Aptamer-Based Nanotechnology for Detection of Plasma Melanoma Markers

描述（由研究者提供）：虽然在理解各种癌症的分子事件方面取得了重大进展，但它们转化为治疗方法的能力有限，而且早期检测仍然是成功治疗的基石。因此，人们对识别个体癌症的敏感和特异性标志物非常感兴趣，特别是在血液中。两个深入研究的领域是循环肿瘤细胞（CTC）的鉴定和定量，以及血浆中敏感和特异性肿瘤标志物的鉴定。我们正在开发一种基于芯片的RNA传感器平台，使用衍生的纳米线（NW）进行CTC的早期检测。该传感器平台依赖于杂交“夹心”的形成，具有优异的特异性（对于2个识别事件的单核苷酸错配辨别）以及灵敏度（数据表明每个单个“夹心”结合诱导约1 kHz的共振频率偏移，这是容易测量的）。该平台是以“自下而上”的方式构建的，我们已经证明，用反义寡核苷酸衍生的纳米线在芯片上整合后完全保留了它们的功能。在这里，我们提出用修饰的DNA适体衍生化NW，特异于临床相关的血浆黑色素瘤肿瘤标志物S100 B和MIA，扩展我们的基于芯片的传感器的功能并显著扩展其多功能性，同时保留多重能力和精致的灵敏度/特异性。本申请的具体目的包括：1）构建并表达S100 B和MIA，并在体外纯化它们。2)使用我们的无引物方案进行适体选择，直到为每种蛋白质鉴定出约5-10个高亲和力适体，并表征结合特性。对适体进行各种修饰以增强稳定性/核酸酶抗性并重新测试结合参数。3)用纯化的S100 B/MIA对点样微阵列的荧光“夹心”结合进行成对测试，以确定识别不同表位的最佳适体对（Ap 1和Ap 2）。4)使用微阵列上的最佳Ap 1/Ap 2对检测加标到分级对照血浆样本中的不同浓度的S100 B和MIA，以确定非特异性结合和~检测限。5)用Ap 1衍生化NW，并使用以下物质进行夹心结合测定：a）荧光标记的Ap 2;和B）与50 nm AuNP共价连接的Ap 2，使用如4）中的加标血浆样品。6)将电流体存款衍生的NW沉积在芯片上，进行如4）中的夹心结合测定，并测量单个NW的共振频率的偏移：基准与常规ELISA。7)使用常规的基于ELISA的试剂定量黑色素瘤患者血浆中的S100 B和MIA水平，用于基准，与来自相同患者样本的CTC水平进行比较，并与AJCC分期相关。将剩余的样品储存起来，以便随后使用基于芯片的传感器进行测试。 公共卫生相关性：该应用专注于检测黑色素瘤患者血液中的肿瘤标志物，旨在开发一种基于纳米技术的平台，在癌症诊断中具有广泛的适用性。待利用的方法可以应用于任何癌症，其中合适的标记物是可用的，将容易地适应于新的标记物，因为它们被发现。该平台旨在同时检测多种不同的肿瘤标志物，我们设想将其开发为早期检测所有主要癌症类型的筛查工具。

DESCRIPTION (provided by investigator): Although major advances have occurred toward understanding the molecular events in various cancers, their translation into therapies has with few exceptions been limited, and early detection remains the cornerstone of successful treatment. Consequently, there is a great deal of interest in identifying sensitive and specific markers for individual cancers, particularly in blood. Two areas of intense investigation are identification and quantification of circulating tumor cells (CTCs), as well as identification of sensitive and specific tumor markers in plasma. We are developing a chip-based RNA sensor platform using derivatized nanowires (NWs) for early detection of CTCs. This sensor platform, which relies on formation of a hybridization "sandwich", possesses excellent specificity (single nucleotide mismatch discrimination for 2 recognition events) as well as sensitivity (data indicate each single "sandwich" binding induces a resonance frequency shift of ~ 1 kHz, which is easily measurable). The platform is being constructed in "bottom-up" fashion, and we have shown that NWs derivatized with antisense oligonucleotides fully retain their functionality after integration on chip. Here we propose to derivatize NWs with modified DNA aptamers, specific for the clinically relevant plasma melanoma tumor markers S100B and MIA, extending the functionality of our chip- based sensor and considerably expanding its versatility while retaining multiplex capabilities and exquisite sensitivity/specificity. The Specific Aims of the application encompass: 1) Make constructs and express S100B and MIA and purify them in vitro. 2) Perform Aptamer selection using our Primer-Free protocol until ~ 5-10 high-affinity aptamers are identified for each protein, and characterize binding properties. Introduce various modifications to aptamers to enhance stability/nuclease resistance and re-test binding parameters. 3) Perform pair-wise testing of fluorescent "sandwich" binding of spotted microarrays with purified S100B/MIA to determine the optimal pair of aptamers (Ap1 and Ap2) recognizing distinct epitopes. 4) Use the optimal pairs of Ap1/Ap2 on microarrays to detect various concentrations of S100B and MIA spiked into fractionated control plasma samples, to determine non-specific binding and ~ limits of detection. 5) Derivatize NWs with Ap1, and perform sandwich binding assays with: a) fluorescently-labeled Ap2; and b) Ap2 covalently linked to 50-nm AuNPs, with spiked plasma samples as in 4). 6) Electrofluidically deposit derivatized NWs on chips, perform sandwich binding assays as in 4), and measure shifts in resonance frequency of individual NWs: Benchmark vs. conventional ELISA. 7) Quantify S100B and MIA levels in plasma from melanoma patients using conventional ELISA-based reagents for benchmarking, compare with CTC levels from the same patient samples, and relate to AJCC Stage. Bank the balance of the samples for subsequent testing with the chip-based sensor. PUBLIC HEALTH RELEVANCE: This application, which is focused on detection of tumor markers in blood from melanoma patients, seeks to develop a nanotechnology-based platform with wide-ranging applicability in cancer diagnostics. The methods to be utilized can be applied to any cancer for which suitable markers are available, will be easily adaptable to new markers as they are discovered. The platform is designed to allow simultaneous detection of a number of different tumor markers, and we envision its development as a screening" tool for early detection of all major cancer types.