Abstract: 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.

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

[1]Chen DM, Lei GF, Peng WL, 2012. Serum cytosolic β-glucosidase levels in neonatal necrotizing enterocolitis. Iran J Pediatr, 22(4):452-456.

[2]de Ovalle S, Cavello I, Brena BM, et al., 2018. Production and characterization of a β-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine. LWT, 87:515-522.

[3]Gómez-Chaparro Moreno JL, Rodríguez Torronteras A, Ruiz González MD, et al., 2016. The β-glucosidase assay: a new diagnostic tool for necrotizing enterocolitis. Sensitivity, specificity, and predictive values. Eur J Pediatr, 175(7):931-941.

[4]Gueguen Y, Chemardin P, Labrot P, et al., 1997. Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava. J Appl Microbiol, 82(4):469-476.

[5]Gurale BP, Dhawane AN, Cui XK, et al., 2016. Indirect detection of glycosidases using amperometry. Anal Chem, 88(8):4248-4253.

[6]Hu R, Zhang S, Wu JW, et al., 2013. Tissue localization and expression difference of endogenous β-glucosidase in digestive system of Musca domestica third instar larvae. Chin J Parasitol Parasit Dis, 31(4):256-261 (in Chinese).

[7]Li F, Mao YD, Wang YF, et al., 2017. Optimization of ultrasonic-assisted enzymatic extraction conditions for improving total phenolic content, antioxidant and antitumor activities in vitro from Trapa quadrispinosa Roxb. residues. Molecules, 22(3):396.

[8]National Development and Reform Commission, 2003. Cellulases, QB 2583-2003. National Standards of People’s Republic of China (in Chinese).

[9]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 wall β-glucosidase. J Photochem Photobiol B, 137:49-54.

[10]Peng J, Ma YH, Chen Y, et al., 2015. Transformation of icariin by immobilized β-glucosidase and snailase. Acta Pharm Sin, 50(12):1652-1659 (in Chinese).

[11]https://doi.org/10.16438/j.0513-4870.2015.12.011

[12]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.

[13]Sharp DC, Steensels J, Shellhammer TH, 2017. The effect of hopping regime, cultivar and β-glucosidase activity on monoterpene alcohol concentrations in wort and beer. J Inst Brew, 123(2):185-191.

[14]Strahsburger E, de Lacey AML, Marotti I, et al., 2017. In vivo assay to identify bacteria with β-glucosidase activity. Electron J Biotechnol, 30:83-87.

[15]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.

[16]Wan LH, Yao Z, Ni F, et al., 2014. Biosynthesis of genipin from gardenoside catalyzed by β-glucosidase in two-phase medium. CIESC J, 65(9):3583-3591 (in Chinese).

[17]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.

[18]Yu HH, Yoshimitsu M, Xu XY, et al., 2017. Investigate optimum conditions and determinate changes of β-glucosidase activity in Scrophularia root under different drying conditions. China J Chin Mater Med, 42(2):274-279 (in Chinese).

[19]https://doi.org/10.19540/j.cnki.cjcmm.20161222.049

[20]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.

[21]Zhang JJ, Shen Z, Xiang Y, et al., 2016. Integration of solution-based assays onto lateral flow device for one-step quantitative point-of-care diagnostics using personal glucose meter. ACS Sens, 1(9):1091-1096.

[22]Zhao LY, Gong JT, Mi WJ, et al., 2017. Determination of β-glucosidase activity in Semen Armeniaceae Amarum. Central South Pharm, 15(2):166-169 (in Chinese).

[23]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.

[24]Zhou X, Huang Z, Yang HW, et al., 2017. β-Glucosidase inhibition sensitizes breast cancer to chemotherapy. Biomed Pharmacother, 91:504-509.

[25]Zuo YF, Zhang W, Wu DL, et al., 2015. Extraction and enzymatic properties of β-glucosidase in Chrysanthemum morifolium. J Anhui Univ Chin Med, 34(5):83-86 (in Chinese).

[26]List of electronic supplementary materials

[27]Data S1 Preparation of the DNS reagent

[28]Data S2 Preparation of the glucose calibration standards

[29]Fig. S1 Experimental protocol design