Smartphone-based mobile detection platform for lung cancer detection in China

摘要 肺癌是中国的首发癌症和癌症死亡原因。在接下来的五年，归因于空气 污染和高烟草消费，中国可能会看到肺癌的发生增加与 据估计，每年有超过800,000例新诊断和700,000例死亡。尽管负担得起的癌症基因特定 中国的药物越来越多，但缺乏有效和负担得起的癌症基因诊断方法。 为了解决这一关键需求，我提出了一种新的廉价、基于智能手机的护理点系统，用于检测罕见的 体液中突变的等位基因使中国农村肺癌的液体活检成为可能。拟议的系统建立在我的 以前在最小仪器和非仪器分子诊断学方面的工作。我的系统会接受原始样本， 例如全血，处理样本，并提供快速检测结果。因为存在着大量丰富的 野生型(WT)-等位基因挑战罕见突变等位基因的检测，我的系统将包括一个样本浓缩步骤，该步骤 利用DNA引导的ArgAerte家族裂解酶来消化和耗尽WT等位基因，同时避免突变 感兴趣的等位基因。这之后将使用多肽进行环介导的等温扩增(LAMP) 核酸(PNA)钳选择性地扩增突变等位基因，但不扩增WT等位基因。用于突变的多重检测 对于等位基因，建议的系统将首先用可编程裂解酶丰富样本，然后让 将样品浓缩到一种新的两级多路等温放大过程(Penn-Ramp)，在以下情况下 点/缺失/插入突变，用PNA钳抑制Penn-Ramp第二代WT等位基因的扩增 舞台。扩增产物将通过生物发光记者和智能手机摄像头进行检测。一款定制的智能手机 应用程序将分析记录的信号；报告测试结果；并在将来将这些结果传输到患者的 医生和记录，并以非身份识别的形式，传输到云中进行时空监控，允许公共卫生官员 确定热点。我们的初步数据表明，我们的方法成功的可能性很高。 我制定了以下三个领域的培训计划：癌症流行病学、癌症诊断和治疗，以及 医疗器械的设计和制造。在这些领域中的每一个领域，我都从 K01咨询委员会成员。咨询委员会由以下领域的国际公认专家组成 工程学、癌症流行病学、肺癌精准医学、液体活检和微生物学已经组装在一起 并将定期开会，评估我的进展并提供指导。 本研究有可能极大地提高中国肺癌的同伴诊断和筛查水平，使 有效的治疗方法。该计划将使我能够通过实地工作、研究、课程学习和 合作，所有这些都将促进和加速我向全球卫生独立调查员的转变。

ABSTRACT Lung cancer is the leading incident cancer and cause of cancer death in China. In the next five years, attributed to air pollution and high tobacco consumption, China will likely see an increase in the occurrence of lung cancer with an estimated of over 800,000 new diagnoses and 700,000 deaths per year. Although affordable cancer genotype-specific drugs are increasingly available in China, effective and affordable diagnostics of cancer genotypes is lacking. To address this critical need, I propose a novel inexpensive, point-of-care smartphone-based system for detecting rare mutant alleles in body fluids to enable liquid biopsy of lung cancer in rural China. The proposed system builds on my prior work with minimally instrumented and un-instrumented molecular diagnostics. My system will accept a raw sample, such as whole blood, process the sample, and provide rapid test results. Since the presence of a large abundance of wildtype (WT)-allele challenges detection of rare mutant alleles, my system will include a sample enrichment step that utilizes DNA guided cleaving enzymes of the Argonaute family to digest and deplete WT alleles while sparing the mutant alleles of interest. This will be followed with a loop mediated isothermal amplification (LAMP) that utilizes peptide nucleic acid (PNA) clamp to selectively amplify mutant alleles, but not WT-alleles. For multiplex detection of mutant alleles, the proposed system will firstly enrich the sample with the programmable cleaving enzyme and then subject the enriched sample to a novel two stage, multiplexed isothermal amplification process (Penn-RAMP), in the case of point/deletion/insertion mutations, with PNA clamps to discourage amplification of WT alleles in Penn-RAMP's second stage. Amplicons will be detected with bioluminescent reporters and a smartphone camera. A custom smartphone application will analyze the recorded signal; report test results; and, in the future, transmit these results to the patient's doctor and records and, in de-identified form, to the cloud for spatiotemporal surveillance, allowing public health officials identify hotspots. Our preliminary data indicates that our approach has high likelihood of success. I have developed a training plan in the following three areas: cancer epidemiology, cancer diagnostics and therapy, and medical device design and fabrication. In each of these areas, I have identified coursework and mentorship support from members of the K01 Advisory Committee. The Advisory Committee composed of internationally recognized experts in engineering, cancer epidemiology, lung cancer precision medicine, liquid biopsy, and microbiology has been assembled and will meet periodically to assess my progress and provide guidance. This research has the potential to greatly improve companion diagnosis and screening of lung cancer in China and enable effective therapies. The program will enable me to obtain new skills through field work, research, coursework, and collaborations, all of which will promote and accelerate my transition to an independent investigator for global health.