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Electrochemical assay of sorbitol dehydrogenase at PEDOT modified electrodes – a new milk biomarker for confirmation of pregnancy in dairy cattle
Analyst ( IF 3.6 ) Pub Date : 2020-09-04 , DOI: 10.1039/d0an01360a Camilo Menezes 1 , Irene Malo-Estepa , Dayle Johnston , Aoife Delaney , Mark Crowe , Michael Diskin , Eithne Dempsey
Analyst ( IF 3.6 ) Pub Date : 2020-09-04 , DOI: 10.1039/d0an01360a Camilo Menezes 1 , Irene Malo-Estepa , Dayle Johnston , Aoife Delaney , Mark Crowe , Michael Diskin , Eithne Dempsey
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A robust electrochemical assay for sorbitol dehydrogenase (SORDH) activity in milk was developed using voltammetry and chronocoulometry at bare and polymer modified transducers. The motivation for the work was to evaluate the potential of SORDH as an early biomarker of bovine pregnancy using milk as sample matrix. SORDH is an enzyme involved in carbohydrate metabolism converting sorbitol, the sugar alcohol form of glucose, into fructose, with NAD+ as a cofactor being simultaneously reduced to NADH. The assay was optimised via direct NADH oxidation on glassy carbon and screen printed carbon electrodes followed by electropolymerisation of 3,4-ethylenedioxythiophene (EDOT) monomer to form an NADH responsive PEDOT surface which operated well in undiluted milk samples. Assay conditions such as incubation time and temperature were optimised resulting in a 3 min assay at 37 °C in the presence of 10 mM NAD+ and 20 mM sorbitol co-substrates, enabling NADH electro oxidation (linear range 0.25–5 mM, sensitivity 9.17 μC cm−2 mM−1 in undiluted milk). SORDH determination followed over the range 0.31–10 U mL−1 in milk samples with sensitivity 5.45 μC cm−2 U−1 mL with LOD 0.0787 U mL−1. The assay was applied to milk sample testing acquired as part of an approved animal study involving control and breeding cycles of dairy cows with focus on analysis at day 19 post artificial insemination. Significant differences between control and pregnant SORDH levels in whole milk animal samples were found (average values 2.57 and 4.07 ng mL−1 respectively), as verified using a commercial SORDH ELISA optical assay. Finally, progesterone monitoring over days 16–21 of the oestrous cycle employed an optical ELISA assay and confirmed maintenance of progesterone levels from day 19 onwards.
中文翻译：
PEDOT修饰电极上山梨醇脱氢酶的电化学测定——一种用于确认奶牛妊娠的新牛奶生物标志物
使用伏安法和计时库仑法在裸露和聚合物改性换能器上开发了一种用于牛奶中山梨醇脱氢酶 (SORDH) 活性的稳健电化学测定法。这项工作的动机是使用牛奶作为样品基质评估 SORDH 作为牛妊娠早期生物标志物的潜力。SORDH 是一种参与碳水化合物代谢的酶，将山梨醇（葡萄糖的糖醇形式）转化为果糖，同时 NAD +作为辅因子被还原为 NADH。该测定通过优化在玻璃碳和丝网印刷碳电极上直接氧化 NADH，然后电聚合 3,4-乙烯二氧噻吩 (EDOT) 单体，形成 NADH 响应性 PEDOT 表面，该表面在未稀释的牛奶样品中运行良好。优化孵育时间和温度等检测条件，在 37 °C 下在 10 mM NAD +和 20 mM 山梨糖醇共底物存在下进行 3 分钟检测，从而实现 NADH 电氧化（线性范围 0.25–5 mM，灵敏度 9.17 μC cm -2 mM -1在未稀释的牛奶中）。在 0.31–10 U mL -1范围内对牛奶样品进行 SORDH 测定，灵敏度为 5.45 μC cm -2 U -1 mL，LOD 为 0.0787 U mL -1. 该测定法应用于作为已批准动物研究的一部分获得的牛奶样本测试，该研究涉及奶牛的控制和繁殖周期，重点是人工授精后第 19 天的分析。使用商业 SORDH ELISA 光学测定法证实，全乳动物样品中对照和妊娠 SORDH 水平之间存在显着差异（平均值分别为 2.57 和 4.07 ng mL -1 ）。最后，在发情周期的第 16-21 天监测黄体酮采用光学 ELISA 测定，并确认从第 19 天开始维持黄体酮水平。
更新日期：2020-09-05
中文翻译：
PEDOT修饰电极上山梨醇脱氢酶的电化学测定——一种用于确认奶牛妊娠的新牛奶生物标志物
使用伏安法和计时库仑法在裸露和聚合物改性换能器上开发了一种用于牛奶中山梨醇脱氢酶 (SORDH) 活性的稳健电化学测定法。这项工作的动机是使用牛奶作为样品基质评估 SORDH 作为牛妊娠早期生物标志物的潜力。SORDH 是一种参与碳水化合物代谢的酶，将山梨醇（葡萄糖的糖醇形式）转化为果糖，同时 NAD +作为辅因子被还原为 NADH。该测定通过优化在玻璃碳和丝网印刷碳电极上直接氧化 NADH，然后电聚合 3,4-乙烯二氧噻吩 (EDOT) 单体，形成 NADH 响应性 PEDOT 表面，该表面在未稀释的牛奶样品中运行良好。优化孵育时间和温度等检测条件，在 37 °C 下在 10 mM NAD +和 20 mM 山梨糖醇共底物存在下进行 3 分钟检测，从而实现 NADH 电氧化（线性范围 0.25–5 mM，灵敏度 9.17 μC cm -2 mM -1在未稀释的牛奶中）。在 0.31–10 U mL -1范围内对牛奶样品进行 SORDH 测定，灵敏度为 5.45 μC cm -2 U -1 mL，LOD 为 0.0787 U mL -1. 该测定法应用于作为已批准动物研究的一部分获得的牛奶样本测试，该研究涉及奶牛的控制和繁殖周期，重点是人工授精后第 19 天的分析。使用商业 SORDH ELISA 光学测定法证实，全乳动物样品中对照和妊娠 SORDH 水平之间存在显着差异（平均值分别为 2.57 和 4.07 ng mL -1 ）。最后，在发情周期的第 16-21 天监测黄体酮采用光学 ELISA 测定，并确认从第 19 天开始维持黄体酮水平。
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