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Chip-based detection of protein cancer markers.

机译：基于芯片的蛋白质癌症标记物检测。

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This dissertation outlines the design, fabrication, and optimization of microfluidic systems for the ultra-sensitive detection of proteins. In particular, it focuses on measuring the concentration of cancer markers in complex media. The Biobarcode Assay (BCA) was adapted and implemented in a microfluidic device in order to achieve this goal. The assay initially demonstrated by the Mirkin Group at Northwestern University, achieves extraordinary sensitivity through the use of dual functionalized gold nanoparticle probes. These particles are encoded with "barcode" DNA sequences that are unique to the target of interest and antibodies for attaching themselves to the target.; The assay protocol first binds the target protein to either to a magnetic microparticle or to the wall of a microfluidic channel using monoclonal antibodies. The gold probes are then introduced into the device and allowed to attach to the target protein. Once the sandwich is formed, the sample is purified and the barcode DNA is released from the captured nanoparticle probes. The presence of the target protein is determined by identifying the DNA sequence that is released. Because each nanoparticle probe carries several hundred DNA per protein binding event, there is substantial signal amplification.; Using this approach, a chip-based system was developed that achieved a 500 attomolar detection limit for prostate specific antigen (PSA). Further modification and optimization has led to multiplexed detection of PSA and HCG with 10 fM sensitivity. Improvements in the protocol have also reduced the chip-based assay time to below 90 minutes.; Efforts to reduce the complexity and cost of the system led to the investigation of an electrical detection scheme for identifying the barcode DNA. The detector consists of a gap between two electrodes that is patterned with single stranded DNA, complimentary to half of the target sequence. The functionalized gap is then exposed to the target sequence and gold nanoparticles functionalized with the other half of the complimentary sequence. The target DNA immobilizes the gold nanoparticles in the gap. Silver deposition, which is facilitated by these nanoparticles, bridges the gap and leads to conductivity changes. While detection of DNA using simple current measurement was possible in a microfluidic format, this work revealed that the BCA, in its current state, is incompatible with this technique. The reducing agents employed to release the barcode DNA from the nanoparticle probes destroy the gold electrodes, preventing the gap from being bridged.; A similar approach was then employed to directly detect protein targets. Instead of DNA, monoclonal antibodies were patterned in the gap. The detector was then exposed to the protein target and gold nanoparticles functionalized with polyclonal antibodies. The target protein was used to immobilize the nanoparticle probes and silver staining was once again employed to bridge the gap It was determined that this technique can be used to detect protein targets both in buffer and serum samples with a 100 pM sensitivity limit.
机译：本文概述了用于蛋白质超灵敏检测的微流控系统的设计，制备和优化。特别是，它专注于测量复杂培养基中癌症标志物的浓度。为了实现此目标，对生物条形码测定法（BCA）进行了修改并在微流体设备中实施。该方法最初由西北大学的Mirkin集团证明，通过使用双重功能化的金纳米颗粒探针获得了非凡的灵敏度。这些颗粒用感兴趣的靶标特有的“条形码” DNA序列和用于将其自身附着于靶标的抗体编码。该测定方案首先使用单克隆抗体将靶蛋白与磁性微粒或微流通道壁结合。然后将金探针引入装置并使其附着于靶蛋白。一旦形成三明治，就将样品纯化，并从捕获的纳米颗粒探针中释放条形码DNA。通过鉴定释放的DNA序列来确定靶蛋白的存在。因为每个纳米颗粒探针每个蛋白结合事件都携带数百个DNA，所以存在大量的信号放大。使用这种方法，开发了一种基于芯片的系统，该系统对前列腺特异抗原（PSA）的检测极限达到500 attomolar。进一步的修改和优化导致对PSA和HCG的多重检测具有10 fM的灵敏度。协议的改进也将基于芯片的分析时间减少到90分钟以下。为降低系统的复杂性和成本的努力导致了对用于识别条形码DNA的电检测方案的研究。检测器由两个电极之间的缝隙组成，该缝隙由单链DNA图案化，与靶序列的一半互补。然后将功能化的缺口暴露于靶序列，并用互补序列的另一半功能化金纳米颗粒。靶DNA将金纳米颗粒固定在间隙中。这些纳米颗粒促进的银沉积弥合了间隙并导致电导率变化。虽然可以以微流体形式使用简单的电流测量来检测DNA，但这项工作表明，处于当前状态的BCA与该技术不兼容。用于从纳米粒子探针释放条形码DNA的还原剂破坏金电极，防止间隙被桥接。然后采用相似的方法直接检测蛋白质靶标。代替DNA，在缝隙中图案化了单克隆抗体。然后将检测器暴露于蛋白质靶标和用多克隆抗体功能化的金纳米颗粒。目标蛋白用于固定纳米颗粒探针，银染再次用于弥合缺口。确定该技术可用于检测缓冲液和血清样品中的蛋白质靶标，灵敏度极限为100 pM。

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