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2023 Volume 51 Issue 1
2023, 51(1): 1-10
doi: 10.19756/j.issn.0253-3820.221345
Abstract:
As an important format of the miniaturization of high performance liquid chromatography, nanoflow liquid chromatography has been widely used in the fields of life science, biomedicine and microscale analysis, etc, due to its unique advantages such as less consumption of solvent, less demand of sample amount, higher sensitivity and better compatibility with mass spectrometer. As the core of nanoflow liquid chromatography, packed columns are the most commonly used column type, which are prepared by packing spherical chromatographic materials in the capillary tube. Since column is the place where the separation actually takes place, it plays an important role in the resolution and selectivity of chromatographic separations. Therefore, preparation of high resolution microcolumns is of great importance in securing high performance nano-LC separations. There are two key technologies in preparation of nano-LC columns: fritting and packing. In recent years, with the continuous development of fritting and packing technologies, the stability and separation performance of packed nanoflow liquid chromatographic columns have been significantly improved. This review summarized the developments in fritting and packing technologies involved in nanoflow liquid chromatographic columns in the past ten years, and discussed the recent applications in the field of omics research and biopharmaceuticals, as well as its prospects for future developments.
As an important format of the miniaturization of high performance liquid chromatography, nanoflow liquid chromatography has been widely used in the fields of life science, biomedicine and microscale analysis, etc, due to its unique advantages such as less consumption of solvent, less demand of sample amount, higher sensitivity and better compatibility with mass spectrometer. As the core of nanoflow liquid chromatography, packed columns are the most commonly used column type, which are prepared by packing spherical chromatographic materials in the capillary tube. Since column is the place where the separation actually takes place, it plays an important role in the resolution and selectivity of chromatographic separations. Therefore, preparation of high resolution microcolumns is of great importance in securing high performance nano-LC separations. There are two key technologies in preparation of nano-LC columns: fritting and packing. In recent years, with the continuous development of fritting and packing technologies, the stability and separation performance of packed nanoflow liquid chromatographic columns have been significantly improved. This review summarized the developments in fritting and packing technologies involved in nanoflow liquid chromatographic columns in the past ten years, and discussed the recent applications in the field of omics research and biopharmaceuticals, as well as its prospects for future developments.
2023, 51(1): 11-21
doi: 10.19756/j.issn.0253-3820.221339
Abstract:
The origin traceability technique can effectively identify the source of food products, and thus plays an important role in protecting consumers' rights and interests and maintaining the reputation of products. Commonly used detection methods for origin traceability include spectroscopy, mass spectrometry, chromatography, and sensor techniques. However, for foods with complex chemical composition, a single analytical technique usually cannot obtain sufficient chemical information, while data fusion strategy can improve the identification effect in origin traceability by processing the data obtained by different detection techniques, and combining it to obtain more useful information. At present, data fusion strategy has been widely used in the field of food origin traceability. In this paper, the origin traceability techniques including spectroscopy, mass spectrometry, chromatography and sensor techniques were introduced. The current application status of data fusion strategy based on these techniques in food origin traceability was reviewed. The development of data fusion strategy in food origin traceability was prospected, with a view to providing references for the research and development of food origin traceability.
The origin traceability technique can effectively identify the source of food products, and thus plays an important role in protecting consumers' rights and interests and maintaining the reputation of products. Commonly used detection methods for origin traceability include spectroscopy, mass spectrometry, chromatography, and sensor techniques. However, for foods with complex chemical composition, a single analytical technique usually cannot obtain sufficient chemical information, while data fusion strategy can improve the identification effect in origin traceability by processing the data obtained by different detection techniques, and combining it to obtain more useful information. At present, data fusion strategy has been widely used in the field of food origin traceability. In this paper, the origin traceability techniques including spectroscopy, mass spectrometry, chromatography and sensor techniques were introduced. The current application status of data fusion strategy based on these techniques in food origin traceability was reviewed. The development of data fusion strategy in food origin traceability was prospected, with a view to providing references for the research and development of food origin traceability.
2023, 51(1): 22-33
doi: 10.19756/j.issn.0253-3820.221308
Abstract:
In recent years, carbon quantum dots (CQDs) have attracted much attention due to their unique fluorescence properties, cheap and abundant raw materials, green and convenient synthesis process, good water solubility and biocompatibility, etc. Numerous researches have shown that the fluorescence intensity of CQDs can be enhanced or reduced once the CQDs combine with specific metal ions. Therefore, there is a good application prospect of CQDs to construct the metal ion fluorescence chemosensor. However, the performance of conventional CQDs-based probe with insufficient fluorescence intensity, poor sensitivity and limited selectivity hamper their application in the detection of heavy metal ions. Doping heteroatoms can enrich the energy level structure of CQDs by adjusting the charge density and spin density of carbon atoms, thus enhancing the fluorescence intensity of CQDs. Besides, the doped heteroatoms can also introduce abundant function groups to CQDs and provide more active sites to capture heavy metal ions, which is beneficial for the amplification of fluorescence detection signal. In this paper, the preparation of atom-doped CQDs of nitrogen, sulfur, phosphorus, zinc and copper and their application in the detection of heavy metal ions are reviewed, and the influence of atom doping on the performance of CQDs, the detection mechanism and the application effect are analyzed. On this basis, the future research direction in this field is discussed.
In recent years, carbon quantum dots (CQDs) have attracted much attention due to their unique fluorescence properties, cheap and abundant raw materials, green and convenient synthesis process, good water solubility and biocompatibility, etc. Numerous researches have shown that the fluorescence intensity of CQDs can be enhanced or reduced once the CQDs combine with specific metal ions. Therefore, there is a good application prospect of CQDs to construct the metal ion fluorescence chemosensor. However, the performance of conventional CQDs-based probe with insufficient fluorescence intensity, poor sensitivity and limited selectivity hamper their application in the detection of heavy metal ions. Doping heteroatoms can enrich the energy level structure of CQDs by adjusting the charge density and spin density of carbon atoms, thus enhancing the fluorescence intensity of CQDs. Besides, the doped heteroatoms can also introduce abundant function groups to CQDs and provide more active sites to capture heavy metal ions, which is beneficial for the amplification of fluorescence detection signal. In this paper, the preparation of atom-doped CQDs of nitrogen, sulfur, phosphorus, zinc and copper and their application in the detection of heavy metal ions are reviewed, and the influence of atom doping on the performance of CQDs, the detection mechanism and the application effect are analyzed. On this basis, the future research direction in this field is discussed.
2023, 51(1): 34-41
doi: 10.19756/j.issn.0253-3820.221198
Abstract:
Based on the acid-induced discoloration of the spiropyran, a visual detection probe was synthesized for pH detection in fruit acid cosmetics. Generally, the spiropyran shows poor water solubility, and it is difficult to apply the unmodified spiropyran to determination of aqueous system. So in this work, by modifying hydroxyl groups, the pKa value of spiropyran probe was controlled and the water solubility was improved, which broadened the scope of its application. The as-synthesized colorimetric hydrogen ion probe of polyhydroxy spiropyran (SP-OH) was characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, infrared (IR) spectra and high resolution mass spectra. The influences of probe concentration, water content and other cations on absorbance were investigated, ultraviolet-visible (UV-Vis) spectroscopy results showed that there was a linear relationship between the hydrogen ion concentration and the ratio of absorbance at 420 nm and 560 nm, the standard equation was A420 nm/A560 nm=-55.66pH+276.83 (R2=0.996), and the linear range was 3.0-5.0. The probe could quickly and spontaneously transform into colored ring-open form in acidic solution, which was verified by 1H NMR and molecular orbital energy level calculation. The pH value of fruit acid cosmetics was determined by the SP-OH probe. In the range of pH 3.0-5.0, the pH value of the samples could be detected by SP-OH probe within 5 min. The measured results were consistent with those of pH meter, and the relative error was 2.4%.
Based on the acid-induced discoloration of the spiropyran, a visual detection probe was synthesized for pH detection in fruit acid cosmetics. Generally, the spiropyran shows poor water solubility, and it is difficult to apply the unmodified spiropyran to determination of aqueous system. So in this work, by modifying hydroxyl groups, the pKa value of spiropyran probe was controlled and the water solubility was improved, which broadened the scope of its application. The as-synthesized colorimetric hydrogen ion probe of polyhydroxy spiropyran (SP-OH) was characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, infrared (IR) spectra and high resolution mass spectra. The influences of probe concentration, water content and other cations on absorbance were investigated, ultraviolet-visible (UV-Vis) spectroscopy results showed that there was a linear relationship between the hydrogen ion concentration and the ratio of absorbance at 420 nm and 560 nm, the standard equation was A420 nm/A560 nm=-55.66pH+276.83 (R2=0.996), and the linear range was 3.0-5.0. The probe could quickly and spontaneously transform into colored ring-open form in acidic solution, which was verified by 1H NMR and molecular orbital energy level calculation. The pH value of fruit acid cosmetics was determined by the SP-OH probe. In the range of pH 3.0-5.0, the pH value of the samples could be detected by SP-OH probe within 5 min. The measured results were consistent with those of pH meter, and the relative error was 2.4%.
2023, 51(1): 42-52
doi: 10.19756/j.issn.0253-3820.221543
Abstract:
A poly(neutral red)/multi-walled carbon nanotube nanocomposite modified electrochemical sensor was established for ratiometric sensing of estriol. The poly(neutral red) was used as the internal reference electroactive probe, and the oxidation peak current ratio of estriol to poly(neutral red) was used for accurate quantitative detection of estriol. The poly(neutral red)/multi-walled carbon nanotube nanocomposite was demonstrated good electroconductivity and high electrocatalytic performance which could improve the detection sensitivity. The response of the ratiometric sensor was linearly proportion to the estriol concentration in the 0.1-2.0 μmol/L range, with a limit of detection (S/N=3) of 0.08 μmol/L. The electrochemical oxidation reaction of estriol on the poly(neutral red)/multiwalled carbon nanotube nanocomposite/glassy carbon electrode was demonstrated to undergo a two-electron/two-proton process. Moreover, the sensor had high sensitivity, strong anti-interference ability and good stability, and was successfully applied to testriol analysis in pharmaceutical cream, urine and lake water samples.
A poly(neutral red)/multi-walled carbon nanotube nanocomposite modified electrochemical sensor was established for ratiometric sensing of estriol. The poly(neutral red) was used as the internal reference electroactive probe, and the oxidation peak current ratio of estriol to poly(neutral red) was used for accurate quantitative detection of estriol. The poly(neutral red)/multi-walled carbon nanotube nanocomposite was demonstrated good electroconductivity and high electrocatalytic performance which could improve the detection sensitivity. The response of the ratiometric sensor was linearly proportion to the estriol concentration in the 0.1-2.0 μmol/L range, with a limit of detection (S/N=3) of 0.08 μmol/L. The electrochemical oxidation reaction of estriol on the poly(neutral red)/multiwalled carbon nanotube nanocomposite/glassy carbon electrode was demonstrated to undergo a two-electron/two-proton process. Moreover, the sensor had high sensitivity, strong anti-interference ability and good stability, and was successfully applied to testriol analysis in pharmaceutical cream, urine and lake water samples.
2023, 51(1): 53-62
doi: 10.19756/j.issn.0253-3820.221034
Abstract:
Structure-specific nuclease (FEN1) is overexpressed in a variety of cancer cells and is considered as a potential biomarker for cancer diagnosis. Accurate detection of FEN1 is of great significance for the early diagnosis and prognosis of cancer. Fluorescence strategies have been widely used in the detection of FEN1 in recent years due to their simple operation and high sensitivity, but the strategy for detecting FEN1 based on ratiometric fluorescence signals has not been reported. Here, a ratiometric fluorescent probe (FAM-RhB@UiO-66-NH2) based on DNA-functionalized metal-organic frameworks (MOFs) was designed for the first time for detection and imaging of intracellular FEN1. Among them, Rhodamine B (RhB) was encapsulated into MOFs, and the fluorophore FAM-modified DNA strand was combined with MOFs through Zr-O-P interaction to form a nanoprobe based on fluorescence resonance energy transfer (FRET). In the presence of FEN1, the DNA strand was cleaved by the enzyme to induce the destruction of the FRET system, resulting in the change of the fluorescence ratio to achieve the detection of FEN1. Under the optimal experimental conditions, the linear range for detection of FEN1 was 0.01-3.0 U, the linear equation was y=0.1314x+1.61071, and the detection limit was as low as 0.004 U (3σ). By using FAM-RhB@UiO-66-NH2 as probe, intracellular imaging of FEN1 could distinguish cancer cells from normal cells.
Structure-specific nuclease (FEN1) is overexpressed in a variety of cancer cells and is considered as a potential biomarker for cancer diagnosis. Accurate detection of FEN1 is of great significance for the early diagnosis and prognosis of cancer. Fluorescence strategies have been widely used in the detection of FEN1 in recent years due to their simple operation and high sensitivity, but the strategy for detecting FEN1 based on ratiometric fluorescence signals has not been reported. Here, a ratiometric fluorescent probe (FAM-RhB@UiO-66-NH2) based on DNA-functionalized metal-organic frameworks (MOFs) was designed for the first time for detection and imaging of intracellular FEN1. Among them, Rhodamine B (RhB) was encapsulated into MOFs, and the fluorophore FAM-modified DNA strand was combined with MOFs through Zr-O-P interaction to form a nanoprobe based on fluorescence resonance energy transfer (FRET). In the presence of FEN1, the DNA strand was cleaved by the enzyme to induce the destruction of the FRET system, resulting in the change of the fluorescence ratio to achieve the detection of FEN1. Under the optimal experimental conditions, the linear range for detection of FEN1 was 0.01-3.0 U, the linear equation was y=0.1314x+1.61071, and the detection limit was as low as 0.004 U (3σ). By using FAM-RhB@UiO-66-NH2 as probe, intracellular imaging of FEN1 could distinguish cancer cells from normal cells.
2023, 51(1): 63-71
doi: 10.19756/j.issn.0253-3820.221378
Abstract:
The level and rate of insulin secretion are regulated by glucose concentration. Syntaxin 1A (STX-1A) is a core component of soluble N-ethylmaleimide-sensitive factor attachment proteins receptor (SNARE) complex and is essential for mediating the docking and fusion of insulin granules with cell membranes during insulin secretion. However, the mechanism of whether and how glucose regulates STX-1A to affect insulin release is still not well elucidated. To address this issue, the regulatory effect of glucose on STX-1A on INS-1 cell membranes was investigated by direct stochastic optical reconstruction microscopy (dSTORM). It was found that the elevated glucose concentration increased the expression of STX-1A on the cell membrane, and the density and aggregation of STX-1A protein clusters on the cell membrane were also increased. However, the glucotoxicity caused by chronic stimulation with high glucose concentration severely reduced the expression of STX-1A protein on the cell membrane, while the density and aggregation of STX-1A protein clusters on the cell membrane were also significantly reduced. By linking the expression and spatial distribution characteristics of STX-1A on cell membranes to insulin-secreting cell functions, the role of glucose in regulating the functional organization of STX-1A at the molecular level was revealed, which provided new insights into the mechanisms by which SNAREs regulate insulin release.
The level and rate of insulin secretion are regulated by glucose concentration. Syntaxin 1A (STX-1A) is a core component of soluble N-ethylmaleimide-sensitive factor attachment proteins receptor (SNARE) complex and is essential for mediating the docking and fusion of insulin granules with cell membranes during insulin secretion. However, the mechanism of whether and how glucose regulates STX-1A to affect insulin release is still not well elucidated. To address this issue, the regulatory effect of glucose on STX-1A on INS-1 cell membranes was investigated by direct stochastic optical reconstruction microscopy (dSTORM). It was found that the elevated glucose concentration increased the expression of STX-1A on the cell membrane, and the density and aggregation of STX-1A protein clusters on the cell membrane were also increased. However, the glucotoxicity caused by chronic stimulation with high glucose concentration severely reduced the expression of STX-1A protein on the cell membrane, while the density and aggregation of STX-1A protein clusters on the cell membrane were also significantly reduced. By linking the expression and spatial distribution characteristics of STX-1A on cell membranes to insulin-secreting cell functions, the role of glucose in regulating the functional organization of STX-1A at the molecular level was revealed, which provided new insights into the mechanisms by which SNAREs regulate insulin release.
2023, 51(1): 72-83
doi: 10.19756/j.issn.0253-3820.221323
Abstract:
Based on proteomic analysis, the changes of protein species, content and metabolic pathway in the leaves of Amygdalus pedunculata Pall at different growth periods were investigated and clarified. The results showed that 6584 credible proteins were detected by tandem mass tags (TMT) labeled quantitative proteomics, and 1678 differential proteins were screened. The results of GO functional enrichment showed that the differential proteins participated in the biological processes such as defense response and response to biological stimulation, and mainly had molecular functions such as signal receptor activity, protein phosphatase inhibitor activity, glutathione transferase activity and glutathione dehydrogenase (ascorbic acid) activity.The content of organic acids in Amygdalus pedunculata Pall leaves decreased gradually from flowering stage to fruit ripening stage. Lyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that it was mainly related to α-linolenic acid metabolism, glyoxylic acid and dicarboxylic acid metabolism and carbon fixation in photosynthetic organisms. Traumatic acid, ketoglutarate, malic acid, citric acid and 12 related proteins played important roles in the metabolic process of organic acids in the leaves of Amygdalus pedunculata Pall at different growth stages.
Based on proteomic analysis, the changes of protein species, content and metabolic pathway in the leaves of Amygdalus pedunculata Pall at different growth periods were investigated and clarified. The results showed that 6584 credible proteins were detected by tandem mass tags (TMT) labeled quantitative proteomics, and 1678 differential proteins were screened. The results of GO functional enrichment showed that the differential proteins participated in the biological processes such as defense response and response to biological stimulation, and mainly had molecular functions such as signal receptor activity, protein phosphatase inhibitor activity, glutathione transferase activity and glutathione dehydrogenase (ascorbic acid) activity.The content of organic acids in Amygdalus pedunculata Pall leaves decreased gradually from flowering stage to fruit ripening stage. Lyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that it was mainly related to α-linolenic acid metabolism, glyoxylic acid and dicarboxylic acid metabolism and carbon fixation in photosynthetic organisms. Traumatic acid, ketoglutarate, malic acid, citric acid and 12 related proteins played important roles in the metabolic process of organic acids in the leaves of Amygdalus pedunculata Pall at different growth stages.
2023, 51(1): 84-92
doi: 10.19756/j.issn.0253-3820.221521
Abstract:
The redox state of the thiol groups of protein cysteine residues is closely related to the local redox level of cells. When these thiol groups are oxidized or reduced, they can greatly affect protein structure, thereby modulating their biological functions and eventually affecting the biological processes and cell fate. In this study, a strategy aiming at selectively labeling the free thiol group of protein cysteine was proposed. In this method, N-ethylmaleimide (NEM), a thiol reactive reagent, was used to block the free thiol groups on the proteins prior to the routine sample processing in proteomics flow (Reduction of disulfide bond by dithiothreitol, alkylation blockage byiodoacetamide). Therefore, the original free thiol groups, as well as those generated from the reduction treatment by dithiothreitol, were blocked with two different thiol reactive reagents with different molecular weights, leading to specific identification of the original free thiol groups within proteins. By using this strategy, a proteomic investigation was performed on the free thiol groups of mitochondrial proteins in colorectal cancer tissues. A total of 1549 mitochondrial proteins were identified, including protein disulfide-isomerase A3, peroxiredoxin-1, mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial inner membrane protein, mitochondrial acetyl-CoA acyltransferase, malate dehydrogenase,calnexin, mitochondrial aspartate aminotransferase, mitochondrial succinate dehydrogenase [ubiquinone] iron-sulfur subunit, etc. Specially, 348 peptides containing free sulfhydryl groups were identified, belonging to 253 proteins. The proteomics data of the mitochondrial proteins as well as the peptides containing free thiols in colon cancer tissues could provide new ideas for further investigation on the redox targets as well as novel biomarkers in mitochondrial proteins in colon cancer.
The redox state of the thiol groups of protein cysteine residues is closely related to the local redox level of cells. When these thiol groups are oxidized or reduced, they can greatly affect protein structure, thereby modulating their biological functions and eventually affecting the biological processes and cell fate. In this study, a strategy aiming at selectively labeling the free thiol group of protein cysteine was proposed. In this method, N-ethylmaleimide (NEM), a thiol reactive reagent, was used to block the free thiol groups on the proteins prior to the routine sample processing in proteomics flow (Reduction of disulfide bond by dithiothreitol, alkylation blockage byiodoacetamide). Therefore, the original free thiol groups, as well as those generated from the reduction treatment by dithiothreitol, were blocked with two different thiol reactive reagents with different molecular weights, leading to specific identification of the original free thiol groups within proteins. By using this strategy, a proteomic investigation was performed on the free thiol groups of mitochondrial proteins in colorectal cancer tissues. A total of 1549 mitochondrial proteins were identified, including protein disulfide-isomerase A3, peroxiredoxin-1, mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial inner membrane protein, mitochondrial acetyl-CoA acyltransferase, malate dehydrogenase,calnexin, mitochondrial aspartate aminotransferase, mitochondrial succinate dehydrogenase [ubiquinone] iron-sulfur subunit, etc. Specially, 348 peptides containing free sulfhydryl groups were identified, belonging to 253 proteins. The proteomics data of the mitochondrial proteins as well as the peptides containing free thiols in colon cancer tissues could provide new ideas for further investigation on the redox targets as well as novel biomarkers in mitochondrial proteins in colon cancer.
2023, 51(1): 93-101
doi: 10.19756/j.issn.0253-3820.221154
Abstract:
A biosensor based on catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET) was established for detection of enterovirus 71 (EV71). A CHA signal amplification strategy was designed to detect the VP1 gene of EV71, which was consisted of a pair of hairpin probes (H1 and H2). The fluorophores Cy3 and Cy5 were labeled on H1 and H2, respectively. When the VP1 gene was present, the catalytic assembly reaction of H1 and H2 was triggered, Cy3 and Cy5 got closer and FRET occurred, leading to a reduced Cy3 signal and an enhanced Cy5 signal. In the absence of VP1 gene, H1 and H2 existed stably in the reaction system and could not generate FRET. Experimental conditions including reaction buffer concentration, the ratio of H1 to H2, reaction temperature and reaction time were optimized. Under the optimized conditions, the fluorescence intensity ratio of Cy5 to Cy3 showed a good linear relationship with EV71 VP1 gene concentration in the range of 0.5-20 nmol/L, and the detection limit was 73 pmol/L (3σ). The spiked recoveries of throat swab samples ranged from 99.6% to 103.1% with the relative standard deviations of 0.3% to 1.7%, showing good specificity and anti-interference ability. The proposed sensor had a good application prospect in EV71 virus monitoring and early diagnosis of hand, foot and mouth disease.
A biosensor based on catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET) was established for detection of enterovirus 71 (EV71). A CHA signal amplification strategy was designed to detect the VP1 gene of EV71, which was consisted of a pair of hairpin probes (H1 and H2). The fluorophores Cy3 and Cy5 were labeled on H1 and H2, respectively. When the VP1 gene was present, the catalytic assembly reaction of H1 and H2 was triggered, Cy3 and Cy5 got closer and FRET occurred, leading to a reduced Cy3 signal and an enhanced Cy5 signal. In the absence of VP1 gene, H1 and H2 existed stably in the reaction system and could not generate FRET. Experimental conditions including reaction buffer concentration, the ratio of H1 to H2, reaction temperature and reaction time were optimized. Under the optimized conditions, the fluorescence intensity ratio of Cy5 to Cy3 showed a good linear relationship with EV71 VP1 gene concentration in the range of 0.5-20 nmol/L, and the detection limit was 73 pmol/L (3σ). The spiked recoveries of throat swab samples ranged from 99.6% to 103.1% with the relative standard deviations of 0.3% to 1.7%, showing good specificity and anti-interference ability. The proposed sensor had a good application prospect in EV71 virus monitoring and early diagnosis of hand, foot and mouth disease.
2023, 51(1): 102-111
doi: 10.19756/j.issn.0253-3820.221241
Abstract:
A magnetic separation competitive immunosensor based on novel enzyme-linked gold nanocomposite probes (E-GNPs) was constructed for detection of ractopamine (RAC). Through the specific interaction between streptavidin and biotin, horseradish peroxidase (HRP) was labeled on the antibody that was modified on the surface of gold nanoparticles (AuNPs) by electrostatic assembly method to obtain E-GNPs. Ovalbumin (OVA)-RAC hapten coated magnetic beads (MBs) was introduced for a competitive reaction, and quantitative analysis of the target was achieved by a color reaction. With the change of the concentration of target molecule RAC, the color of the enzyme-catalyzed substrate changed, and the concentration of RAC had a linear relationship with the color signal intensity. The UV-vis absorption spectroscopy characterization results showed that one E-GNP could carry 11 HRP molecules, which made the immunosensor highly sensitive. The prepared sensor was successfully used for detection of RAC with a detection limit of 1.75 pg/mL, showing 10 times higher sensitivity than that of traditional ELISA method. Cross-reaction experiments showed that the sensor had good selectivity for RAC, and could be applied to real samples such as pork, beef and mutton, with spiked recoveries ranging from 88.3% to 103.4%. This analytical method provided a new idea for rapid screening of RAC in animal-derived foods.
A magnetic separation competitive immunosensor based on novel enzyme-linked gold nanocomposite probes (E-GNPs) was constructed for detection of ractopamine (RAC). Through the specific interaction between streptavidin and biotin, horseradish peroxidase (HRP) was labeled on the antibody that was modified on the surface of gold nanoparticles (AuNPs) by electrostatic assembly method to obtain E-GNPs. Ovalbumin (OVA)-RAC hapten coated magnetic beads (MBs) was introduced for a competitive reaction, and quantitative analysis of the target was achieved by a color reaction. With the change of the concentration of target molecule RAC, the color of the enzyme-catalyzed substrate changed, and the concentration of RAC had a linear relationship with the color signal intensity. The UV-vis absorption spectroscopy characterization results showed that one E-GNP could carry 11 HRP molecules, which made the immunosensor highly sensitive. The prepared sensor was successfully used for detection of RAC with a detection limit of 1.75 pg/mL, showing 10 times higher sensitivity than that of traditional ELISA method. Cross-reaction experiments showed that the sensor had good selectivity for RAC, and could be applied to real samples such as pork, beef and mutton, with spiked recoveries ranging from 88.3% to 103.4%. This analytical method provided a new idea for rapid screening of RAC in animal-derived foods.
2023, 51(1): 112-119
doi: 10.19756/j.issn.0253-3820.221420
Abstract:
A method was developed for screening protein binders from complicated ginseng samples by a streptavidin-coated 96-well plate format coupled with ultra-high performance liquid chromatography-orbitrap mass spectrometry (UHPLC-Orbitrap-MS). The Ricinus communis agglutinin 120 (RCA 120) coated 96-well plate and lysozyme coated 96-well plate (as control) were incubated with oligosaccharide standards respectively, and the compounds with the decreased peak areas in experimental group compared to those in the control group were detected as binders by UHPLC-ESI-MS. The factors such as incubation time, incubation temperature, and buffer, which might affect the binding affinity and reproducibility, were optimized. The potential of the approach was examined using the extracts of white ginseng, American ginseng and red ginseng. Three disaccharides and two trisaccharides were screened out from the extracts of white ginseng. As for the extracts of American ginseng, two disaccharides and two trisaccharides were observed to bind to RCA120. While no binders were detected in the extracts of red ginseng. Significant differences were observed in the relative binding degrees (RBDs) of the detected oligosaccharides in white ginseng and American ginseng binding to RCA 120. To our knowledge, it's the first time to reveal the differences and analogies in RCA 120-binding capabilities of oligosaccharides between the extracts of white ginseng and American ginseng, indicating the efficiency of the developed method for analysis of complicated samples.
A method was developed for screening protein binders from complicated ginseng samples by a streptavidin-coated 96-well plate format coupled with ultra-high performance liquid chromatography-orbitrap mass spectrometry (UHPLC-Orbitrap-MS). The Ricinus communis agglutinin 120 (RCA 120) coated 96-well plate and lysozyme coated 96-well plate (as control) were incubated with oligosaccharide standards respectively, and the compounds with the decreased peak areas in experimental group compared to those in the control group were detected as binders by UHPLC-ESI-MS. The factors such as incubation time, incubation temperature, and buffer, which might affect the binding affinity and reproducibility, were optimized. The potential of the approach was examined using the extracts of white ginseng, American ginseng and red ginseng. Three disaccharides and two trisaccharides were screened out from the extracts of white ginseng. As for the extracts of American ginseng, two disaccharides and two trisaccharides were observed to bind to RCA120. While no binders were detected in the extracts of red ginseng. Significant differences were observed in the relative binding degrees (RBDs) of the detected oligosaccharides in white ginseng and American ginseng binding to RCA 120. To our knowledge, it's the first time to reveal the differences and analogies in RCA 120-binding capabilities of oligosaccharides between the extracts of white ginseng and American ginseng, indicating the efficiency of the developed method for analysis of complicated samples.
2023, 51(1): 120-129
doi: 10.19756/j.issn.0253-3820.221162
Abstract:
N-terminal pro-brain natriuretic peptide (NT-proBNP) is an essential biomarker for diagnosis of heart failure. Currently, since reference materials of NT-proBNP are unavailable globally, value transfer activities are hard to implement from the higher level to the end-users, which makes terminal clinical testing results inconsistent and incomparable. To achieve standard detection of NT-proBNP, in this work, recombinant NT-proBNP was chosen as a target during traceable approach development. Firstly, the immune capacity and relative molecular weight were tested and varied through Western Blot assay and mass spectrometric methods. Next, two kinds of isotope dilution mass spectrometry were adopted for the absolute quantification of NT-proBNP. The results suggested that the protein hydrolysis reaction reached equilibrium in 48 h. NT-proBNP was calculated to be 79.62 μg/g with an relative standard deviation (RSD) of 0.89% by detection of valine, isoleucine and arginine. On the other hand, the isotope dilution mass spectrometry method was used to analyze signature peptides produced by enzymatic digestion. NT-proBNP was calculated to be 76.04 μg/g with an RSD of 1.85% by detection of the two peptides under the conditions of enzymatic digestion for 24h and at mass ratio of 25∶1 of NT-proBNP to protease. The quantitative analysis results from the two methods were consistent with each other. As the amino acids applied in quantifications were SI-traceable primary reference materials, all the results were also traceable. Such approaches were candidates for NT-proBNP reference material development.
N-terminal pro-brain natriuretic peptide (NT-proBNP) is an essential biomarker for diagnosis of heart failure. Currently, since reference materials of NT-proBNP are unavailable globally, value transfer activities are hard to implement from the higher level to the end-users, which makes terminal clinical testing results inconsistent and incomparable. To achieve standard detection of NT-proBNP, in this work, recombinant NT-proBNP was chosen as a target during traceable approach development. Firstly, the immune capacity and relative molecular weight were tested and varied through Western Blot assay and mass spectrometric methods. Next, two kinds of isotope dilution mass spectrometry were adopted for the absolute quantification of NT-proBNP. The results suggested that the protein hydrolysis reaction reached equilibrium in 48 h. NT-proBNP was calculated to be 79.62 μg/g with an relative standard deviation (RSD) of 0.89% by detection of valine, isoleucine and arginine. On the other hand, the isotope dilution mass spectrometry method was used to analyze signature peptides produced by enzymatic digestion. NT-proBNP was calculated to be 76.04 μg/g with an RSD of 1.85% by detection of the two peptides under the conditions of enzymatic digestion for 24h and at mass ratio of 25∶1 of NT-proBNP to protease. The quantitative analysis results from the two methods were consistent with each other. As the amino acids applied in quantifications were SI-traceable primary reference materials, all the results were also traceable. Such approaches were candidates for NT-proBNP reference material development.
2023, 51(1): 130-138
doi: 10.19756/j.issn.0253-3820.221213
Abstract:
A novel fluorescent probe CPS based on coumarin-derived acylhydrazone was designed and synthesized by using 7-diethylamino coumarin as report group. The results showed that probe CPS had obviously "On-Off" fluorescence response to Cu2+ in the methanol solution, and the stoichiometry of CPS-Cu2+ complex was estimated to be 2∶1. Meanwhile, a fluorescence sensor based on the complex CPS-Cu2+ showed high selectivity and sensitivity towards glyphosate, and the detection limit of glyphosate was 64.8 nmol/L (10.95 ng/mL). The mechanism of fluorescence sensor to detect glyphosate was speculated through Job's plot, molar ratio method and mass spectrometry. In addition, the applicability of fluorescence sensor in detection of glyphosate was verified by the standard recovery tests in practical water samples.
A novel fluorescent probe CPS based on coumarin-derived acylhydrazone was designed and synthesized by using 7-diethylamino coumarin as report group. The results showed that probe CPS had obviously "On-Off" fluorescence response to Cu2+ in the methanol solution, and the stoichiometry of CPS-Cu2+ complex was estimated to be 2∶1. Meanwhile, a fluorescence sensor based on the complex CPS-Cu2+ showed high selectivity and sensitivity towards glyphosate, and the detection limit of glyphosate was 64.8 nmol/L (10.95 ng/mL). The mechanism of fluorescence sensor to detect glyphosate was speculated through Job's plot, molar ratio method and mass spectrometry. In addition, the applicability of fluorescence sensor in detection of glyphosate was verified by the standard recovery tests in practical water samples.
2023, 51(1): 139-145
doi: 10.19756/j.issn.0253-3820.221161
Abstract:
Three different pH-sensitive colorants including cyanidin cation (CC), bromothymol blue/methyl red (BTB/MR) and bromocresol purple (BCP) were respectively incorporated into polyvinyl alcohol (PVA) matrix to prepare intelligent indicator films by electrospinning for visual meat freshness monitoring. The morphology of films captured by scanning electron microscopy indicated that colorants had been successfully dispersed into PVA matrix and could be more sensitive due to the high specific surface area offered by the nanofiber structure. Each indicator film exhibited an obvious color response to volatile ammonia with a color difference value (△E) greater than the lowest distinguishable value (△E=7) when the mass fraction of colorant was increased to 3%. Color changes of intelligent indicator films during the minced pork freshness monitoring could be clearly observed by naked eyes and the corresponding △E calculated according to the lightness (L), redness (a) and yellowness (b) of indicator films and total volatile basic nitrogen (TVB-N) contents determined by an automated Kjeldahl apparatus were positively correlated, which suggested that the intelligent indicator films could accurately indicate the freshness and held great promise in the application of rapid and real-time monitoring of meat freshness.
Three different pH-sensitive colorants including cyanidin cation (CC), bromothymol blue/methyl red (BTB/MR) and bromocresol purple (BCP) were respectively incorporated into polyvinyl alcohol (PVA) matrix to prepare intelligent indicator films by electrospinning for visual meat freshness monitoring. The morphology of films captured by scanning electron microscopy indicated that colorants had been successfully dispersed into PVA matrix and could be more sensitive due to the high specific surface area offered by the nanofiber structure. Each indicator film exhibited an obvious color response to volatile ammonia with a color difference value (△E) greater than the lowest distinguishable value (△E=7) when the mass fraction of colorant was increased to 3%. Color changes of intelligent indicator films during the minced pork freshness monitoring could be clearly observed by naked eyes and the corresponding △E calculated according to the lightness (L), redness (a) and yellowness (b) of indicator films and total volatile basic nitrogen (TVB-N) contents determined by an automated Kjeldahl apparatus were positively correlated, which suggested that the intelligent indicator films could accurately indicate the freshness and held great promise in the application of rapid and real-time monitoring of meat freshness.
