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

项目总结/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， 死亡率4- 5%。DKA病理是胰岛素缺乏的结果，这导致了过度生产 乙酰乙酸酯（AcAc）、β-羟基丁酸酯（BHB）和丙酮形式的酮体。电流 DKA的诊断策略依赖于i）用硝普钠试纸条进行尿液检测， AcAc，和ii）用酶电化学生物传感器进行血液测试以检测BHB。不幸的是， 这些测试与众所周知的化学限制有关，导致假阳性和假阴性 结果此外，患者认为尿液和血液测试是不愉快的、耗时的、侵入性的， 甚至是痛苦的这项拨款申请将集中在开发一种非侵入性和病人友好的 无酶电化学呼吸测醉器，其化学选择性地分析呼吸中的丙酮水平， DKA患者人们普遍认为，DKA期间产生的小尺寸酮体 使它们能够排放到支气管内腔中以挥发性有机物的形式呼出 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1250 ppm。在这项提案中，我们的目标是取代传统上使用的酶， 作为电化学生物传感器中的生物识别元件，具有我们的点击化学氨基氧基（-ONH 2）或 肼（-NHNH 2）涂层，已知其与羰基化合物快速、有效和化学选择性地反应， VOC，如丙酮。该项目建立在Laulhé集团的研究优势之上， 在合成和电化学羰基代谢物的选择性衍生化方面拥有丰富的经验 方法论和工程背景。我们将检验呼吸中的丙酮可以 容易使用氨氧基或肼涂层捕获，并且所得加合物可以电化学 在低氧化电位下氧化以获得电化学信号。电流的强度和电势 通过氧化产生的将提供定性和定量的信息， 呼吸样本我们提出以下三个目标： 目的1：设计合成含氨基氧和肼的羰基化合物涂层库 诱捕 目的2：使用丙酮和恒电位仪对涂层进行体外测试，用于电化学研究。 目标3：设计一个呼气测醉器原型。 该提案的主要影响将是无酶呼吸测醉器的概念验证 糖尿病酮症酸中毒的诊断和管理的原型。

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.