Paper-Based Device for Rapid Detection of Cerebrospinal Fluid Leakage

项目摘要 摘要脑脊髓液漏是耳鼻喉科手术中常见的并发症。它有 据估计，高达13.8%的内窥镜颅底手术会导致CSF泄漏。在急性情况下， 诸如磁共振成像（MRI）或计算机断层摄影（CT）的成像技术用于 考核临床高度怀疑的患者可能会被直接带到手术室， 管理，包括识别泄漏部位，并使用天然组织或生物相容性 修补受影响部位的材料。然而，在临床或术后检测CSF渗漏通常是困难的。 医院环境，因为术后患者有分泌物并不罕见，因此区分 来自含有CSF的那些的正常分泌物可能是具有挑战性的。未能识别和修复泄漏可能会 导致严重的并发症，如脑膜炎、脑干疝和死亡。目前没有 经过验证的，可用的测试，允许医生，谁是担心脑脊液泄漏，以廉价和非- 侵入性地排除泄漏的存在因此，医生必须回到相同的诊断模式 用于急性环境。然而，这些诊断测试的昂贵和侵入性使得它们 在一个看起来很好的病人身上很难证明。已经开发了替代方法， CSF渗漏检测，如β-2转铁蛋白电泳或酶联免疫吸附试验 （ELISA）;然而，由于成本高和获得结果的时间长，它们很少使用。最近，研究人员 研究了β-微量蛋白（β-TRANSPROIN，简称TRANSPROIN）的检测，发现它与β-2转铁蛋白在 CSF的敏感性和特异性。虽然在研究环境中使用，但这种蛋白质尚未用于 临床检测 该项目的主要目标是开发下一代快速、廉价和简单的诊断设备 用于检测脑脊液渗漏该装置将包括使用含水双- 相系统（ATPS）、用于信号放大的比色酶和快速侧流免疫测定 (LFA)用于检测。传统的LFA对于在相关的温度下检测BMPTP不够灵敏。 浓度的为了克服这一障碍，ATPS可用于将靶蛋白浓缩几倍。 在LFA检测之前的数量级。通过使用纸微流体，我们已经证明，我们的 该设备可以同时无缝地浓缩和检测目标蛋白。改善LFA 检测限更进一步，我们已经证明了使用ATPS作为一种新的方法的可行性， 顺序地将信号增强试剂输送通过检测区。我们将开发原型 我们的设备使用两种方法：一个“低挂水果”两阶段平台，和一个“高风险，高回报” 一级平台一旦完全开发，该设备将允许临床医生更迅速地检测和治疗CSF 泄漏，以及用于防止患者谁，否则似乎从接受昂贵和侵入性 研究和程序。此外，这种装置不限于耳鼻喉科领域，而是还可以 发现用于识别脊髓或地球仪的损伤，并可能在排除脑脊液泄漏中发挥作用。 术后神经外科患者，具有较低的预测试泄漏概率。

PROJECT SUMMARY   Cerebrospinal fluid (CSF) leak is a common complication of numerous procedures in otolaryngology. It has been estimated that up to 13.8% of endoscopic skull base surgeries result in CSF leaks. In the acute setting, imaging techniques such as magnetic resonance imaging (MRI) or computer tomography (CT) are used for assessment. Patients with high enough clinical suspicion may be taken directly to the operating room for management, which involves identifying the site of the leak and using either native tissue or biocompatible materials to patch the affected site. It is often difficult, however, to detect CSF leaks in clinical or postoperative hospital settings, as it is not unusual for postoperative patients to have secretions, and therefore distinguishing normal secretions from those containing CSF can be challenging. Failure to recognize and repair a leak can result in severe complications, such as meningitis, brainstem herniation, and death. Currently, there are no proven, available tests that allow a physician, who is concerned about a CSF leak, to cheaply and non- invasively rule out the presence of a leak. As a result, physicians must return to the same diagnostic modalities that are used in the acute setting. However, the costly and invasive nature of these diagnostic tests make them difficult to justify in a patient who seems otherwise well. Alternative methods have been developed for the detection of CSF leaks, such as beta-2 transferrin electrophoresis or enzyme-linked immunosorbent assay (ELISA); however, they are rarely used due to high cost and long time-to-result. More recently, researchers have looked into the detection of beta-trace protein (TP) and found it to be comparable to beta-2 transferrin in sensitivity and specificity for CSF. Although used in a research setting, this protein has yet to be used for clinical detection. The main goal of this project is to develop a next generation, rapid, inexpensive and simple diagnostic device for detection of CSF leaks. The device will incorporate a sample pre-concentration step using the aqueous two- phase system (ATPS), colorimetric enzymes for signal amplification, and a rapid lateral-flow immunoassay (LFA) for detection. The traditional LFA is not sensitive enough for the detection of TP at the relevant concentrations. To overcome this barrier, the ATPS can be used to concentrate the target protein by several orders of magnitude prior to LFA detection. By using paper microfluidics, we have demonstrated that our device can simultaneously and seamlessly concentrate and detect target proteins. To improve the LFA detection limit even further, we have demonstrated the feasibility of using the ATPS as a novel method of sequentially delivering signal enhancement reagents across a detection zone. We will develop the prototypes of our device using two approaches: a “low-hanging fruit” two-stage platform, and a “high-risk, high-reward” one-stage platform. Once fully developed, the device will allow clinicians to more rapidly detect and treat CSF leaks, as well as be used to prevent patients who otherwise appear well from receiving expensive and invasive studies and procedures. Furthermore, such a device is not limited to the field of otolaryngology, but may also find use in identifying injuries to the spinal cord or globe, and may have a role in ruling out a CSF leak in postoperative neurosurgical patients with a low pretest probability of having a leak.