Sample-free onsite detection of cocaine using microneedles via laser-treated skin

 描述（由申请人提供）：滥用可卡因、大麻和海洛因等非法药物仍然是全国严重的公共卫生问题，并与惊人的经济和社会后果有关。可卡因仍然是医院急诊中记录最多的非法药物，也是吸毒相关死亡的主要原因， 美国的由于高达23%的致命受伤司机在药物测试中发现阳性结果，国家药物管制政策办公室强调了美国对药物驾驶的主要关注。尿液分析是最常见的可卡因测试类型，尽管尿液收集对其实用性提出了挑战。还有其他基于生物可卡因的可卡因测试，如血液和唾液测试，但不实用，因为血液样本需要高级别的医疗管理人员和专门的分析设备。另一种替代方法是使用表面改性微针（MN）通过皮肤进行微创诊断。然而，由于皮肤中大多数血液生物标志物的浓度太低而不能被MN阵列捕获，因此该测试具有低灵敏度和高变化。在这里，我们提出了一种新的探针MN为基础的测定结合使用短暂的激光照射在一个微小的皮肤区域积累血液生物标志物，如可卡因到上层真皮。该样品累积方法使得能够使用插入激光处理的皮肤中的探针-MN容易地原位检测和定量生物标志物。STTR第一阶段的目标是表征和验证探针-MN阵列在体外和体内的灵敏度和特异性，并将检测可靠性与血浆和尿液样本中可卡因的常规分析进行比较。具体目的如下：目的1：确定可卡因特异性结合MN探针的最佳设计，并进行可卡因的体外检测。我们将开发一个方案的表面修饰的MN与Cy 3标记的可卡因特异性适体和Cy 5标记的中和剂。将根据体内试验的迭代反馈优化MN的长度（范围为50 - 300 um）。MN阵列的灵敏度、特异性和响应时间将通过体外试验进行评价。目的2：验证可卡因在小鼠和大鼠皮肤上检测的可行性和一致性。可卡因特异性MN探针将在小鼠和大鼠中进行评估，以证明所提出的微创MN可卡因检测结合使用临床安全剂量的激光在皮肤上的短暂照射的可行性。将根据可卡因的常规血浆和尿液分析测定确定灵敏度、特异性和可靠性。整个STTR项目的最终目标是开发和商业化微创，无样本，原位可卡因检测工具包，包括 设备和可卡因特异性探针-MN阵列，用于在社区、工作场所或急诊室和诊所中对人体进行快速现场可卡因检测。

 DESCRIPTION (provided by applicant): The abuse of illicit drugs such as cocaine, marijuana and heroin remains a critical public health concern throughout the country and is associated with staggering economic and social consequences. Cocaine remains the most frequently recorded illicit drug in emergency hospital visits and the leading cause of drug- related deaths in the United States. Since as high as 23% of fatally injured drivers were found positive results in drug tests, the Office of National Drug Control Policy emphasizes the major concern of drugged driving in the United States. Urinalysis is the most common type of cocaine test, though urine collection has challenged its practicality. There are other biological specimen-based cocaine tests that have been explored such as blood and saliva tests but are not practical since blood samples need high-level medical administrators and specialized analytic equipment. Another alternative is the minimally invasive diagnosis through skin using surface modified microneedles (MNs). However, since concentrations of most blood biomarkers in the skin are too low to be captured by the MN array, the test suffers from low sensitivity and high variation. Here we propose a novel probe-MN-based assay in conjunction of using brief laser illumination on a tiny skin area to accumulate blood biomarker, such as cocaine into the upper dermis. This sample accumulation method enables the biomarker to be readily detected and quantified in situ using the probe-MNs inserted into the laser- treated skin. The objective for the STTR Phase I is to characterize and validate sensitivity and specificity of the probe-MN array in vitro and in vivo, and compare detection reliability with conventional analysis of cocaine in plasma and in urine samples. The specific aims are as follows: Aim 1: Determine optimal design of cocaine-specific binding MN probe and characterize cocaine assay in vitro. We will develop a protocol of surface modification of MNs with Cy3-labeled cocaine-specific aptamer and Cy5- labeled neutralizer. Length of the MNs ranging from 50 - 300um will be optimized based on iterative feedback from in vivo test. Sensitivity, specificity, and response time of the MNs array will be evaluated by in vitro tests. Aim 2: Demonstrate the feasibility and consistence of upper dermal cocaine detection in mice and rats. The cocaine-specific MN probe will be evaluated in mice and rats to demonstrate feasibility of the proposed minimally invasive MN-based cocaine detection in conjunction of using a brief illumination of a clinically safe dose of laser on the skin. Sensitiviy, specificity, and reliability will be determined with respect to conventional assays of plasma and urinalysis of cocaine. The ultimate goal of the entire STTR project is to develop and commercialize the minimally invasive, sample- free, in situ cocaine detection toolkit consisting of device and cocaine-specific probe-MN arrays for prompt onsite cocaine detection in humans in community, work places, or emergency rooms and clinics.