一种基于血糖仪的简便且微量的β-葡萄糖苷酶活性检测方法（英文）
|
摘要
目的:β-葡萄糖苷酶活性是新生儿坏死性小肠结肠炎早期诊断和药用植物有效成分改变的重要指标。现有活性检测方法操作时间长,试剂消耗大,且需要昂贵的仪器设备和专业的技术人员,不利于β-葡萄糖苷酶的快速、实时检测。本文尝试构建一种基于血糖仪的酶活性检测方法,为实际样品中β-葡萄糖苷酶活性的快速检测提供科学依据。创新点:首次将便携式血糖仪引入β-葡萄糖苷酶活性检测中,通过酶反应条件优化、方法学考察、实际样品中的应用以及现有方法的比较,建立β-葡萄糖苷酶活性检测新方法。方法:采用单因素试验,以酶反应溶液、pH值、反应温度、反应时间和底物浓度为指标,优化酶活性测定条件;通过考察精密度、稳定性、重复性、加样回收率、专一性等指标验证方法的可行性;通过实际样品中新方法的应用以及与现有二硝基水杨酸(DNS)法和4-硝基苯基-β-D-吡喃葡萄糖苷(pN PG)法的比较,研究新方法在实际样品检测中的适用性。结论:酶活性测定最优条件为以柠檬酸-柠檬酸钠缓冲液(0.05 mol/L,pH 5.0)为反应溶液,0.03 g/mL水杨苷为底物,在50°C条件下,反应30 min。新方法具有良好的精密度、准确性、重复性、稳定性和耐用性,且不受β-葡聚糖酶、蜗牛酶、β-半乳糖苷酶、半纤维素酶和葡萄糖醛酸的影响,专一性较强。β-葡萄糖苷酶在缓冲液和大鼠血清中线性关系良好,线性范围分别为0.0873～1.5498U/m L和0.4076～2.9019U/m L。该方法已成功应用于苦杏仁、燀苦杏仁、酶制剂以及黑曲霉培养的一系列β-葡萄糖苷酶活性检测中,结果与现有方法所测的基本相符。
β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873–1.5498 U/mL and 0.4076–2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid(DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring. è?a
β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873–1.5498 U/mL and 0.4076–2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid(DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring. è?a
引文
Chen DM,Lei GF,Peng WL,2012.Serum cytosolicβ-glucosidase levels in neonatal necrotizing enterocolitis.Iran J Pediatr,22(4):452-456.
de Ovalle S,Cavello I,Brena BM,et al.,2018.Production and characterization of aβ-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine.LWT,87:515-522.https://doi.org/10.1016/j.lwt.2017.09.026
Gómez-Chaparro Moreno JL,Rodríguez Torronteras A,Ruiz González MD,et al.,2016.Theβ-glucosidase assay:a new diagnostic tool for necrotizing enterocolitis.Sensitivity,specificity,and predictive values.Eur J Pediatr,175(7):931-941.https://doi.org/10.1007/s00431-016-2724-8
Gueguen Y,Chemardin P,Labrot P,et al.,1997.Purification and characterization of an intracellularβ-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava.J Appl Microbiol,82(4):469-476.https://doi.org/10.1046/j.1365-2672.1997.00136.x
Gurale BP,Dhawane AN,Cui XK,et al.,2016.Indirect detection of glycosidases using amperometry.Anal Chem,88(8):4248-4253.https://doi.org/10.1021/acs.analchem.5b03943
Hu R,Zhang S,Wu JW,et al.,2013.Tissue localization and expression difference of endogenousβ-glucosidase in digestive system of Musca domestica third instar larvae.Chin J Parasitol Parasit Dis,31(4):256-261(in Chinese).
Li F,Mao YD,Wang YF,et al.,2017.Optimization of ultrasonicassisted enzymatic extraction conditions for improving total phenolic content,antioxidant and antitumor activities in vitro from Trapa quadrispinosa Roxb.residues.Molecules,22(3):396.https://doi.org/10.3390/molecules22030396
National Development and Reform Commission,2003.Cellulases,QB 2583-2003.National Standards of People’s Republic of China(in Chinese).
Patro L,Mohapatra PK,Biswal UC,et al.,2014.Dehydration induced loss of photosynthesis in Arabidopsis leaves during senescence is accompanied by the reversible enhancement in the activity of cell wallβ-glucosidase.JPhotochem Photobiol B,137:49-54.https://doi.org/10.1016/j.jphotobiol.2014.03.018
Peng J,Ma YH,Chen Y,et al.,2015.Transformation of icariin by immobilizedβ-glucosidase and snailase.Acta Pharm Sin,50(12):1652-1659(in Chinese).https://doi.org/10.16438/j.0513-4870.2015.12.011
Rather MY,Mishra S,Chand S,2010.β-Glucosidase catalyzed synthesis of octyl-β-D-glucopyranoside using whole cells of Pichia etchellsii in micro aqueous media.J Biotechnol,150(4):490-496.https://doi.org/10.1016/j.jbiotec.2010.09.933
Sharp DC,Steensels J,Shellhammer TH,2017.The effect of hopping regime,cultivar andβ-glucosidase activity on monoterpene alcohol concentrations in wort and beer.JInst Brew,123(2):185-191.https://doi.org/10.1002/jib.418
Strahsburger E,de Lacey AML,Marotti I,et al.,2017.In vivo assay to identify bacteria withβ-glucosidase activity.Electron J Biotechnol,30:83-87.https://doi.org/10.1016/j.ejbt.2017.08.010
Su J,Xu J,Chen Y,et al.,2013.Sensitive detection of copper(II)by a commercial glucometer using click chemistry.Biosens Bioelectron,45:219-222.https://doi.org/10.1016/j.bios.2013.01.069
Wan LH,Yao Z,Ni F,et al.,2014.Biosynthesis of genipin from gardenoside catalyzed byβ-glucosidase in two-phase medium.CIESC J,65(9):3583-3591(in Chinese).https://doi.org/10.3969/j.issn.0438-1157.2014.09.037
Wang ZZ,Chen ZW,Gao N,et al.,2015.Transmutation of personal glucose meters into portable and highly sensitive microbial pathogen detection platform.Small,11(37):4970-4975.https://doi.org/10.1002/smll.201500944
Yu HH,Yoshimitsu M,Xu XY,et al.,2017.Investigate optimum conditions and determinate changes ofβ-glucosidase activity in Scrophularia root under different drying conditions.China J Chin Mater Med,42(2):274-279(in Chinese).https://doi.org/10.19540/j.cnki.cjcmm.20161222.049
Zhang J,Tang Y,Teng LM,et al.,2015.Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode.Biosens Bioelectron,68:232-238.https://doi.org/10.1016/j.bios.2015.01.001
Zhang JJ,Shen Z,Xiang Y,et al.,2016.Integration of solutionbased assays onto lateral flow device for one-step quantitative point-of-care diagnostics using personal glucose meter.ACS Sens,1(9):1091-1096.https://doi.org/10.1021/acssensors.6b00270
Zhao LY,Gong JT,Mi WJ,et al.,2017.Determination ofβ-glucosidase activity in Semen Armeniaceae Amarum.Central South Pharm,15(2):166-169(in Chinese).https://doi.org/10.7539/j.issn.1672-2981.2017.02.007
Zhao YT,Chen X,Lin S,et al.,2017.Integrated immunochromatographic strip with glucometer readout for rapid quantification of phosphorylated proteins.Anal Chim Acta,964:1-6.https://doi.org/10.1016/j.aca.2017.01.011
Zhou X,Huang Z,Yang HW,et al.,2017.β-Glucosidase inhibition sensitizes breast cancer to chemotherapy.Biomed Pharmacother,91:504-509.https://doi.org/10.1016/j.biopha.2017.04.113
Zuo YF,Zhang W,Wu DL,et al.,2015.Extraction and enzymatic properties ofβ-glucosidase in Chrysanthemum morifolium.J Anhui Univ Chin Med,34(5):83-86(in Chinese).https://doi.org/10.3969/j.issn.2095-7246.2015.05.024
de Ovalle S,Cavello I,Brena BM,et al.,2018.Production and characterization of aβ-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine.LWT,87:515-522.https://doi.org/10.1016/j.lwt.2017.09.026
Gómez-Chaparro Moreno JL,Rodríguez Torronteras A,Ruiz González MD,et al.,2016.Theβ-glucosidase assay:a new diagnostic tool for necrotizing enterocolitis.Sensitivity,specificity,and predictive values.Eur J Pediatr,175(7):931-941.https://doi.org/10.1007/s00431-016-2724-8
Gueguen Y,Chemardin P,Labrot P,et al.,1997.Purification and characterization of an intracellularβ-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava.J Appl Microbiol,82(4):469-476.https://doi.org/10.1046/j.1365-2672.1997.00136.x
Gurale BP,Dhawane AN,Cui XK,et al.,2016.Indirect detection of glycosidases using amperometry.Anal Chem,88(8):4248-4253.https://doi.org/10.1021/acs.analchem.5b03943
Hu R,Zhang S,Wu JW,et al.,2013.Tissue localization and expression difference of endogenousβ-glucosidase in digestive system of Musca domestica third instar larvae.Chin J Parasitol Parasit Dis,31(4):256-261(in Chinese).
Li F,Mao YD,Wang YF,et al.,2017.Optimization of ultrasonicassisted enzymatic extraction conditions for improving total phenolic content,antioxidant and antitumor activities in vitro from Trapa quadrispinosa Roxb.residues.Molecules,22(3):396.https://doi.org/10.3390/molecules22030396
National Development and Reform Commission,2003.Cellulases,QB 2583-2003.National Standards of People’s Republic of China(in Chinese).
Patro L,Mohapatra PK,Biswal UC,et al.,2014.Dehydration induced loss of photosynthesis in Arabidopsis leaves during senescence is accompanied by the reversible enhancement in the activity of cell wallβ-glucosidase.JPhotochem Photobiol B,137:49-54.https://doi.org/10.1016/j.jphotobiol.2014.03.018
Peng J,Ma YH,Chen Y,et al.,2015.Transformation of icariin by immobilizedβ-glucosidase and snailase.Acta Pharm Sin,50(12):1652-1659(in Chinese).https://doi.org/10.16438/j.0513-4870.2015.12.011
Rather MY,Mishra S,Chand S,2010.β-Glucosidase catalyzed synthesis of octyl-β-D-glucopyranoside using whole cells of Pichia etchellsii in micro aqueous media.J Biotechnol,150(4):490-496.https://doi.org/10.1016/j.jbiotec.2010.09.933
Sharp DC,Steensels J,Shellhammer TH,2017.The effect of hopping regime,cultivar andβ-glucosidase activity on monoterpene alcohol concentrations in wort and beer.JInst Brew,123(2):185-191.https://doi.org/10.1002/jib.418
Strahsburger E,de Lacey AML,Marotti I,et al.,2017.In vivo assay to identify bacteria withβ-glucosidase activity.Electron J Biotechnol,30:83-87.https://doi.org/10.1016/j.ejbt.2017.08.010
Su J,Xu J,Chen Y,et al.,2013.Sensitive detection of copper(II)by a commercial glucometer using click chemistry.Biosens Bioelectron,45:219-222.https://doi.org/10.1016/j.bios.2013.01.069
Wan LH,Yao Z,Ni F,et al.,2014.Biosynthesis of genipin from gardenoside catalyzed byβ-glucosidase in two-phase medium.CIESC J,65(9):3583-3591(in Chinese).https://doi.org/10.3969/j.issn.0438-1157.2014.09.037
Wang ZZ,Chen ZW,Gao N,et al.,2015.Transmutation of personal glucose meters into portable and highly sensitive microbial pathogen detection platform.Small,11(37):4970-4975.https://doi.org/10.1002/smll.201500944
Yu HH,Yoshimitsu M,Xu XY,et al.,2017.Investigate optimum conditions and determinate changes ofβ-glucosidase activity in Scrophularia root under different drying conditions.China J Chin Mater Med,42(2):274-279(in Chinese).https://doi.org/10.19540/j.cnki.cjcmm.20161222.049
Zhang J,Tang Y,Teng LM,et al.,2015.Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode.Biosens Bioelectron,68:232-238.https://doi.org/10.1016/j.bios.2015.01.001
Zhang JJ,Shen Z,Xiang Y,et al.,2016.Integration of solutionbased assays onto lateral flow device for one-step quantitative point-of-care diagnostics using personal glucose meter.ACS Sens,1(9):1091-1096.https://doi.org/10.1021/acssensors.6b00270
Zhao LY,Gong JT,Mi WJ,et al.,2017.Determination ofβ-glucosidase activity in Semen Armeniaceae Amarum.Central South Pharm,15(2):166-169(in Chinese).https://doi.org/10.7539/j.issn.1672-2981.2017.02.007
Zhao YT,Chen X,Lin S,et al.,2017.Integrated immunochromatographic strip with glucometer readout for rapid quantification of phosphorylated proteins.Anal Chim Acta,964:1-6.https://doi.org/10.1016/j.aca.2017.01.011
Zhou X,Huang Z,Yang HW,et al.,2017.β-Glucosidase inhibition sensitizes breast cancer to chemotherapy.Biomed Pharmacother,91:504-509.https://doi.org/10.1016/j.biopha.2017.04.113
Zuo YF,Zhang W,Wu DL,et al.,2015.Extraction and enzymatic properties ofβ-glucosidase in Chrysanthemum morifolium.J Anhui Univ Chin Med,34(5):83-86(in Chinese).https://doi.org/10.3969/j.issn.2095-7246.2015.05.024