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2019 Volume 47 Issue 7
2019, 47(7): 965-975
doi: 10.19756/j.issn.0253-3820.191044
Abstract:
Commercialization is the current major theme of microfluidics. Since lab-on-printed circuit board (lab-on-PCB) can integrate PCB technology and polymer microfluidic manufacturing process, a low-cost, standardized and mass productive platform has been formed to promote commercialization efficiently. In this paper, we summarized the research of lab-on-PCB in the past 20 years and reviewed the progress from structures, materials and fabrication methods according to the analysis of typical lab-on-PCB architectures. Besides, the current applications and commercialization, the shortcomings and problems to be solved, and future directions were also discussed and prospected.
Commercialization is the current major theme of microfluidics. Since lab-on-printed circuit board (lab-on-PCB) can integrate PCB technology and polymer microfluidic manufacturing process, a low-cost, standardized and mass productive platform has been formed to promote commercialization efficiently. In this paper, we summarized the research of lab-on-PCB in the past 20 years and reviewed the progress from structures, materials and fabrication methods according to the analysis of typical lab-on-PCB architectures. Besides, the current applications and commercialization, the shortcomings and problems to be solved, and future directions were also discussed and prospected.
2019, 47(7): 976-984
doi: 10.19756/j.issn.0253-3820.181677
Abstract:
A vacuum ultraviolet single-photon ionization mass spectrometer, which has the merits of broad-spectrum soft ionization, low fragment peak, low matrix influence, high sensitivity, and short response time, is fit for the use of online analysis of mixed volatile organic compounds. In this review, the theory and structural composition of the single-photon ionization mass spectrometer are described, and the current research progress of sampling systems, ionization sources, and mass spectrometers are summarized. Then, a summary is provided on how online analysis has been applied to the fields of atmospheric composition observation, atmospheric chemistry research, environmental monitoring, food detection, etc. Finally, the direction and trends for future work are proposed.
A vacuum ultraviolet single-photon ionization mass spectrometer, which has the merits of broad-spectrum soft ionization, low fragment peak, low matrix influence, high sensitivity, and short response time, is fit for the use of online analysis of mixed volatile organic compounds. In this review, the theory and structural composition of the single-photon ionization mass spectrometer are described, and the current research progress of sampling systems, ionization sources, and mass spectrometers are summarized. Then, a summary is provided on how online analysis has been applied to the fields of atmospheric composition observation, atmospheric chemistry research, environmental monitoring, food detection, etc. Finally, the direction and trends for future work are proposed.
2019, 47(7): 985-991
doi: 10.19756/j.issn.0253-3820.181793
Abstract:
For the past two decades, gas chromatography-mass spectrometry (GC-MS), the "gold standard" method, was developed rapidly in terms of the analysis and detection. Among them, the miniaturization of the ion trap mass analyzer developed rapidly, while the quadrupole mass analyzer was relatively slow due to its maximum mass range with the low level. In this work, a small quadrupole mass analyzer with a diameter of 6 mm and a length of 115 mm was designed. Based on this, a small quadrupole mass spectrometer was built and combined with gas chromatography. The test results of mass range, sensitivity, quantitative repeatability and qualitative ability of the instrument proved that the maximum mass range of the miniature quadrupole mass analyzer was more than 500 Th under the unit mass resolution. Based on this miniature quadrupole mass analyzer, the RSD of quantitative reproducibility of perfluoronaphthalene (1 ng) was better than 3%. In the range of 0.1 μg to 0.001 μg, the methamphetamine indicated that the quality and the signal intensity were in the relationship of y=3.19662×107x1.24606 (R2=0.988). The 16 common drugs at ng level could be accurately identified. The combination of quadrupole mass analyzer and ion pump with wider mass range was expected to enhance the application of small quadrupole mass spectrometer in the field of emergency, drug attack, military counter-terrorism, aerospace and others.
For the past two decades, gas chromatography-mass spectrometry (GC-MS), the "gold standard" method, was developed rapidly in terms of the analysis and detection. Among them, the miniaturization of the ion trap mass analyzer developed rapidly, while the quadrupole mass analyzer was relatively slow due to its maximum mass range with the low level. In this work, a small quadrupole mass analyzer with a diameter of 6 mm and a length of 115 mm was designed. Based on this, a small quadrupole mass spectrometer was built and combined with gas chromatography. The test results of mass range, sensitivity, quantitative repeatability and qualitative ability of the instrument proved that the maximum mass range of the miniature quadrupole mass analyzer was more than 500 Th under the unit mass resolution. Based on this miniature quadrupole mass analyzer, the RSD of quantitative reproducibility of perfluoronaphthalene (1 ng) was better than 3%. In the range of 0.1 μg to 0.001 μg, the methamphetamine indicated that the quality and the signal intensity were in the relationship of y=3.19662×107x1.24606 (R2=0.988). The 16 common drugs at ng level could be accurately identified. The combination of quadrupole mass analyzer and ion pump with wider mass range was expected to enhance the application of small quadrupole mass spectrometer in the field of emergency, drug attack, military counter-terrorism, aerospace and others.
2019, 47(7): 992-997
doi: 10.19756/j.issn.0253-3820.191039
Abstract:
A quartz crystal microbalance (QCM) instrument was developed on the basis of difference frequency technique. The experimental data showed that the difference frequency between the reference crystal and the detection crystal was in the range of ±10 to ±30 kHz, the accuracy was less than 0.0028% (relative error of the difference frequency data) and the precision was less than 0.2825% (the difference frequency theoretical value and measurement value error). In this work, the direct digital synthesizer (DDS) digital generator was used to generate an adjustable reference frequency source, and the difference frequency value was within the optimal range. A portable quartz crystal microbalance analyzer was based on Arduino microcomputer. The instrument had a 3.5-inch LCD screen to display the dynamic curve, and the SD card synchronously stored the data. The reference frequency could be adjusted according to the experimental conditions. The average frequency drift values of gas phase and pure water were less than 0.13 Hz/min and 0.23 Hz/min, which indicated that the instrument had good stability. The linear relationship of the instrument to different density of sodium chloride was 0.9891. The response experiment with different viscosity glycerol showed that Δf and (ηlρl)1/2 had a linear relationship, which meant that the instrument had good responsiveness. At the same time, the instrument was used in combination with electrochemical workstations for on-line detection of Cu deposition process. The mass response value was 0.61 ng/Hz, which was 82.4% of the theoretical value.
A quartz crystal microbalance (QCM) instrument was developed on the basis of difference frequency technique. The experimental data showed that the difference frequency between the reference crystal and the detection crystal was in the range of ±10 to ±30 kHz, the accuracy was less than 0.0028% (relative error of the difference frequency data) and the precision was less than 0.2825% (the difference frequency theoretical value and measurement value error). In this work, the direct digital synthesizer (DDS) digital generator was used to generate an adjustable reference frequency source, and the difference frequency value was within the optimal range. A portable quartz crystal microbalance analyzer was based on Arduino microcomputer. The instrument had a 3.5-inch LCD screen to display the dynamic curve, and the SD card synchronously stored the data. The reference frequency could be adjusted according to the experimental conditions. The average frequency drift values of gas phase and pure water were less than 0.13 Hz/min and 0.23 Hz/min, which indicated that the instrument had good stability. The linear relationship of the instrument to different density of sodium chloride was 0.9891. The response experiment with different viscosity glycerol showed that Δf and (ηlρl)1/2 had a linear relationship, which meant that the instrument had good responsiveness. At the same time, the instrument was used in combination with electrochemical workstations for on-line detection of Cu deposition process. The mass response value was 0.61 ng/Hz, which was 82.4% of the theoretical value.
2019, 47(7): 998-1005
doi: 10.19756/j.issn.0253-3820.181702
Abstract:
Adhesive tape combined with the core-shell SERS nanoprobe was used to recognize the latent fingerprints on the surface of the object, which provided an effective solution for the imaging of latent fingerprint on large immobile object and surface object. First, Au@pNTP@SiO2 SERS probes modified by aptamers specific to lysozyme was prepared and characterized by TEM/EDS. The SERS enhancement activity was also tested. Then the probe was incubated with the aged-latent fingerprints on the surface of different object. Afterwards, the dried latent fingerprint with modified SERS probes was transferred on the adhesive tape and imaged under the confocal Raman microscope. The results showed that the size distribution of the core-shell SERS probe was uniform, mostly concentrated in 60 nm, leading to excellent stability and dispersity. The SERS probe also had enhanced SERS effect, strong SERS intensity and reproducibility with the characteristic peak at 723, 855, 1081, 1343 and 1572 cm-1, which was consistent with the previous reports. When applied this SERS probe into the tape-transferred latent fingerprint's imaging, not only the first-level structure (pattern) of fingerprint but also the second structure of fingerprint such as the ridge interruption and ridge fork could be clearly seen. The successful development of the SERS imaging of transferred latent fingerprint on adhesive tapes combined with the aptamer-functionalized SERS probe provides an effective method for recognizing latent fingerprints in forensic science, which also has the potential application in the other areas such as food safety and public safety testing.
Adhesive tape combined with the core-shell SERS nanoprobe was used to recognize the latent fingerprints on the surface of the object, which provided an effective solution for the imaging of latent fingerprint on large immobile object and surface object. First, Au@pNTP@SiO2 SERS probes modified by aptamers specific to lysozyme was prepared and characterized by TEM/EDS. The SERS enhancement activity was also tested. Then the probe was incubated with the aged-latent fingerprints on the surface of different object. Afterwards, the dried latent fingerprint with modified SERS probes was transferred on the adhesive tape and imaged under the confocal Raman microscope. The results showed that the size distribution of the core-shell SERS probe was uniform, mostly concentrated in 60 nm, leading to excellent stability and dispersity. The SERS probe also had enhanced SERS effect, strong SERS intensity and reproducibility with the characteristic peak at 723, 855, 1081, 1343 and 1572 cm-1, which was consistent with the previous reports. When applied this SERS probe into the tape-transferred latent fingerprint's imaging, not only the first-level structure (pattern) of fingerprint but also the second structure of fingerprint such as the ridge interruption and ridge fork could be clearly seen. The successful development of the SERS imaging of transferred latent fingerprint on adhesive tapes combined with the aptamer-functionalized SERS probe provides an effective method for recognizing latent fingerprints in forensic science, which also has the potential application in the other areas such as food safety and public safety testing.
2019, 47(7): 1006-1013
doi: 10.19756/j.issn.0253-3820.181330
Abstract:
Telomerase is usually activated in tumour or cancel cells. The detection of telomerase activity is of great significance for early diagnosis of human humour and cancers as well as screening telomerase-target anticancer therapy. Based on the fluorescence resonance energy transfer (FRET) between cationic conjugated polymer- poly[(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorenylene phenylene (PFP) and SYBR Green I (SG), a simple and fast telomerase activity assay strategy was developed. In the presence of telomerase, the primer was extended, generating a special DNA with many repeat sequences-(GGTTAG)n. Then, the telomerase elongated products are fixedly connected with magnetic beads through the specific combination of streptavidin (STV) with biotin. A specific DNA probe-(CTAACC)2 which is matched with two repeat sequences, is used to hybridize with telomerase elongated product. After the telomerase elongated products form double strands DNA (dsDNA), a fluorescent dye SG is added to the hybridization solution and embed into the dsDNA. PFP is added finally. PFP, with positive charges, is a water soluble cationic polymer. It can adsorb on the dsDNA by electrostatic interaction, generating FRET from PFP to SG. So, quantitative determination of telomerase activity can be achieved by detection of FRET efficiency. The telomerase activity in 3×105 Hela cells has been obviously detected. To improve the detection sensitivity, hybridization chain reaction (HCR) as signal amplification technology has been used to couple with FRET. The telomerase activity in 6×104 Hela cells has been detected. The detection sensitivity is improved higher almost one order of magnitude.
Telomerase is usually activated in tumour or cancel cells. The detection of telomerase activity is of great significance for early diagnosis of human humour and cancers as well as screening telomerase-target anticancer therapy. Based on the fluorescence resonance energy transfer (FRET) between cationic conjugated polymer- poly[(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorenylene phenylene (PFP) and SYBR Green I (SG), a simple and fast telomerase activity assay strategy was developed. In the presence of telomerase, the primer was extended, generating a special DNA with many repeat sequences-(GGTTAG)n. Then, the telomerase elongated products are fixedly connected with magnetic beads through the specific combination of streptavidin (STV) with biotin. A specific DNA probe-(CTAACC)2 which is matched with two repeat sequences, is used to hybridize with telomerase elongated product. After the telomerase elongated products form double strands DNA (dsDNA), a fluorescent dye SG is added to the hybridization solution and embed into the dsDNA. PFP is added finally. PFP, with positive charges, is a water soluble cationic polymer. It can adsorb on the dsDNA by electrostatic interaction, generating FRET from PFP to SG. So, quantitative determination of telomerase activity can be achieved by detection of FRET efficiency. The telomerase activity in 3×105 Hela cells has been obviously detected. To improve the detection sensitivity, hybridization chain reaction (HCR) as signal amplification technology has been used to couple with FRET. The telomerase activity in 6×104 Hela cells has been detected. The detection sensitivity is improved higher almost one order of magnitude.
2019, 47(7): 1014-1020
doi: 10.19756/j.issn.0253-3820.181791
Abstract:
A double-quenching molecular beacons (MB) was designed based on dual quenching of organic quencher Black Hole Quencher 1 (BHQ-1) and guanine (G) bases to fluorophore 6-carboxyfluorescein group (FAM). A dual color fluorescence quantitative detection method for specific single-stranded DNA sequences has been developed based on MB and nucleic acid dye hoechst 33258. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In this MB, FAM and BHQ-1 are respectively selected as fluorophore and organic quencher, three continuous nucleotides with G base are connected with BHQ-1, the bases of the stem in MB is completely designed as C-G base pairs, and the base sequence of the loop in MB is designed as the complementary sequence of target DNA. In the absence of target DNA, the MBs is the stem-loop structure, the FAM is close to BHQ-1 and G bases, the fluorescence of FAM is dually quenched by BHQ-1 and G bases, and the fluorescence of FAM is very weak. In addition, the C-G base pairs of MB cannot be combined with Hoechst 33258, so the fluorescence of Hoechst 33258 is also very weak. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strand structure, the stem-loop structure of MB is destroyed, and the FAM is separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. At the same time, the nucleic acid dye Hoechst 33258 binds to the A-T base pair in double-stranded DNA, and its fluorescent signal is significantly enhanced. Thus, a dual color fluorescence quantitative detection for the target DNA can be realized through the fluorescence enhancement of FAM and Hoechst 33258. Under optimized conditions, the total fluorescence intensities of FAM and Hoechst 33258 exhibit a good linear dependence on concentration of target DNA in the range of 0.05-8.0 nmol/L, and the regression equation is ΔIT=192.2C + 115.08 (R2=0.9938) with detection limit of 20 pmol/L (3σ, n=9). The method has simple operation, high sensitivity, good selectivity and low detection limit.
A double-quenching molecular beacons (MB) was designed based on dual quenching of organic quencher Black Hole Quencher 1 (BHQ-1) and guanine (G) bases to fluorophore 6-carboxyfluorescein group (FAM). A dual color fluorescence quantitative detection method for specific single-stranded DNA sequences has been developed based on MB and nucleic acid dye hoechst 33258. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In this MB, FAM and BHQ-1 are respectively selected as fluorophore and organic quencher, three continuous nucleotides with G base are connected with BHQ-1, the bases of the stem in MB is completely designed as C-G base pairs, and the base sequence of the loop in MB is designed as the complementary sequence of target DNA. In the absence of target DNA, the MBs is the stem-loop structure, the FAM is close to BHQ-1 and G bases, the fluorescence of FAM is dually quenched by BHQ-1 and G bases, and the fluorescence of FAM is very weak. In addition, the C-G base pairs of MB cannot be combined with Hoechst 33258, so the fluorescence of Hoechst 33258 is also very weak. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strand structure, the stem-loop structure of MB is destroyed, and the FAM is separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. At the same time, the nucleic acid dye Hoechst 33258 binds to the A-T base pair in double-stranded DNA, and its fluorescent signal is significantly enhanced. Thus, a dual color fluorescence quantitative detection for the target DNA can be realized through the fluorescence enhancement of FAM and Hoechst 33258. Under optimized conditions, the total fluorescence intensities of FAM and Hoechst 33258 exhibit a good linear dependence on concentration of target DNA in the range of 0.05-8.0 nmol/L, and the regression equation is ΔIT=192.2C + 115.08 (R2=0.9938) with detection limit of 20 pmol/L (3σ, n=9). The method has simple operation, high sensitivity, good selectivity and low detection limit.
2019, 47(7): 1021-1028
doi: 10.19756/j.issn.0253-3820.191153
Abstract:
The simultaneous and sensitive detection of metabolites of DNA (uric acid (UA), xanthine (XA) and hypoxanthine (HX)) is of great significance for the early diagnosis and prevention of related diseases caused by abnormal metabolism. In this study, hydroxyl functionalized metal-organic framework (OH-MOFs) and electrochemically reduced graphene oxide (ERGO) nano-functional interface was designed and constructed based on the synergic effect of Fe terephthalate OH-MOFs (OH-MIL-101(Fe) and ERGO for simultaneous detection of UA, XA and HX in blood. Nano-composites were prepared by ultrasonic mixing method, and were characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and UV-Vis spectroscopic methods. By casting the surface of glassy carbon electrode (GCE) with the composite and electrochemical reduction of the modified electrode, OH-MOFs-ERGO/GCE was obtained. Under the optimum conditions, the oxidation peak currents of UA, XA and HX were linearly correlated to their concentrations in the ranges of 0.20-1150 μmol/L, 0.15-800 μmol/L, and 0.40-600 μmol/L, respectively. The detection limits for UA, XA and HX were 0.12, 0.10 and 0.20 μmol/L (S/N=3), respectively. The recoveries of UA, XA and HX in real serum samples were between 99.5% and 105.8%. The proposed electrode was expected to provide a simple and easy detection method for the physiological and pathological study of purine metabolism.
The simultaneous and sensitive detection of metabolites of DNA (uric acid (UA), xanthine (XA) and hypoxanthine (HX)) is of great significance for the early diagnosis and prevention of related diseases caused by abnormal metabolism. In this study, hydroxyl functionalized metal-organic framework (OH-MOFs) and electrochemically reduced graphene oxide (ERGO) nano-functional interface was designed and constructed based on the synergic effect of Fe terephthalate OH-MOFs (OH-MIL-101(Fe) and ERGO for simultaneous detection of UA, XA and HX in blood. Nano-composites were prepared by ultrasonic mixing method, and were characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and UV-Vis spectroscopic methods. By casting the surface of glassy carbon electrode (GCE) with the composite and electrochemical reduction of the modified electrode, OH-MOFs-ERGO/GCE was obtained. Under the optimum conditions, the oxidation peak currents of UA, XA and HX were linearly correlated to their concentrations in the ranges of 0.20-1150 μmol/L, 0.15-800 μmol/L, and 0.40-600 μmol/L, respectively. The detection limits for UA, XA and HX were 0.12, 0.10 and 0.20 μmol/L (S/N=3), respectively. The recoveries of UA, XA and HX in real serum samples were between 99.5% and 105.8%. The proposed electrode was expected to provide a simple and easy detection method for the physiological and pathological study of purine metabolism.
2019, 47(7): 1029-1034
doi: 10.19756/j.issn.0253-3820.191176
Abstract:
A homogeneous electrochemical biosensor for miRNA-21 detection based on DNA-templated click chemistry and catalytic hairpin assembly was developed. 5-Amino-2,3-dicyano-1,4-naphthoquinone (ADNQ) and 2-furanpropanoic acid (FPA) were linked to two suitable probes of hairpin structure, respectively. In the presence of miRNA-21, the catalytic hairpin assembly between the two probes could be triggered. Therefore, ADNQ and FPA linked to the probes got very close to each other and reacted by a Diels-Alder reaction to change the quinone structure of ADNQ intoto ketone, making it no longer electro-active. Thus, the current difference (ΔI) could be used to indicate the concentration of miRNA-21. The feasibility and detecting performance of the proposed biosensor were also examined by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and polyacrylamide gel electrophoresis (PAGE). The biosensor was successfully applied to sensitively detect miRNA-21 in a linear concentration range from 0.1-1.0×104 pmol/L with a detection limit of 0.037 pmol/L (S/N=3). The biosensor was suitable for detection of miRNA-21 in blood samples.
A homogeneous electrochemical biosensor for miRNA-21 detection based on DNA-templated click chemistry and catalytic hairpin assembly was developed. 5-Amino-2,3-dicyano-1,4-naphthoquinone (ADNQ) and 2-furanpropanoic acid (FPA) were linked to two suitable probes of hairpin structure, respectively. In the presence of miRNA-21, the catalytic hairpin assembly between the two probes could be triggered. Therefore, ADNQ and FPA linked to the probes got very close to each other and reacted by a Diels-Alder reaction to change the quinone structure of ADNQ intoto ketone, making it no longer electro-active. Thus, the current difference (ΔI) could be used to indicate the concentration of miRNA-21. The feasibility and detecting performance of the proposed biosensor were also examined by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and polyacrylamide gel electrophoresis (PAGE). The biosensor was successfully applied to sensitively detect miRNA-21 in a linear concentration range from 0.1-1.0×104 pmol/L with a detection limit of 0.037 pmol/L (S/N=3). The biosensor was suitable for detection of miRNA-21 in blood samples.
2019, 47(7): 1035-1044
doi: 10.19756/j.issn.0253-3820.191093
Abstract:
A method was developed for investigation of regulatory role of transcription factors (TFs) in trastuzumab resistance of gastric cancer cells by liquid chromatography-mass spectrometry coupled with concatenated tandem array of transcription factor response elements (catTFRE) pull down proteomic technique. A synthetic DNA containing catTFRE was used as an affinity reagent, on which a biotin was labeled to become a DNA decoy, and then TFs were enriched via catTFRE pull down and detected by liquid chromatography-mass spectrometry. Intensity based absolute quantification (iBAQ) was applied to quantify TFs, and the TFs with a significant change in DNA binding activity were filtered out. Signaling pathways regulated by TFs, family classification, regulatory network and TFs-targets analysis were done based on WebGestalt (2017) database. The result showed that, 359 TFs were detected and quantified, and comparing with parental cells, 61 TFs in the trastuzumab resistance gastric cancer cells showed a remarkable activity change in DNA binding, of which 48 TFs showed increased activity and 13 showed decreased activity. The activated TFs belonged to bZIP, bHLH, homebox, HMG box, Zine finger, etc. KEGG pathway enrichment analysis demonstrated that multiple pathways including cancer, MAPK, Wnt, TGF-β, apoptosis changed prominently in trastuzumab resistant gastric cells, TFs-targets analysis revealed that TFs including TCF7L2, TCF7, FOXC1, JUN, MYC, FOS, ELK4, NFκB2, DDIT3 and ATF2 had an important regulatory effect on epithelial-mesenchymal transformation (EMT), Wnt and MAPK signaling pathways which contributed to trastuzumab-resistance in gastric cancer cells. This implicated that the targeting signaling pathways regulated by these TFs could possibly reverse trastuzumab resistance in gastric cancer.
A method was developed for investigation of regulatory role of transcription factors (TFs) in trastuzumab resistance of gastric cancer cells by liquid chromatography-mass spectrometry coupled with concatenated tandem array of transcription factor response elements (catTFRE) pull down proteomic technique. A synthetic DNA containing catTFRE was used as an affinity reagent, on which a biotin was labeled to become a DNA decoy, and then TFs were enriched via catTFRE pull down and detected by liquid chromatography-mass spectrometry. Intensity based absolute quantification (iBAQ) was applied to quantify TFs, and the TFs with a significant change in DNA binding activity were filtered out. Signaling pathways regulated by TFs, family classification, regulatory network and TFs-targets analysis were done based on WebGestalt (2017) database. The result showed that, 359 TFs were detected and quantified, and comparing with parental cells, 61 TFs in the trastuzumab resistance gastric cancer cells showed a remarkable activity change in DNA binding, of which 48 TFs showed increased activity and 13 showed decreased activity. The activated TFs belonged to bZIP, bHLH, homebox, HMG box, Zine finger, etc. KEGG pathway enrichment analysis demonstrated that multiple pathways including cancer, MAPK, Wnt, TGF-β, apoptosis changed prominently in trastuzumab resistant gastric cells, TFs-targets analysis revealed that TFs including TCF7L2, TCF7, FOXC1, JUN, MYC, FOS, ELK4, NFκB2, DDIT3 and ATF2 had an important regulatory effect on epithelial-mesenchymal transformation (EMT), Wnt and MAPK signaling pathways which contributed to trastuzumab-resistance in gastric cancer cells. This implicated that the targeting signaling pathways regulated by these TFs could possibly reverse trastuzumab resistance in gastric cancer.
2019, 47(7): 1045-1053
doi: 10.19756/j.issn.0253-3820.181762
Abstract:
Long term inorganic arsenic exposure can cause many diseases and health damage, and the biological mechanism underneath remains unclear. Globally, over 200 million people are under the threat of arsenic pollution of drinking water. In this study, we performed metabolomics analysis of rat urine samples which were exposed to inorganic arsenic through drinking water for a long term. The results indicated that long-term arsenic exposure could cause significant urinary metabolome change, especially for the high-dose exposure groups. Totally 36 kinds of differential metabolites were identified, in which 14 were related with amino acid metabolism, including tryptophan, arginine, proline, lysine, β-alanine, cysteine, methionine, glycine, serine and threonine, and 11 were related with lipid metabolism, including sphingolipids and fatty acids. The expression and post-translational modifications of proteins were also influenced. Such biological process mutations are the key molecular indicators of arsenic toxicological mechanisms.
Long term inorganic arsenic exposure can cause many diseases and health damage, and the biological mechanism underneath remains unclear. Globally, over 200 million people are under the threat of arsenic pollution of drinking water. In this study, we performed metabolomics analysis of rat urine samples which were exposed to inorganic arsenic through drinking water for a long term. The results indicated that long-term arsenic exposure could cause significant urinary metabolome change, especially for the high-dose exposure groups. Totally 36 kinds of differential metabolites were identified, in which 14 were related with amino acid metabolism, including tryptophan, arginine, proline, lysine, β-alanine, cysteine, methionine, glycine, serine and threonine, and 11 were related with lipid metabolism, including sphingolipids and fatty acids. The expression and post-translational modifications of proteins were also influenced. Such biological process mutations are the key molecular indicators of arsenic toxicological mechanisms.
2019, 47(7): 1054-1060
doi: 10.19756/j.issn.0253-3820.191070
Abstract:
When strontium (Sr) isotopic analysis is carried out for geological samples of high rubidium (Rb)/Sr ratios, it is difficult to completely remove the isobaric interference of Rb on Sr isotopic analysis by using traditional strong acid cation exchange resin for a single separation and purification. Actually, one purification procedure performed twice is necessary to eliminate the interference of 87Rb on the testing of 87Sr/86Sr. Although the purification procedure could ensure a good effect on the separation and purification of Sr, the process is tedious and time-consuming, which is not suitable for simultaneous separation and purification of Sr from large quantity of geological samples with high Rb/Sr ratios. In this study, the conventional twice separation process was optimized, and an experimental method for rapid separation of Sr from geological samples with high Rb/Sr ratios was established. The Sr solution after purified by AG50 resin was added to a new AG50 resin directly to purify Rb and Sr again. The whole separation process would be accomplished quickly and efficiently. The method combined with high sensitive thermal ionization mass spectrometry (TIMS) is completely satisfied to high precision determination of 87Sr/86Sr isotope ratios for the samples of high Rb/Sr ratios. Through the application of the new separation method with a large number of geological samples of high Rb/Sr ratios, it is proved that the experimental process is stable and reliable and showing crucial application value.
When strontium (Sr) isotopic analysis is carried out for geological samples of high rubidium (Rb)/Sr ratios, it is difficult to completely remove the isobaric interference of Rb on Sr isotopic analysis by using traditional strong acid cation exchange resin for a single separation and purification. Actually, one purification procedure performed twice is necessary to eliminate the interference of 87Rb on the testing of 87Sr/86Sr. Although the purification procedure could ensure a good effect on the separation and purification of Sr, the process is tedious and time-consuming, which is not suitable for simultaneous separation and purification of Sr from large quantity of geological samples with high Rb/Sr ratios. In this study, the conventional twice separation process was optimized, and an experimental method for rapid separation of Sr from geological samples with high Rb/Sr ratios was established. The Sr solution after purified by AG50 resin was added to a new AG50 resin directly to purify Rb and Sr again. The whole separation process would be accomplished quickly and efficiently. The method combined with high sensitive thermal ionization mass spectrometry (TIMS) is completely satisfied to high precision determination of 87Sr/86Sr isotope ratios for the samples of high Rb/Sr ratios. Through the application of the new separation method with a large number of geological samples of high Rb/Sr ratios, it is proved that the experimental process is stable and reliable and showing crucial application value.
2019, 47(7): 1061-1067
doi: 10.19756/j.issn.0253-3820.191064
Abstract:
Microbes in the reclaimed water can form biofilms, which thus increases difficulties to the post-use of the reclaimed water. In this work, the effects of exogenous protein on biofilm formation was elaborated, and the mechanism was revealed by further exploring the micro-structure and characteristics of the biofilms. The results showed that there was a positive relationship of biofilm formation to experimental protein concentration in the range of 0-8.0 mg/L in typical effluents. The Zeta potential of bacterial cell suspensions decreased 35% upon the addition of 8 mg/L protein. The contact angle of bacterial cell suspensions decreased gradually with the increase of protein concentration, indicating that the protein could modify the cell surface to be more hydrophobic. In addition, exogenous protein could bridge with the functional groups on the cell surface, resulting in a weakened bacterial hydrophilicity and an enhanced cells aggregation. Extracellular polysaccharides produced under the stimulation could promote the maturation of biofilms. This study supplied some important information on advanced treatment of effluents after biological treatment.
Microbes in the reclaimed water can form biofilms, which thus increases difficulties to the post-use of the reclaimed water. In this work, the effects of exogenous protein on biofilm formation was elaborated, and the mechanism was revealed by further exploring the micro-structure and characteristics of the biofilms. The results showed that there was a positive relationship of biofilm formation to experimental protein concentration in the range of 0-8.0 mg/L in typical effluents. The Zeta potential of bacterial cell suspensions decreased 35% upon the addition of 8 mg/L protein. The contact angle of bacterial cell suspensions decreased gradually with the increase of protein concentration, indicating that the protein could modify the cell surface to be more hydrophobic. In addition, exogenous protein could bridge with the functional groups on the cell surface, resulting in a weakened bacterial hydrophilicity and an enhanced cells aggregation. Extracellular polysaccharides produced under the stimulation could promote the maturation of biofilms. This study supplied some important information on advanced treatment of effluents after biological treatment.
2019, 47(7): 1068-1074
doi: 10.19756/j.issn.0253-3820.191204
Abstract:
Human coagulation factor 7a (FVⅡa) is the core factor for exogenous coagulation pathway. Developing safe, effective and economical coagulants or anticoagulants is still a challenge. In this study, a single-stranded DNA (ssDNA) aptamer for FVⅡa was isolated based on systematic evolution of ligands by exponential enrichment (SELEX), which was based on the isolation of the sequences bound to the target protein, which would be absorbed on nitrocellulose membrane. The aptamer was isolated after 11 rounds of selection. The apparent dissociation constant of the aptamer to FVⅡa was 102 nmol/L. The aptamer-based fluorescent assay for FVⅡa was established, exhibing simplicity and efficiency. The quantitative detection range of FVⅡa by this assay was 10-30 nmol/L, providing a new strategy for detection of FVⅡa. The non-denaturation polyacrylamide gel electrophoresis and the aptamer-based fluorescent assay results both showed that the aptamer had good selectivity toward FVⅡa.
Human coagulation factor 7a (FVⅡa) is the core factor for exogenous coagulation pathway. Developing safe, effective and economical coagulants or anticoagulants is still a challenge. In this study, a single-stranded DNA (ssDNA) aptamer for FVⅡa was isolated based on systematic evolution of ligands by exponential enrichment (SELEX), which was based on the isolation of the sequences bound to the target protein, which would be absorbed on nitrocellulose membrane. The aptamer was isolated after 11 rounds of selection. The apparent dissociation constant of the aptamer to FVⅡa was 102 nmol/L. The aptamer-based fluorescent assay for FVⅡa was established, exhibing simplicity and efficiency. The quantitative detection range of FVⅡa by this assay was 10-30 nmol/L, providing a new strategy for detection of FVⅡa. The non-denaturation polyacrylamide gel electrophoresis and the aptamer-based fluorescent assay results both showed that the aptamer had good selectivity toward FVⅡa.
2019, 47(7): 1075-1081
doi: 10.19756/j.issn.0253-3820.181761
Abstract:
Domoic acid (DA), a type of hydrophilic toxin produced by marine diatoms, can cause memory loss, which poses a serious threat to marine culture and even human health. In this study, a new method was developed for the highly efficient accumulation and detection of trace DA in seawater by high performance liquid chromatography-ultraviolet detection/mass spectrometry (HPLC-UV/MS). In this method, a solid phase extraction disk made of sulfonated styrene-vinyl benzene copolymer was used for DA enrichment and purification from acidified seawater samples. A C18 chromatographic column was employed for DA separation using acetonitrile-water solution (90%, V/V) containing 2 mmol/L ammonium acetate and 0.1% (V/V) formic acid as mobile phase. Detection wavelength was set at 242 nm. Under optimal conditions, the average recovery could still maintain at 89.3% with good precision (relative standard deviation (RSD) ≤ 4.0%) while enrichment factor could reach up to 2000. The limit of detection (S/N=3) was 2.5 ng/L. The results showed that the HPLC-UV detection developed here could meet the sensitivity requirements of DA determination in real seawater samples. This method was used to determine the seawater samples obtained from East China Sea, and it was found that the DA content in two station seawater samples was up to 2.7 μg/L. This method was efficient in determination of the concentration of DA in coastal aquaculture seawater, with easy-operation and high sensitivity.
Domoic acid (DA), a type of hydrophilic toxin produced by marine diatoms, can cause memory loss, which poses a serious threat to marine culture and even human health. In this study, a new method was developed for the highly efficient accumulation and detection of trace DA in seawater by high performance liquid chromatography-ultraviolet detection/mass spectrometry (HPLC-UV/MS). In this method, a solid phase extraction disk made of sulfonated styrene-vinyl benzene copolymer was used for DA enrichment and purification from acidified seawater samples. A C18 chromatographic column was employed for DA separation using acetonitrile-water solution (90%, V/V) containing 2 mmol/L ammonium acetate and 0.1% (V/V) formic acid as mobile phase. Detection wavelength was set at 242 nm. Under optimal conditions, the average recovery could still maintain at 89.3% with good precision (relative standard deviation (RSD) ≤ 4.0%) while enrichment factor could reach up to 2000. The limit of detection (S/N=3) was 2.5 ng/L. The results showed that the HPLC-UV detection developed here could meet the sensitivity requirements of DA determination in real seawater samples. This method was used to determine the seawater samples obtained from East China Sea, and it was found that the DA content in two station seawater samples was up to 2.7 μg/L. This method was efficient in determination of the concentration of DA in coastal aquaculture seawater, with easy-operation and high sensitivity.
2019, 47(7): 1082-1089
doi: 10.19756/j.issn.0253-3820.181500
Abstract:
A non-targeted ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry (UHPLC-LTQ/Orbitrap-MS) metabolomics method was established to explore the potential urinary biomarkers of biliary atresia(BA), and to provide scientific basis for understanding the pathogenesis and early screening of BA. Urine samples from 32 BA infants and 40 normal controls were collected, and metabolic profiles of these urine samples were obtained. Finally, 32 kinds of endogenous differential metabolites were identified as potential biomarkers for BA based on principle component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Alanine, aspartic acid and glutamate, arginine and proline, D-glutamate and D-glutamine, histidine, glycine, serine and threonine metabolism pathway were involved in BA, suggesting that there was extensive disorder of amino acid metabolism in the progression of BA. Among them, glutamic acid, L-glutamine, citrulline, proline, glycocyamine, 5'-methylthioadenosine and epsilon-(gamma-Glutamyl)-lysine had obvious upward or downward trends, which are involved in a series of pathological mechanism of BA.
A non-targeted ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry (UHPLC-LTQ/Orbitrap-MS) metabolomics method was established to explore the potential urinary biomarkers of biliary atresia(BA), and to provide scientific basis for understanding the pathogenesis and early screening of BA. Urine samples from 32 BA infants and 40 normal controls were collected, and metabolic profiles of these urine samples were obtained. Finally, 32 kinds of endogenous differential metabolites were identified as potential biomarkers for BA based on principle component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Alanine, aspartic acid and glutamate, arginine and proline, D-glutamate and D-glutamine, histidine, glycine, serine and threonine metabolism pathway were involved in BA, suggesting that there was extensive disorder of amino acid metabolism in the progression of BA. Among them, glutamic acid, L-glutamine, citrulline, proline, glycocyamine, 5'-methylthioadenosine and epsilon-(gamma-Glutamyl)-lysine had obvious upward or downward trends, which are involved in a series of pathological mechanism of BA.
2019, 47(7): 1090-1097
doi: 10.19756/j.issn.0253-3820.181492
Abstract:
A total of 38 kinds of major, minor and trace elements in marine sediment were analyzed by a combination technique of wavelength dispersive and energy dispersion X-ray fluorescence spectrometer (WD-EDXRF). The sample was prepared by powder pressing, and the stream sediments, soil, rock and marine sediment certified reference materials (CRMs) were used to establish calibration curves. The light and trace elements with heavy overlapped spectral lines were analyzed by WDXRF, and the other major and minor elements that without heavy overlapped spectral lines were analyzed by EDXRF. The matrix effect was corrected by Alpha empirical coefficient method and scattered rays internal standard method. The spectral overlapping interference of elements was accurately deducted by multiple regression. With the proposed methodology, the detection limits of minor elements were mostly below 18 μg/g, and those of light elements (Na, Mg) were lower than 15 μg/g, which were suitable for element determination in most marine sediments. Precision of the proposed method was checked by analyzing the stream sediment CRMs (GBW07309), and the relative standard deviations (RSD, n=10) of the determination results were lower than 9.41% except for individual elements. The quality of the analytical procedure was demonstrated at a 95% confidence level, in which the estimated analytical uncertainties agreed with those from the RM's certificates of analysis. The WD-EDXRF method was applied to the determination of major and minor elements in different types of marine sediments collected from the Indian Ocean, and the relative error of some elements were less than 10%, between the values analyzed by ICP-OES, ICP-MS and WD-EDXRF. In a word, WD-EDXRF is a well-established analytical technique for multi-element determination in various sample types, especially for marine sediment, and it will be a new technical support to investigate the marine resource and ecology.
A total of 38 kinds of major, minor and trace elements in marine sediment were analyzed by a combination technique of wavelength dispersive and energy dispersion X-ray fluorescence spectrometer (WD-EDXRF). The sample was prepared by powder pressing, and the stream sediments, soil, rock and marine sediment certified reference materials (CRMs) were used to establish calibration curves. The light and trace elements with heavy overlapped spectral lines were analyzed by WDXRF, and the other major and minor elements that without heavy overlapped spectral lines were analyzed by EDXRF. The matrix effect was corrected by Alpha empirical coefficient method and scattered rays internal standard method. The spectral overlapping interference of elements was accurately deducted by multiple regression. With the proposed methodology, the detection limits of minor elements were mostly below 18 μg/g, and those of light elements (Na, Mg) were lower than 15 μg/g, which were suitable for element determination in most marine sediments. Precision of the proposed method was checked by analyzing the stream sediment CRMs (GBW07309), and the relative standard deviations (RSD, n=10) of the determination results were lower than 9.41% except for individual elements. The quality of the analytical procedure was demonstrated at a 95% confidence level, in which the estimated analytical uncertainties agreed with those from the RM's certificates of analysis. The WD-EDXRF method was applied to the determination of major and minor elements in different types of marine sediments collected from the Indian Ocean, and the relative error of some elements were less than 10%, between the values analyzed by ICP-OES, ICP-MS and WD-EDXRF. In a word, WD-EDXRF is a well-established analytical technique for multi-element determination in various sample types, especially for marine sediment, and it will be a new technical support to investigate the marine resource and ecology.
2019, 47(7): 1098-1105
doi: 10.19756/j.issn.0253-3820.181711
Abstract:
A method for non-target screening of pesticide residues in dairy products by ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) was developed. A database of pesticide residue standards was established for the experimental application of data-dependent acquisition mass spectrometry technology. Subsequently, the fragmentation mechanism of eight classes of 100 pesticides was studied, and the characteristic fracture fragments of different classes of pesticides were obtained. By combining pesticide fragmentation with the pesticide residue standard database, a non-directional screening method based on data-independent acquisition mass spectrometry was developed. Finally, the established method was applied to the non-directional analysis of pesticide residues in 93 batches of pasteurized milk and ultra-high temperature instant sterilized milk. As a result, pre-undetected azoxystrobin (2.3 μg/kg) and chlorthiamide (6.1 μg/kg) were detected in the samples, with the relative standard deviations of 2.3% and 1.2%. The high-resolution non-target screening analysis method established here can provide effective technical means for rapid screening and identification of pesticides in dairy products.
A method for non-target screening of pesticide residues in dairy products by ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) was developed. A database of pesticide residue standards was established for the experimental application of data-dependent acquisition mass spectrometry technology. Subsequently, the fragmentation mechanism of eight classes of 100 pesticides was studied, and the characteristic fracture fragments of different classes of pesticides were obtained. By combining pesticide fragmentation with the pesticide residue standard database, a non-directional screening method based on data-independent acquisition mass spectrometry was developed. Finally, the established method was applied to the non-directional analysis of pesticide residues in 93 batches of pasteurized milk and ultra-high temperature instant sterilized milk. As a result, pre-undetected azoxystrobin (2.3 μg/kg) and chlorthiamide (6.1 μg/kg) were detected in the samples, with the relative standard deviations of 2.3% and 1.2%. The high-resolution non-target screening analysis method established here can provide effective technical means for rapid screening and identification of pesticides in dairy products.
2019, 47(7): 1106-1113
doi: 10.19756/j.issn.0253-3820.191167
Abstract:
Human milk oligosaccharides are a kind of natural prebiotics that exist in human milk. They play an irreplaceable role in the growth of infantile intestinal flora, the improvement of immune system and the resistance to pathogen infection. The function of human milk oligosaccharides is closely related to their structures which are complex with various linkages. Besides, the oligosaccharides have a large number of isomers, the number and complexity of which increase dramatically with the degrees of their polymerization. Therefore, the structural analysis of oligosaccharide isomers with a high degree of polymerization is one of the difficulties in the study of human milk oligosaccharides, and is of great significance for the study of the biological function of oligosaccharides. In this study, electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q/TOF-MS) was used to analyze the structures of eighteen high-degree polymerized human milk oligosaccharides in a negative ion mode, and three of them were firstly reported. Differences of fragment ions between the isomers were discussed, and the cleavage rules of oligosaccharide isomers were summarized. This study provided a scientific basis for the structural analysis of the complex isomers of highly polymerized human milk oligosaccharides and the discovery of novel sugar structures.
Human milk oligosaccharides are a kind of natural prebiotics that exist in human milk. They play an irreplaceable role in the growth of infantile intestinal flora, the improvement of immune system and the resistance to pathogen infection. The function of human milk oligosaccharides is closely related to their structures which are complex with various linkages. Besides, the oligosaccharides have a large number of isomers, the number and complexity of which increase dramatically with the degrees of their polymerization. Therefore, the structural analysis of oligosaccharide isomers with a high degree of polymerization is one of the difficulties in the study of human milk oligosaccharides, and is of great significance for the study of the biological function of oligosaccharides. In this study, electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q/TOF-MS) was used to analyze the structures of eighteen high-degree polymerized human milk oligosaccharides in a negative ion mode, and three of them were firstly reported. Differences of fragment ions between the isomers were discussed, and the cleavage rules of oligosaccharide isomers were summarized. This study provided a scientific basis for the structural analysis of the complex isomers of highly polymerized human milk oligosaccharides and the discovery of novel sugar structures.
2019, 47(7): 1114-1120
doi: 10.19756/j.issn.0253-3820.181720
Abstract:
Rapid identification of chemical constituents responsible for defined bioactivities from traditional Chinese medicines (TCMs) is a great challenge. A strategy for rapid identification of the chemical ingredients from the extracts of the rhizoma of Curcuma kwangsiensis with supercritical fluid extraction (SFE) was proposed using ultra-performance convergence chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPC2-QTOF-MS) combined with UNIFI library. In this study, a total of 47 chemical compounds which were separated in 7 min were tentatively identified, including 42 sesquiterpenes. There were 37 chemical constituents in 8 groups of isomers in the analysis. The strategy which can rapidly, effectively, and comprehensively screen and identify components from TCMs has potential application in TCMs analysis as an alternative method besides UPLC-QTOF-MS and GC-MS.
Rapid identification of chemical constituents responsible for defined bioactivities from traditional Chinese medicines (TCMs) is a great challenge. A strategy for rapid identification of the chemical ingredients from the extracts of the rhizoma of Curcuma kwangsiensis with supercritical fluid extraction (SFE) was proposed using ultra-performance convergence chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPC2-QTOF-MS) combined with UNIFI library. In this study, a total of 47 chemical compounds which were separated in 7 min were tentatively identified, including 42 sesquiterpenes. There were 37 chemical constituents in 8 groups of isomers in the analysis. The strategy which can rapidly, effectively, and comprehensively screen and identify components from TCMs has potential application in TCMs analysis as an alternative method besides UPLC-QTOF-MS and GC-MS.
