PRADA: Portable Reusable Accurate Diagnostics with nanoAntennas for Multiplexed Biomarker Screening of Preterm Labor

项目摘要/摘要： 自发性早产(妊娠37周)是新生儿和婴儿的主要原因。 全球婴儿死亡率，约占所有婴儿死亡人数的60%。PTL的生理病理仍很差 由于与风险因素相关的异质性，包括产妇年龄、多胎 怀孕，以及其他并存疾病。确定PTL高危女性的现行临床标准 包括宫颈长度评估和宫颈阴道液中胎儿纤维连接蛋白的筛查。然而， 这些方法是不够的，而且往往无法确定可能早产的妇女。最近的病人 元数据研究已经确定了多个与PTL相关的生物标记物，但风险评分基于单个 生物标记物的阳性预测值很低，在预测PTL方面效果不佳。这个 拟议研究的科学前提是，临床上迫切需要准确的诊断 多路生物标志物检测功能，同时经济实惠，便于常规床边操作 对所有患者进行筛查。进一步通过使用 来自相同患者的相同样本类型将最大限度地减少试验间和试验内的差异，这仍然是 当前床边诊断中的挑战。 这项研究的目的是用一种创新的生物诊断产品Prada来解决这一未得到满足的需求， 它结合了高灵敏度、特异度、多重、使用低样本量和低成本。普拉达， 便携式可重复使用的纳米天线精确诊断，由磁性微珠捕获探头组成 带有多克隆抗体的功能化，以及带有条形码的近红外共振金纳米星“天线” 表面增强拉曼光谱检测生物标志物 (SERS)。Prada已经成功地展示了对3个PTL生物标志物的多重检测，其中包括α- 胎蛋白(AFP)、促肾上腺皮质激素释放激素(CRH)与粒细胞巨噬细胞集落刺激 妊娠患者血清中的GM-CSF因子。Prada达到了超灵敏的检测极限(LOD，1.7 pg/ml Prada显示，与商业Luminex和ELISA相比，Prada是风险分层的理想选择 低得多的LOD。此外，普拉达还可以重复使用(由于使用了磁珠)，从而降低了总体成本 允许&gt；15次使用单个微流控设备。这项工作将利用创新的普拉达来 定量检测包括C反应蛋白在内的6个预测PTL的血清标志物 C反应蛋白、甲胎蛋白、IL-10、IL-6、CRH和GM-CSF以Luminex和ELISA为基准(目标1)。此外，我们 将验证与18-35岁患者血清中的PradA的多路复用 单胎妊娠和孕龄19-36周组(目标2)。产妇风险分值将为 将患者分为高风险组和低风险组，并将评分与他们的 现有诊断以确定Prada的准确性。我们的中心假设是普拉达将使 强大的床边诊断功能，可准确预测PTL的发病，识别需要立即治疗的患者 治疗，并最大限度地减少对低风险人群的不必要干预。 Prada在临床上是可翻译的，因为金纳米颗粒已经在临床试验中(NCT00436410， NCT00356980)，并集成到商业化验(第一反应妊娠试验)中，SERS具有 在商用传感器(Oxonica、Renishaw Diagnostics等)中展示了实用性。这个有影响力的项目将 利用PI(Bardhan)在纳米颗粒诊断和SERS方面的专业知识，以及互补的 共同研究人员Leon Bellan博士(微流体)、Jeff Reese博士(PTL机制)和J. 迈克尔·牛顿(PTL患者治疗)。

Project Summary/Abstract: Spontaneous preterm labor (PTL, <37 weeks gestation) is the leading cause of neonatal and infant mortality globally, representing ~60% of all infant deaths. The physiopathology of PTL remains poorly understood due to the heterogeneities associated with the risk factors including maternal age, multiple gestation, and co-morbidities among others. Current clinical standard to identify women at high risk of PTL include cervical length assessment, and screening for fetal fibronectin in the cervicovaginal fluid. However, these approaches are inadequate and often fail to identify women who may deliver preterm. Recent patient metadata studies have identified multiple biomarkers correlated to PTL but risk scoring based on single biomarkers has shown low positive predictive values and have been ineffective in PTL prediction. The scientific premise of the proposed study is that there is an urgent clinical need for accurate diagnostics with multiplexed biomarker detection capabilities that are simultaneously affordable to facilitate routine bedside screening for all patients. Further by benchmarking the biodiagnostic against commercial assays using the same sample type from the same patients will minimize inter- and intra-assay variation, which remains a challenge in current bedside diagnostics. The objective of this study is to address this unmet need with an innovative biodiagnostic, PRADA, which combines high sensitivity, specificity, multiplexing, use of low sample volumes, and low cost. PRADA, portable reusable accurate diagnostics with nanoantennas, consists of magnetic microbeads capture probes functionalized with polyclonal antibodies, and near-infrared resonant gold nanostar “antennas” barcoded with Raman tags and labeled with peptides to detect the biomarkers via surface-enhanced Raman spectroscopy (SERS). PRADA has successfully demonstrated multiplexed detection of 3 PTL biomarkers including alpha- fetoprotein (AFP), corticotrophin releasing hormone (CRH) and granulocyte macrophage colony-stimulating factor (GM-CSF) in pregnant patient serum. PRADA achieved ultrasensitive limit of detection (LOD, 1.7 pg/ml GM-CSF) ideal for risk stratification, and when compared to commercial Luminex and ELISA, PRADA showed far lower LODs. Further, PRADA is also reusable (due to the use of magnetic beads), lowering the overall cost by allowing >15 uses of a single microfluidic device. This work will leverage innovative PRADA to quantitatively detect 6 serum biomarkers which are the most predictive of PTL including C-reactive protein (CRP), AFP, IL-10, IL-6, CRH, and GM-CSF benchmarked against Luminex and ELISA (Aim 1). Further, we will validate multiplexing with PRADA in serum of patients recruited for this study in the 18 – 35 years age group with singleton pregnancy and gestation age 19 – 36 weeks (Aim 2). A maternal risk score will be established categorizing patients into high and low risk groups, and the scoring will be compared to their existing diagnosis to determine the accuracy of PRADA. Our central hypothesis is PRADA will enable a robust bedside diagnosis that accurately predicts the onset of PTL, identifies patients who need immediate treatment, and minimizes unnecessary intervention for those at low risk. PRADA is clinically translatable as gold nanoparticles are already in clinical trials (NCT00436410, NCT00356980) and integrated in commercial assays (First response pregnancy tests), and SERS has demonstrated utility in commercial sensors (Oxonica, Renishaw Diagnostics etc.). This impactful project will leverage the expertise of the PI (Bardhan) in nanoparticle diagnostics and SERS, with the complementary expertise of co-investigators Dr. Leon Bellan (microfluidics), Dr. Jeff Reese (mechanisms of PTL), and Dr. J. Michael Newton (PTL patient treatment).