to study atmospheric radical reactions. In the work, a commercial VUV discharge lamp with photon energy at 10.6 eV was installed as light source and a cage-shaped photoionization source was employed to efficiently extract and focus ions. With this spectrometer, a limit of detection (LOD) of 0.03 μg/L (S/N=3) for benzene detection was obtained. Then a microwave discharge flow tube was employed as reactor to produce free radicals and model their atmospheric reactions. The concentration of the methyl radical was measured with its self-reaction kinetics within the flow tube. As a representative example, the reaction of CH3 and NO was investigated by VUVPI-TOFMS. Both the stable species and radicals involved in the reaction were probed and the reaction rate constant at room temperature and 300 Pa pressure was measured to be k(CH3+NO)=1.2×10-12 cm3/(molecule·s).
2020, 48(1): 34-39
doi: 10.19756/j.issn.0253-3820.191317
Abstract:
The peptide portion of the sialylglycopeptide (SGP) in egg yolk is composed of six amino acids residues (KVANKT), in which asparagine (N) is modified by sialylated complex N-glycan. SGP is mainly obtained from egg yolk, and currently, the methods for separation and purification of SGP are cumbersome, expensive, and difficult to scale production. In this study, a simple and low-cost method for separation and purification of sialylated glycopeptide from egg yolk was developed based on the hydrophilic interaction chromatography by using medical absorbent cotton as stationary phase. Firstly, 50 egg yolks were treated with phenol to obtain crude sialylated glycopeptide. The obtained glycopeptide was then formulated into a 150 mg/mL solution and loaded onto the prepared cotton hydrophilic chromatographic column, followed by the treatment with 100%, 95%, 85% and 75% acetonitrile aqueous solution to remove the impurity. Finally, the sialyglycopeptide was obtained by eluting with deionized water. By this way, 300 mg SGP was obtained and its purity was identified by high-performance liquid chromatography (HPLC) to be 95%. The glycan composition and glycosylation site of SGP were further validated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS).
The peptide portion of the sialylglycopeptide (SGP) in egg yolk is composed of six amino acids residues (KVANKT), in which asparagine (N) is modified by sialylated complex N-glycan. SGP is mainly obtained from egg yolk, and currently, the methods for separation and purification of SGP are cumbersome, expensive, and difficult to scale production. In this study, a simple and low-cost method for separation and purification of sialylated glycopeptide from egg yolk was developed based on the hydrophilic interaction chromatography by using medical absorbent cotton as stationary phase. Firstly, 50 egg yolks were treated with phenol to obtain crude sialylated glycopeptide. The obtained glycopeptide was then formulated into a 150 mg/mL solution and loaded onto the prepared cotton hydrophilic chromatographic column, followed by the treatment with 100%, 95%, 85% and 75% acetonitrile aqueous solution to remove the impurity. Finally, the sialyglycopeptide was obtained by eluting with deionized water. By this way, 300 mg SGP was obtained and its purity was identified by high-performance liquid chromatography (HPLC) to be 95%. The glycan composition and glycosylation site of SGP were further validated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS).
2020, 48(1): 40-48
doi: 10.19756/j.issn.0253-3820.191318
Abstract:
MicroRNA (miRNA) detection is of considerable significance in the diagnosis and treatment of cancer. The expression level of cancer-related miRNA differs significantly in various cancer cell types, and it remains challenging to detect miRNA at both high and low levels accurately using a conventional sensor with fixed limit of detection and narrow linear detecting range. Herein, three kinds of gold nanoparticle-quantum dots composite were constructed by reasonable DNA sequence design. By stacking these nanocomposites sequentially, three kinds of sensors of single, double and multiple amplifications based on entropy-driven catalytic circuits were successfully established. The product of the upper nanocomposite was served as the catalyst of the downstream nanocomposite to activate disassembly, inducing high-order amplification. With stacking a nucleic acid circuit of the sensor, the detection limit of the sensor was reduced by one order of magnitude. Finally, the nanosensor had a detection limit of fmol/L. By stacking entropy-driving nucleic acid circuits, a novel, dynamically tunable nanosensor was established for quantitatively detecting miRNA with different expression levels in cancer cells.
MicroRNA (miRNA) detection is of considerable significance in the diagnosis and treatment of cancer. The expression level of cancer-related miRNA differs significantly in various cancer cell types, and it remains challenging to detect miRNA at both high and low levels accurately using a conventional sensor with fixed limit of detection and narrow linear detecting range. Herein, three kinds of gold nanoparticle-quantum dots composite were constructed by reasonable DNA sequence design. By stacking these nanocomposites sequentially, three kinds of sensors of single, double and multiple amplifications based on entropy-driven catalytic circuits were successfully established. The product of the upper nanocomposite was served as the catalyst of the downstream nanocomposite to activate disassembly, inducing high-order amplification. With stacking a nucleic acid circuit of the sensor, the detection limit of the sensor was reduced by one order of magnitude. Finally, the nanosensor had a detection limit of fmol/L. By stacking entropy-driving nucleic acid circuits, a novel, dynamically tunable nanosensor was established for quantitatively detecting miRNA with different expression levels in cancer cells.
2020, 48(1): 49-56
doi: 10.19756/j.issn.0253-3820.191618
Abstract:
Ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry was used in plasma metabolomics study of patients with coronary heart disease and coronary heart disease-type 2 diabetes mellitus. The aim was to explore the metabolic characteristics of coronary heart disease and coronary heart disease-type 2 diabetes mellitus, which would be useful for the optimization of clinical treatment plans based on metabolomics. A total of 78 endogenous metabolites were analyzed in plasma. On this basis, combined with partial least squares-discriminant analysis and variable importance projection, 20 characteristic metabolites were screened to distinguish healthy control from patients with coronary heart disease, 35 metabolites were screened for discrimination of patients with coronary heart disease and coronary heart disease-type 2 diabetes mellitus, 37 metabolites were screened to distinguish healthy control from patients with coronary heart disease-type 2 diabetes mellitus. Metabolic pathway analysis showed that amino acid metabolism played an important role in the physiological metabolism of these two types of patients, and fatty acid metabolism was likely to be associated with the complication of type 2 diabetes mellitus.
Ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry was used in plasma metabolomics study of patients with coronary heart disease and coronary heart disease-type 2 diabetes mellitus. The aim was to explore the metabolic characteristics of coronary heart disease and coronary heart disease-type 2 diabetes mellitus, which would be useful for the optimization of clinical treatment plans based on metabolomics. A total of 78 endogenous metabolites were analyzed in plasma. On this basis, combined with partial least squares-discriminant analysis and variable importance projection, 20 characteristic metabolites were screened to distinguish healthy control from patients with coronary heart disease, 35 metabolites were screened for discrimination of patients with coronary heart disease and coronary heart disease-type 2 diabetes mellitus, 37 metabolites were screened to distinguish healthy control from patients with coronary heart disease-type 2 diabetes mellitus. Metabolic pathway analysis showed that amino acid metabolism played an important role in the physiological metabolism of these two types of patients, and fatty acid metabolism was likely to be associated with the complication of type 2 diabetes mellitus.
2020, 48(1): 57-65
doi: 10.19756/j.issn.0253-3820.191343
Abstract:
Continuous phase co-flow can increase the throughput and control flexibility of step emulsification, and has an important effect on droplet microfluidic. This study built a multi-channel step emulsification device to generate Janus droplets and to study the effects of both continuous phase co-flow and dispersed phase flow on the generation of Janus droplets. Firstly, Janus droplets were successfully prepared by the device, and the generation stability was optimized by using glass capillaries instead of microcapillary film based on their different wall contact angles at the channel outlet. Secondly, the effect on the generation of Janus droplet was studied by changing the flows of the continuous and dispersed phases, respectively. The result showed that the Janus droplets generation stability was almost independent of the two phases flow rates. When the continuous phase co-flow rate increased, the resulted Janus droplet diameter decreased, and the generation frequency increased. The droplet diameter decreased by 61% with the co-flow rate increasing from 0.01 mL/h to 30 mL/h. When the flow rate of dispersed phase increased, both the diameter and generation frequency of the generated Janus droplet increased. The droplet diameter increased by 63% as the dispersed phase flow increased from 0.1 mL/h to 3.0 mL/h. Within the scope of this study, the diameter and the generation frequency of the two components Janus droplet could be controlled in 638-1640 μm and 0.02-2.2 Hz, respectively. In addition, three-component droplets were generated successfully and steadily by a three-component droplet generation chip which was prepared based on the two-component droplet generation device. By changing the two phase flow rates, the droplet diameter and frequency could also be tuned on-line. When the co-flow rate increased from 1 mL/h to 30 mL/h, the droplet diameter decreased by 45%, and when the dispersed phase flow increased from 0.1 mL/h to 5 mL/h, the droplet diameter increased by 89%. The generation frequencies increased accordingly. Additionally, it was found that the co-flow effect had a greater impact on the generation of three-component droplet due to the result that the three-component droplet diameter changed greater than the two-component droplet when changing both the continuous and dispersed phase flows. Therefore, in this study, two-component and three-component droplets were generated by co-flowing step emulsification, which could be controlled on-line by changing the co-flow rate. The results of this study provided a basis and method for micro and trace research of chemical and analysis industry.
Continuous phase co-flow can increase the throughput and control flexibility of step emulsification, and has an important effect on droplet microfluidic. This study built a multi-channel step emulsification device to generate Janus droplets and to study the effects of both continuous phase co-flow and dispersed phase flow on the generation of Janus droplets. Firstly, Janus droplets were successfully prepared by the device, and the generation stability was optimized by using glass capillaries instead of microcapillary film based on their different wall contact angles at the channel outlet. Secondly, the effect on the generation of Janus droplet was studied by changing the flows of the continuous and dispersed phases, respectively. The result showed that the Janus droplets generation stability was almost independent of the two phases flow rates. When the continuous phase co-flow rate increased, the resulted Janus droplet diameter decreased, and the generation frequency increased. The droplet diameter decreased by 61% with the co-flow rate increasing from 0.01 mL/h to 30 mL/h. When the flow rate of dispersed phase increased, both the diameter and generation frequency of the generated Janus droplet increased. The droplet diameter increased by 63% as the dispersed phase flow increased from 0.1 mL/h to 3.0 mL/h. Within the scope of this study, the diameter and the generation frequency of the two components Janus droplet could be controlled in 638-1640 μm and 0.02-2.2 Hz, respectively. In addition, three-component droplets were generated successfully and steadily by a three-component droplet generation chip which was prepared based on the two-component droplet generation device. By changing the two phase flow rates, the droplet diameter and frequency could also be tuned on-line. When the co-flow rate increased from 1 mL/h to 30 mL/h, the droplet diameter decreased by 45%, and when the dispersed phase flow increased from 0.1 mL/h to 5 mL/h, the droplet diameter increased by 89%. The generation frequencies increased accordingly. Additionally, it was found that the co-flow effect had a greater impact on the generation of three-component droplet due to the result that the three-component droplet diameter changed greater than the two-component droplet when changing both the continuous and dispersed phase flows. Therefore, in this study, two-component and three-component droplets were generated by co-flowing step emulsification, which could be controlled on-line by changing the co-flow rate. The results of this study provided a basis and method for micro and trace research of chemical and analysis industry.
2020, 48(1): 66-73
doi: 10.19756/j.issn.0253-3820.191281
Abstract:
A new sandwich structure toluene gas sensor was designed by transferring LaFe1-xPdxO3 nanofiber that was prepared by electrospinning method onto silicon substrate with platinum-inter digital electrodes by dip-coating method. The crystalline phase and microstructure of LaFe1-xPdxO3 nanofibers were characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM). Sensitive mechanism and electrochemical characteristic of LaFe1-xPdxO3 nanofibers were analyzed by X-ray photoelectron spectroscopy (XPS). These characteristic tests for the toluene gas sensor were carried out to obtain the optimal sensitive performance, temperature, relative humidity, dynamic response, selectivity and stability. The result showed that the toluene gas sensor based on LaFe0.9Pd0.1O3 nanofiber had a good linear relationship with toluene in the concentration range of 1.0-30 μg/m3 at operating temperature of 200℃. The dynamic response time and recover time of sensor to 30 μg/m3 toluene were 3.8 and 1.8 s, respectively. This kind of sensor showed good anti-disturbance to such gases as benzene, xylene, ethanol, formaldehyde and acetone. Besides, the toluene gas sensor response was attenuated only about 0.3, when it was applied continually on automobile about 50 days.
A new sandwich structure toluene gas sensor was designed by transferring LaFe1-xPdxO3 nanofiber that was prepared by electrospinning method onto silicon substrate with platinum-inter digital electrodes by dip-coating method. The crystalline phase and microstructure of LaFe1-xPdxO3 nanofibers were characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM). Sensitive mechanism and electrochemical characteristic of LaFe1-xPdxO3 nanofibers were analyzed by X-ray photoelectron spectroscopy (XPS). These characteristic tests for the toluene gas sensor were carried out to obtain the optimal sensitive performance, temperature, relative humidity, dynamic response, selectivity and stability. The result showed that the toluene gas sensor based on LaFe0.9Pd0.1O3 nanofiber had a good linear relationship with toluene in the concentration range of 1.0-30 μg/m3 at operating temperature of 200℃. The dynamic response time and recover time of sensor to 30 μg/m3 toluene were 3.8 and 1.8 s, respectively. This kind of sensor showed good anti-disturbance to such gases as benzene, xylene, ethanol, formaldehyde and acetone. Besides, the toluene gas sensor response was attenuated only about 0.3, when it was applied continually on automobile about 50 days.
2020, 48(1): 74-82
doi: 10.19756/j.issn.0253-3820.191442
Abstract:
A fluorescent aptasensor for detection of dimethoate pesticide based on aptamer regulated carbon dots was fabricated. In the presence of dimethoate, SS24-S-35 aptamer could alter the quenching efficiency of silver nanoparticles (AgNPs) on the fluorescence of carbon dots prepared from ethylenediamine and citric acid, and the degree of fluorescence quenching was proportional to the concentration of dimethoate molecules. Therefore, this strategy used the proposed aptasensor to quantitatively detect dimethoate pesticide by fluorescent assay. To improve the detection performance of the as-prepared aptasensor, the fluorescence quenching mechanism of AgNPs on carbon dots was fully discussed, and some parameters such as the types of carbon dots and the amount of AgNPs were investigated. Under optimal conditions, the aptasensor could detect dimethoate in a linear range from 6 μg/L to 200 μg/L (R2=0.984), with a limit of detection (LOD) as low as 2.24 μg/L (3σ/S). Moreover, the further studies confirmed that other pesticides had negligible effect on detection of dimethoate, indicating the excellent specificity of aptasensor for dimethoate detection. The aptasensor was succesfully used in detection of dimethoate pesticide in real agriculture products with recovery of 85.1%-105.0%, which indicated that the proposed aptasensor had potential application in the detection of pesticide residues.
A fluorescent aptasensor for detection of dimethoate pesticide based on aptamer regulated carbon dots was fabricated. In the presence of dimethoate, SS24-S-35 aptamer could alter the quenching efficiency of silver nanoparticles (AgNPs) on the fluorescence of carbon dots prepared from ethylenediamine and citric acid, and the degree of fluorescence quenching was proportional to the concentration of dimethoate molecules. Therefore, this strategy used the proposed aptasensor to quantitatively detect dimethoate pesticide by fluorescent assay. To improve the detection performance of the as-prepared aptasensor, the fluorescence quenching mechanism of AgNPs on carbon dots was fully discussed, and some parameters such as the types of carbon dots and the amount of AgNPs were investigated. Under optimal conditions, the aptasensor could detect dimethoate in a linear range from 6 μg/L to 200 μg/L (R2=0.984), with a limit of detection (LOD) as low as 2.24 μg/L (3σ/S). Moreover, the further studies confirmed that other pesticides had negligible effect on detection of dimethoate, indicating the excellent specificity of aptasensor for dimethoate detection. The aptasensor was succesfully used in detection of dimethoate pesticide in real agriculture products with recovery of 85.1%-105.0%, which indicated that the proposed aptasensor had potential application in the detection of pesticide residues.
2020, 48(1): 83-89
doi: 10.19756/j.issn.0253-3820.191576
Abstract:
A method for detecting cysteine based on fluorescenceresonance energy transfer (FRET) between DNA quantum dots (QDs) and polydopamine (PDA) was developed. Since the fluorescence emitted by DNA QDs was absorbed by the PDA molecule, FRET occurred, resulting in fluorescence quenching of DNA QDs, and leaving the DNA QDs in a fluorescent "off" state. In the presence of cysteine, the spontaneous oxidative polymerization from dopamine (DA) to PDA was blocked, the fluorescence of DNA QDs was restored, and the fluorescence was "on" state. Based on this, a cysteine fluorescence sensor was developed. The sensor had good selectivity to cysteine, and there was no interference between common amino acids and small biothiol molecules. The linear equation was y=0.0181x-0.0185 in linear range of 10.0-100.0 μmol/L. The limit of detection (LOD) was 1.7 μmol/L (S/N=3). The method was successfully applied to determination of cysteine in human urine samples, with recoveries of 98.6%-105.9%.
A method for detecting cysteine based on fluorescenceresonance energy transfer (FRET) between DNA quantum dots (QDs) and polydopamine (PDA) was developed. Since the fluorescence emitted by DNA QDs was absorbed by the PDA molecule, FRET occurred, resulting in fluorescence quenching of DNA QDs, and leaving the DNA QDs in a fluorescent "off" state. In the presence of cysteine, the spontaneous oxidative polymerization from dopamine (DA) to PDA was blocked, the fluorescence of DNA QDs was restored, and the fluorescence was "on" state. Based on this, a cysteine fluorescence sensor was developed. The sensor had good selectivity to cysteine, and there was no interference between common amino acids and small biothiol molecules. The linear equation was y=0.0181x-0.0185 in linear range of 10.0-100.0 μmol/L. The limit of detection (LOD) was 1.7 μmol/L (S/N=3). The method was successfully applied to determination of cysteine in human urine samples, with recoveries of 98.6%-105.9%.
2020, 48(1): 90-96
doi: 10.19756/j.issn.0253-3820.191355
Abstract:
Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) has been more and more used in the qualitative investigation of organic materials in cultural relics, owing to its high sensitivity and wide range of application. However, there are few systematical studies on identifying species of different protein materials by Py-GC/MS. Therefore, porcine blood, animal glue and egg, which are all commonly used as binding media in ancient Chinese artworks and objects, such as polychrome, architecture and furniture, have been investigated in the present work by Py-GC/MS, and the characteristic components of each protein have been summarized. Based on the established Py-GC/MS procedure, it's found that some nitrogen-free pyrolysis products can be served as characteristic components for distinguishing porcine blood, animal glue and egg white. Nitrogen-contained pyrolysis products of porcine blood, animal glue and egg white can be mainly divided into pyrroles, nitriles, prolines and indoles, and the relative content of the four classes of products in different protein is distinctly different, so it can also be served as feature information for distinguishing porcine blood, animal glue and egg white. In addition, pyrolysis products of egg yolk are mainly fatty acids. The established Py-GC/MS procedure and summarized data were successfully applied to the identification of protein binding medium in two historical samples of Qin Dynasty, which were collected from the mortar of Polychrome Lacquered Table with "Kui Long" Pattern and the ground layer of gilding painting on the eaves of West Wei-Fang of Hall of Mental Cultivation respectively. Research results of the present work are easy to spread, and it's not only suitable for the rapid and accurate identification of different proteins in cultural relics, but also has reference significance in the research of other organic materials used in cultural relics.
Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) has been more and more used in the qualitative investigation of organic materials in cultural relics, owing to its high sensitivity and wide range of application. However, there are few systematical studies on identifying species of different protein materials by Py-GC/MS. Therefore, porcine blood, animal glue and egg, which are all commonly used as binding media in ancient Chinese artworks and objects, such as polychrome, architecture and furniture, have been investigated in the present work by Py-GC/MS, and the characteristic components of each protein have been summarized. Based on the established Py-GC/MS procedure, it's found that some nitrogen-free pyrolysis products can be served as characteristic components for distinguishing porcine blood, animal glue and egg white. Nitrogen-contained pyrolysis products of porcine blood, animal glue and egg white can be mainly divided into pyrroles, nitriles, prolines and indoles, and the relative content of the four classes of products in different protein is distinctly different, so it can also be served as feature information for distinguishing porcine blood, animal glue and egg white. In addition, pyrolysis products of egg yolk are mainly fatty acids. The established Py-GC/MS procedure and summarized data were successfully applied to the identification of protein binding medium in two historical samples of Qin Dynasty, which were collected from the mortar of Polychrome Lacquered Table with "Kui Long" Pattern and the ground layer of gilding painting on the eaves of West Wei-Fang of Hall of Mental Cultivation respectively. Research results of the present work are easy to spread, and it's not only suitable for the rapid and accurate identification of different proteins in cultural relics, but also has reference significance in the research of other organic materials used in cultural relics.
2020, 48(1): 97-103
doi: 10.19756/j.issn.0253-3820.191398
Abstract:
In analysis of strontium (Sr) isotope in geological samples with high rubidium (Rb)/Sr ratios, a large number of matrix elements (Na, K, etc.) and isobaric interference of Rb on Sr isotopic analysis were effectively removed by coprecipitation method using NaOH. The influence of reaction temperature, reaction time and alkali concentration were investigated, and the optimum reaction conditions were determined finally. The results showed that the removal rate of Rb, Na, K, Al was over 99% and the recovery of Sr was better than 96% by coprecipitation for 1 h using 2 mL of NaOH with a mass-to-volume ratio of 5% at 100℃. After coprecipitation, high purity Sr was obtained by a single separation and purification using AG50W×12 cation exchange resin. NaOH (99.99%) without adding excess matrix elements and the total procedure blanks of Sr was less than 182 pg. With this method, Sr isotope was separated from eight standard rock samples with different Rb/Sr ratios (JR-2, BCR-2, BHVO-2, W-2, AGV-2, JG-3, JA-3, GSP-2), and then high precision determination of Sr isotope ratios was obtained by highly sensitive thermal ionization mass spectrometer (TIMS). The result showed that the coprecipitation-cation exchange resin separation process had many advantages such as wide application range, stability and reliability, low consumption of inorganic acids and short reaction time. Preconcentration of Sr by the coprecipitation method provided a new separation way for Sr isotope determination in geological samples with high Rb/Sr ratios, showing an important application value.
In analysis of strontium (Sr) isotope in geological samples with high rubidium (Rb)/Sr ratios, a large number of matrix elements (Na, K, etc.) and isobaric interference of Rb on Sr isotopic analysis were effectively removed by coprecipitation method using NaOH. The influence of reaction temperature, reaction time and alkali concentration were investigated, and the optimum reaction conditions were determined finally. The results showed that the removal rate of Rb, Na, K, Al was over 99% and the recovery of Sr was better than 96% by coprecipitation for 1 h using 2 mL of NaOH with a mass-to-volume ratio of 5% at 100℃. After coprecipitation, high purity Sr was obtained by a single separation and purification using AG50W×12 cation exchange resin. NaOH (99.99%) without adding excess matrix elements and the total procedure blanks of Sr was less than 182 pg. With this method, Sr isotope was separated from eight standard rock samples with different Rb/Sr ratios (JR-2, BCR-2, BHVO-2, W-2, AGV-2, JG-3, JA-3, GSP-2), and then high precision determination of Sr isotope ratios was obtained by highly sensitive thermal ionization mass spectrometer (TIMS). The result showed that the coprecipitation-cation exchange resin separation process had many advantages such as wide application range, stability and reliability, low consumption of inorganic acids and short reaction time. Preconcentration of Sr by the coprecipitation method provided a new separation way for Sr isotope determination in geological samples with high Rb/Sr ratios, showing an important application value.
2020, 48(1): 104-112
doi: 10.19756/j.issn.0253-3820.191367
Abstract:
Shellfish toxins are a kind of important marine pollutant. In this study, a novel ionic liquid bonded silica gel material (silica-[SOIM] [PF6]) was prepared by grafting octyl-functionalized ionic liquid onto silica gel, which was characterized and analyzed by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and elemental analysis. Solid phase extraction cartridge of silica-[SOIM] [PF6], coupled with LC-MS/MS technique was used to extract and determine the concentration of shellfish toxins[Okadaic acid (OA), Dinophysistoxin-1 (DTX-1) and Dinophysistoxin-2 (DTX-2)] in seawater for the first time. It was found that the solid phase extraction material silica-[SOIM] [PF6] had multiple interactions with the 3 kinds of shellfish toxins, such as hydrophobic and ion-exchange. The method was optimized in aspects of volume and pH of the loaded sample solution, as well as the usage of rinsing and eluting reagents. Results indicated that the silica-[SOIM] [PF6] had good extraction effect on target shellfish toxins in seawater, which was better than or equivalent to the performance of commercial extraction materials. The method had a good linearity (R2>0.995) in the concentration range of shellfish toxins from 0.02 μg/L to 2.50 μg/L, with limit of detection (LOD) of 0.01 μg/L and limit of quantification (LOQ) of 0.05 μg/L. The average recoveries for 3 kinds of shellfish toxins were 93.0%-116.0% at different spiking levels in blank seawater. Simultaneously, the material had good reproducibility, with the relative standard deviation (RSD) of less than 15% intra-and inter-batch. This method was accurate, sensitive, simple and reliable, and could be used for the extraction and detection of shellfish toxins in actual samples.
Shellfish toxins are a kind of important marine pollutant. In this study, a novel ionic liquid bonded silica gel material (silica-[SOIM] [PF6]) was prepared by grafting octyl-functionalized ionic liquid onto silica gel, which was characterized and analyzed by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and elemental analysis. Solid phase extraction cartridge of silica-[SOIM] [PF6], coupled with LC-MS/MS technique was used to extract and determine the concentration of shellfish toxins[Okadaic acid (OA), Dinophysistoxin-1 (DTX-1) and Dinophysistoxin-2 (DTX-2)] in seawater for the first time. It was found that the solid phase extraction material silica-[SOIM] [PF6] had multiple interactions with the 3 kinds of shellfish toxins, such as hydrophobic and ion-exchange. The method was optimized in aspects of volume and pH of the loaded sample solution, as well as the usage of rinsing and eluting reagents. Results indicated that the silica-[SOIM] [PF6] had good extraction effect on target shellfish toxins in seawater, which was better than or equivalent to the performance of commercial extraction materials. The method had a good linearity (R2>0.995) in the concentration range of shellfish toxins from 0.02 μg/L to 2.50 μg/L, with limit of detection (LOD) of 0.01 μg/L and limit of quantification (LOQ) of 0.05 μg/L. The average recoveries for 3 kinds of shellfish toxins were 93.0%-116.0% at different spiking levels in blank seawater. Simultaneously, the material had good reproducibility, with the relative standard deviation (RSD) of less than 15% intra-and inter-batch. This method was accurate, sensitive, simple and reliable, and could be used for the extraction and detection of shellfish toxins in actual samples.
2020, 48(1): 113-120
doi: 10.19756/j.issn.0253-3820.191535
Abstract:
A three-dimensional gallnut tannin/ordered mesoporous silica composite 1.2-PT-10-KIT-6 was prepared by one-step method with tetraethyl orthosilicate as silicon source, gallnut tannin as adsorption source and glutaraldehyde as crosslinking agent. Transmission electron microscopy, small angle X-ray diffraction, N2 adsorption and Fourier transform infrared spectroscopy were used to characterize the materials. All the materials had a three-dimensional cubic structure similar to KIT-6. The maximum adsorption capacity of gallium reached 186.73 mg/g at pH 10, which conformed to Langmuir monolayer adsorption, and the correlation coefficient R2 was 0.99. In the binary system of Ga/Ge and Ga/As, the selectivity factor was more than 1.5, which could realize the selective separation of gallium. The adsorption mechanism was ion exchange between phenolic hydroxyl group and Ga(OH)4- and the O atom on phenolic hydroxyl group chelated with Ga(OH)4- to form a chelate. After six regeneration cycles, the recovery was over 95%.
A three-dimensional gallnut tannin/ordered mesoporous silica composite 1.2-PT-10-KIT-6 was prepared by one-step method with tetraethyl orthosilicate as silicon source, gallnut tannin as adsorption source and glutaraldehyde as crosslinking agent. Transmission electron microscopy, small angle X-ray diffraction, N2 adsorption and Fourier transform infrared spectroscopy were used to characterize the materials. All the materials had a three-dimensional cubic structure similar to KIT-6. The maximum adsorption capacity of gallium reached 186.73 mg/g at pH 10, which conformed to Langmuir monolayer adsorption, and the correlation coefficient R2 was 0.99. In the binary system of Ga/Ge and Ga/As, the selectivity factor was more than 1.5, which could realize the selective separation of gallium. The adsorption mechanism was ion exchange between phenolic hydroxyl group and Ga(OH)4- and the O atom on phenolic hydroxyl group chelated with Ga(OH)4- to form a chelate. After six regeneration cycles, the recovery was over 95%.
2020, 48(1): 121-128
doi: 10.19756/j.issn.0253-3820.191376
Abstract:
Tobacco (Nicotiana tabacum L.) is a model plant for plant science research and one of the important economic crops, and waterlogging stress is one of the most important factors affecting the quality and yield of Nicotiana tabacum. In this study, the instrument parameters such as the type of extractant, the flow rate of extractant reagent, the electrospray voltage, the pressure of sample auxiliary, the pressure of extractant reagent and the temperature of ion transfer tube were optimized by serine, which was common in Nicotiana tabacum as an optimized ion. A total of 22 metabolites of Nicotiana tabacum were obtained under waterlogging stress by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment, including serine, proline, glutamic, D,L-alanyl-glycine, leucine, asparagine, histidine, catechol, maltol, cinnamic acid, caffeic acid, ferulic acid, resveratrol, kaempferol, quercetin chlorogenic acid, nicotine, nornicotine, phenylacetaldehyde, citral, malic acid adn β-damascenone. In response to waterlogged stress, the contents of proline, glutamic acid, serine, histidine about primary metabolites in Nicotiana tabacum were increased, and the contents of secondary metabolites such as chlorogenic acid, ferulic acid, caffeic acid, catechol, phenylacetaldehyde, nicotine and nornicotine were also increased. The results exhibited the metabolites changes of Nicotiana tabacum under waterlogging stress, and thus provided a theoretical basis for further understanding the response to waterlogging stress in Nicotiana tabacum. Accordingly, the application of ND-EESI-MS in plant in-situ detection had many advantages such as high sensitivity, wide detection range, high throughput and rapid detection (less than 1 minute for detection of a single sample). Also it provided a new plant metabolomics technique for enriching the metabolic mechanism of Nicotiana tabacum in response to waterlogging stress.
Tobacco (Nicotiana tabacum L.) is a model plant for plant science research and one of the important economic crops, and waterlogging stress is one of the most important factors affecting the quality and yield of Nicotiana tabacum. In this study, the instrument parameters such as the type of extractant, the flow rate of extractant reagent, the electrospray voltage, the pressure of sample auxiliary, the pressure of extractant reagent and the temperature of ion transfer tube were optimized by serine, which was common in Nicotiana tabacum as an optimized ion. A total of 22 metabolites of Nicotiana tabacum were obtained under waterlogging stress by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment, including serine, proline, glutamic, D,L-alanyl-glycine, leucine, asparagine, histidine, catechol, maltol, cinnamic acid, caffeic acid, ferulic acid, resveratrol, kaempferol, quercetin chlorogenic acid, nicotine, nornicotine, phenylacetaldehyde, citral, malic acid adn β-damascenone. In response to waterlogged stress, the contents of proline, glutamic acid, serine, histidine about primary metabolites in Nicotiana tabacum were increased, and the contents of secondary metabolites such as chlorogenic acid, ferulic acid, caffeic acid, catechol, phenylacetaldehyde, nicotine and nornicotine were also increased. The results exhibited the metabolites changes of Nicotiana tabacum under waterlogging stress, and thus provided a theoretical basis for further understanding the response to waterlogging stress in Nicotiana tabacum. Accordingly, the application of ND-EESI-MS in plant in-situ detection had many advantages such as high sensitivity, wide detection range, high throughput and rapid detection (less than 1 minute for detection of a single sample). Also it provided a new plant metabolomics technique for enriching the metabolic mechanism of Nicotiana tabacum in response to waterlogging stress.
2020, 48(1): 129-136
doi: 10.19756/j.issn.0253-3820.191501
Abstract:
The directed evolution technology was used for recombination of glycosidase ABQ to transform triterpenoid saponins with different structures, including Esculentoside A, Notoginsenoside R1 and Saikosaponin A, and the biotransformation process of the triterpenoid saponins was investigated by rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC/Q-TOF-MS). The result showed that ABQ had high activities for Esculentoside A, Notoginsenoside R1, Saikosaponin A, however, it showed different substrates selectivity for the three saponins. Besides, ABQ could catalyze the hydrolysis of C-3 lateral glucose of Esculentoside A and the C-20 lateral glucose of Notoginsenoside R1 to produce Esculentoside B and Notoginsenoside R2, repectively. And the recombinant glycosidase ABQ could catalyze the hydrolysis of C-3 lateral glucose of Saikosaponin A to produce Prosaikogenin F and then further catalyzed the hydrolysis of C-3 fucoside to produce Saikogenin F. In brief, the directed evolution technology could improve enzyme activity and environmental adaptability, and the chromatography-mass spectrometry could be used to evaluate the role of recombinant enzymes by real-time detection of prototypes and products.
The directed evolution technology was used for recombination of glycosidase ABQ to transform triterpenoid saponins with different structures, including Esculentoside A, Notoginsenoside R1 and Saikosaponin A, and the biotransformation process of the triterpenoid saponins was investigated by rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC/Q-TOF-MS). The result showed that ABQ had high activities for Esculentoside A, Notoginsenoside R1, Saikosaponin A, however, it showed different substrates selectivity for the three saponins. Besides, ABQ could catalyze the hydrolysis of C-3 lateral glucose of Esculentoside A and the C-20 lateral glucose of Notoginsenoside R1 to produce Esculentoside B and Notoginsenoside R2, repectively. And the recombinant glycosidase ABQ could catalyze the hydrolysis of C-3 lateral glucose of Saikosaponin A to produce Prosaikogenin F and then further catalyzed the hydrolysis of C-3 fucoside to produce Saikogenin F. In brief, the directed evolution technology could improve enzyme activity and environmental adaptability, and the chromatography-mass spectrometry could be used to evaluate the role of recombinant enzymes by real-time detection of prototypes and products.
2020, 48(1): 137-144
doi: 10.19756/j.issn.0253-3820.191543
Abstract:
A metal ion ligands and molecular imprinted electrochemical sensors with double recognition patterns were prepared using molecular imprinting technique and with copper ion coordination unit as the double recognition mode. L-cystine (L-cys) was firstly assembled on the surface of graphene (rGO) and gold nanoparticles (AuNPs) modified glassy carbon electrode by AuS bond, and then the Cu-THR complex was assembled on the electrode surface by the coordination of L-cys and thrombin (Tn) by layer-by-layer self-assembly method. Thionine was used as monomer and electropolymerization method was used to form the imprinted membrane. The composite nanomaterials were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The performance of the sensor was studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Under the optimum conditions, the sensor showed an excellent linear relationship with thrombin concentration in the range of 2.0×10-9-5.0×10-7 g/L. The linear equation was -ΔI (μA)=17.73+1.84lgc (g/L). The kinetic adsorption model of THR molecularly imprinted electrochemical sensor was investigated, and the measured imprinting factor β of the imprinted sensor was 4.32, with the binding rate constant k of 5.68 s. The sensor had good stability and reproducibility, and could be used to detect thrombin in practice.
A metal ion ligands and molecular imprinted electrochemical sensors with double recognition patterns were prepared using molecular imprinting technique and with copper ion coordination unit as the double recognition mode. L-cystine (L-cys) was firstly assembled on the surface of graphene (rGO) and gold nanoparticles (AuNPs) modified glassy carbon electrode by AuS bond, and then the Cu-THR complex was assembled on the electrode surface by the coordination of L-cys and thrombin (Tn) by layer-by-layer self-assembly method. Thionine was used as monomer and electropolymerization method was used to form the imprinted membrane. The composite nanomaterials were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The performance of the sensor was studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Under the optimum conditions, the sensor showed an excellent linear relationship with thrombin concentration in the range of 2.0×10-9-5.0×10-7 g/L. The linear equation was -ΔI (μA)=17.73+1.84lgc (g/L). The kinetic adsorption model of THR molecularly imprinted electrochemical sensor was investigated, and the measured imprinting factor β of the imprinted sensor was 4.32, with the binding rate constant k of 5.68 s. The sensor had good stability and reproducibility, and could be used to detect thrombin in practice.
2020, 48(1): 145-152
doi: 10.19756/j.issn.0253-3820.191349
Abstract:
A molecularly imprinted electrochemical sensor for specific recognition of malachite green (MG) was prepared by electropolymerization of o-phenylenediamine on the surface of porous carbon nanofiber (PCNF) modified glassy carbon electrode with MG as template molecule. The electrochemical performance of the sensor was characterized by differential pulse voltammetry (DPV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The experimental parameters, such as the amount of PCNF, molar ratio of template molecule to functional monomer, scan cycle of CV, eluent, elution time, pH value of sample solution and incubation time, were optimized. Under optimal conditions, the peak current was linear to MG concentration in the range of 0.10-10.0 nmol/L, with a detection limit of 0.042 nmol/L. The prepared sensor possessed excellent selectivity, reproducibility and stability, and was applied to the detection of MG in commercially available fresh grass carp and aquaculture water samples with recoveries of 82.0%-96.5%.
A molecularly imprinted electrochemical sensor for specific recognition of malachite green (MG) was prepared by electropolymerization of o-phenylenediamine on the surface of porous carbon nanofiber (PCNF) modified glassy carbon electrode with MG as template molecule. The electrochemical performance of the sensor was characterized by differential pulse voltammetry (DPV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The experimental parameters, such as the amount of PCNF, molar ratio of template molecule to functional monomer, scan cycle of CV, eluent, elution time, pH value of sample solution and incubation time, were optimized. Under optimal conditions, the peak current was linear to MG concentration in the range of 0.10-10.0 nmol/L, with a detection limit of 0.042 nmol/L. The prepared sensor possessed excellent selectivity, reproducibility and stability, and was applied to the detection of MG in commercially available fresh grass carp and aquaculture water samples with recoveries of 82.0%-96.5%.
