Development of an Enzyme-Free Electrochemical Breathalyzer for the Diagnosis and Management of Diabetic Ketoacidosis

项目摘要/摘要 糖尿病酮症酸中毒(DKA)是一种危及生命的疾病，每年影响20万患者，并有 死亡率为4-5%。DKA的病理是胰岛素缺乏导致生产过剩的结果。 以乙酰乙酸酯(AcAc)、β-羟基丁酸酯(BHB)和丙酮形式存在的酮体。当前 DKA的诊断策略依赖于：1)硝普钠纸条尿检 ACAC，以及ii)用酶电化学生物传感器进行血液检测以检测BHB。不幸的是， 这些测试与众所周知的导致假阳性和假阴性的化学限制有关 结果。此外，患者认为尿液和血液测试令人不快、耗时、有创和 甚至是令人痛苦的。这项拨款申请将集中于开发一种非侵入性和患者友好的 化学选择性分析呼气中丙酮水平的无酶电化学呼气测定仪 DKA患者。人们普遍认为，在DKA过程中产生的酮体的小尺寸 使它们能够以挥发性有机物质的形式排入支气管腔并呼出 化合物(VOCs)。特别是，丙酮是DKA的生物标志物，因为它是呼吸中的主要VOC 患者的浓度高达1250ppm。在这项提议中，我们的目标是取代传统使用的酶 作为生物识别元件在电化学生物传感器中使用我们的点击化学氨氧基(-ONH2)或 肼(-NHNH2)涂层，众所周知，它与羰基反应迅速、有效并具有化学选择性 丙酮等挥发性有机化合物。该项目建立在劳莱集团研究实力的基础上，该集团拥有 在羰基代谢物的选择性衍生化、合成和电化学方面有丰富经验 方法论和工程背景。我们将检验呼气中的丙酮可以是 使用氨氧基或联氨涂层很容易捕获，生成的加合物可以电化学 在低氧化电位下氧化以获得电化学信号。海流的强度和潜力 将提供有关丙酮水平的定性和定量信息 呼气样本。我们提出了以下三个目标： 目的1：设计和合成含氨氧基和联氨的羰基涂料文库 诱捕。 目的2：使用丙酮和恒电位仪对涂层进行体外测试，以用于电化学研究。 目标3：设计一个酒精测定仪的原型。 这项提案的主要影响将是对无酶呼气测定仪的概念验证 用于诊断和治疗糖尿病酮症酸中毒的原型。

PROJECT SUMMARY/ABSTRACT Diabetic ketoacidosis (DKA) is a life-threatening condition that affects 200,000 patients annually and has a mortality rate of 4–5%. DKA pathology is the result of an insulin deficiency, which leads to an overproduction of ketone bodies in the form of acetoacetate (AcAc), β-hydroxybutyrate (BHB), and acetone. Current strategies for the diagnosis of DKA rely on i) urine testing with nitroprusside paper strips for the detection of AcAc, and ii) blood testing with enzymatic electrochemical biosensors for the detection of BHB. Unfortunately, these tests are associated with well-known chemical limitations that lead to false-positive and false-negative results. Additionally, patients perceive urine and blood tests as unpleasant, time-consuming, invasive, and even distressing. This grant application will focus on the development of a noninvasive and patient-friendly enzyme-free electrochemical breathalyzer that chemoselectively analyzes acetone levels in the breath of patients with DKA. It is widely recognized that the small size of the ketone bodies generated during DKA enables them to be discharged into the endobronchial cavity to be exhaled in the form of volatile organic compounds (VOCs). Acetone, in particular, is a biomarker for DKA since it is the major VOC in the breath of patients at concentrations as high as 1250 ppm. In this proposal, we aim to replace enzymes traditionally used as biological recognition element in electrochemical biosensors with our click chemistry aminooxy (–ONH2) or hydrazine (–NHNH2) coatings, which are known to react rapidly, efficiently, and chemoselectively with carbonyl VOCs such as acetone. This project builds on the research strengths of the Laulhé Group, which has extensive experience in selective derivatization of carbonyl metabolites, synthetic and electrochemical methodology, and an engineering background. We will test the hypothesis that acetone in breath can be readily trapped using aminooxy or hydrazine coatings and that the resulting adducts can be electrochemically oxidized at low oxidation potentials to obtain an electrochemical signal. Intensity and potential of the current generated through oxidation will provide qualitative and quantitative information about the levels of acetone in the breath sample. We propose the following 3 aims: Aim 1: Design and synthesize libraries of aminooxy- and hydrazine-containing coatings for carbonyl trapping. Aim 2: In vitro testing of coatings using acetone and potentiostat for electrochemical studies. Aim 3: Design a breathalyzer prototype. The primary impact of this proposal will be the proof-of-concept validation of an enzyme-free breathalyzer prototype for the diagnosis and management of diabetic ketoacidosis.