Smartphone Enabled Point-of-Care Detection of Serum Markers of Liver Cancer

摘要 肝细胞癌(HCC)是全球第六大常见癌症，在中低度和 中等收入国家(LMIC)。仅中国一人就占到了肝癌诊断的50%。手术切除是 主要也是唯一的治疗选择，但只有30%-40%的新诊断患者是此类患者的候选对象 治疗。这是因为肝细胞癌的发展通常是沉默的，诊断相对较晚， 当疾病已经转移或长得太大而不能手术切除时。因此，早期诊断 是获得积极临床结果的关键，因为在早期阶段-当肿瘤很小和 非转移性--将导致受影响患者的治愈率显著增加。在这一假设的推动下， 这项建议的目标是开发一种基于血液的肝细胞癌护理点检测(POCT) (肝细胞癌)。我们开发的POCT，“D4 POCT”，非常适合LMIC的基于血液的检测 设置。D4 POCT使用聚合物刷涂抗体(Ab)微阵列，几乎没有 背景结合，产生定量结果，并在30分钟内达到皮摩尔灵敏度。全 试剂是喷墨打印的，储存在D4 POCT盒上，不需要冷藏。在直接 将指纹血样应用到试剂盒上，自动捕获和检测分析物，生成 一种可量化的信号，通过将盒式磁带放在一个小设备中获得，该设备以磁性方式连接到SMART 手机，它通过车载应用程序对微阵列进行图像和分析。我们将开发多路D4 POCT 可同时检测甲胎蛋白、Dickkopf-1和骨桥蛋白 将为肝细胞癌的诊断提供较高的敏感性和特异性。我们选择的标记物是基于最近的临床 有证据表明，AFP、DKK-1和OPN作为一个多路复用板同时测量 为肝细胞癌的早期准确诊断提供了一种有前景的策略。这一建议的创新之处在于 几十年来首次完全自下而上的重新设计临床诊断的“黄金标准”--三明治 免疫分析-作为一种独立的护理点微阵列分析，可以通过 智能手机。该项目将由浙江杜克大学的一个调查小组进行 省人民医院和我们的行业合作伙伴，Immoor，一家领先的诊断公司， 获得了这项技术的权利。在杜克大学通过具体会议初步验证D4 POCT之后 性能里程碑，我们将展示在LMIC中使用的技术功能和临床潜力 设置在浙江省人民医院在广州中国使用患者衍生的手指棒 血样。该项目的影响是广泛的：超越了一种新的、低成本和临床验证的 用于肝癌的护理点诊断技术，这项诊断技术可用于针对任何 可获得抗体对的分析物，因此广泛适用于用于以下目的的所有免疫分析 癌症或其他疾病的诊断和预后。

ABSTRACT Hepatocellular cancer (HCC) is the sixth most common cancer worldwide, and is highly prevalent in low- and middle-income countries (LMIC). China alone accounts for >50% of HCC diagnoses. Surgical removal is the primary and only curative option, yet only 30-40% of newly diagnosed patients are candidates for such treatment. This is because the development of HCC is usually silent and diagnosis occurs relatively late, when the disease has either metastasized or grown too large for surgical removal. Therefore, early diagnosis is the key to achieving positive clinical outcomes, as removal at early-stage —when the tumor is small and not metastatic— would result in a marked increase in cures in affected patients. Motivated by this hypothesis, the objective of this proposal is to develop a blood-based point-of-care test (POCT) for hepatocellular cancer (HCC). The POCT we have developed, the “D4 POCT”, is ideally suited for blood-based testing in LMIC settings. The D4 POCT uses polymer brush-coated antibody (Ab) microarrays that have virtually no background binding, yields quantitative results, and achieves picomolar sensitivity within 30 minutes. All reagents are inkjet-printed and stored on D4 POCT cassettes, which do not require refrigeration. Upon direct application of fingerstick blood onto a cassette, analyte capture and detection occur automatically, generating a quantifiable signal obtained by placing the cassette in a small device that magnetically attaches to a smart phone, which images and analyzes microarrays via on-board App. We will develop a multiplexed D4 POCT capable of simultaneously detecting alpha-fetoprotein (AFP), dickkopf-1 (DKK-1) and osteopontin (OPN) that will provide high sensitivity and specificity for HCC diagnosis. Our choice of markers is based on recent clinical evidence showing that the simultaneous measurement of AFP, DKK-1, and OPN as a multiplexed panel provides a promising strategy for early and accurate diagnosis of HCC. The innovation of this proposal is the first complete bottom-up redesign in decades of the “gold standard” clinical diagnostic—the sandwich immunoassay—as a self-contained point-of-care microarray assay that can be imaged and interrogated by a smart phone. This project will be carried out by a consortium of investigators from Duke University, Zhejiang Provincial People's hospital and our industry partner, Immucor, a leading diagnostics company that has acquired the rights to this technology. After initial validation of the D4 POCT at Duke by meeting specific performance milestones, we will demonstrate technical functionality and clinical potential for use in LMIC settings at the Zhejiang Provincial People's hospital in Guangzhou China using patient-derived finger-stick blood samples. The impact of this project is broad: beyond a new, low-cost, and clinically validated technology for the point-of-care diagnosis of HCC, this diagnostic technology can be utilized to target any analyte for which Ab pairs are available, and is hence broadly applicable to all immunoassays used for diagnosis and prognosis for cancer or other diseases.