Aptamer-Based Nanotechnology for Detection of Plasma Melanoma Markers

描述（由研究者提供）：尽管在了解各种癌症的分子事件方面取得了重大进展，但将其转化为治疗方法的过程中几乎没有例外，早期发现仍然是成功治疗的基石。因此，人们对识别个别癌症的敏感和特异性标记物，特别是血液中的标记物非常感兴趣。两个领域的深入研究是循环肿瘤细胞（CTCs）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，使用衍生纳米线（NWs）进行ctc的早期检测。该传感器平台依赖于杂交“三明治”的形成，具有优异的特异性（对2个识别事件的单核苷酸错配辨别）和灵敏度（数据表明每个“三明治”结合引起约1 kHz的共振频移，易于测量）。该平台正在以“自下而上”的方式构建，我们已经证明，反义寡核苷酸衍生的NWs在芯片上集成后完全保留其功能。在这里，我们建议用修饰的DNA适体衍生NWs，特异性针对临床相关的血浆黑色素瘤肿瘤标记物S100B和MIA，扩展我们基于芯片的传感器的功能，并大大扩展其多功能性，同时保留多重功能和精致的灵敏度/特异性。申请的具体目的包括：1)制备S100B和MIA的构建物并进行体外表达和纯化。2)使用我们的无引物协议进行适配体选择，直到为每个蛋白质鉴定出约5-10个高亲和力适配体，并表征其结合特性。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)对斑点微阵列与纯化S100B/MIA的荧光“三明治”结合进行配对测试，以确定识别不同表位的最佳适配体对（Ap1和Ap2）。4)利用微阵列上的最佳Ap1/Ap2对检测不同浓度的S100B和MIA加入到分离的对照血浆样品中，确定非特异性结合和检测限。5)用Ap1衍生NWs，并使用：a)荧光标记的Ap2进行夹心结合试验；b) Ap2共价连接到50 nm的AuNPs，与加尖血浆样品（如4）。6)电流体沉积衍生的NWs在芯片上，执行三明治结合测定（如4），并测量单个NWs的共振频率变化：基准与常规ELISA。7)使用传统的elisa试剂定量黑色素瘤患者血浆中的S100B和MIA水平作为基准，与来自同一患者样本的CTC水平进行比较，并与AJCC分期相关。将样品的余额存入银行，以便随后使用基于芯片的传感器进行测试。

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.