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CLC number: Q556.2; R284.1

On-line Access: 2019-03-01

Received: 2018-08-06

Revision Accepted: 2018-10-11

Crosschecked: 2019-01-10

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Journal of Zhejiang University SCIENCE B 2019 Vol.20 No.3 P.264-272

10.1631/jzus.B1800416


A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity


Author(s):  Min-Yi Jin, Tong Zhang, Yi-Shun Yang, Yue Ding, Jun-Song Li, Gao-Ren Zhong

Affiliation(s):  Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; more

Corresponding email(s):   zhangtdmj@hotmail.com

Key Words:  Glucometer-based assay, β, -Glucosidase, Activity detection


Min-Yi Jin, Tong Zhang, Yi-Shun Yang, Yue Ding, Jun-Song Li, Gao-Ren Zhong. A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity[J]. Journal of Zhejiang University Science B, 2019, 20(3): 264-272.

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author="Min-Yi Jin, Tong Zhang, Yi-Shun Yang, Yue Ding, Jun-Song Li, Gao-Ren Zhong",
journal="Journal of Zhejiang University Science B",
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publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1800416"
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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

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(pNPG)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~ 1.5498 U/mL和0.4076~2.9019 U/mL.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.

关键词:血糖仪;β-葡萄糖苷酶;活性检测

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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[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

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