Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, Massachusetts, USA.
Nat Protoc. 2011 Oct 20;6(11):1761-74. doi: 10.1038/nprot.2011.400.
This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3-4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml(-1).
本方案描述了用于制造条码水凝胶微球的核心方法，包括蛋白质靶标的捕获和标记以及用于多重检测的微粒的快速微流扫描。由聚乙二醇制成的多功能水凝胶颗粒是一种敏感、无固定化和生物惰性的悬浮阵列，可用于蛋白质的多重测量。每种颗粒类型都带有独特的图形代码，由微球的晶圆结构中的未聚合孔组成; 该代码用于识别通过颗粒的单独探针区域共价结合的抗体探针。蛋白质检测方案可分为三个步骤：(i) 通过微流控流延光刻以每小时 16,000 个颗粒的速度合成颗粒; (ii) 在 3-4 小时的测定中，使用抗体夹心技术在颗粒内捕获和标记蛋白质靶标; (iii) 流动扫描程序，以每秒 25 个颗粒的速度检测条码并定量相应的靶标。通过使用所描述的技术，可以可重复地合成单探针或多探针颗粒，并用于可定制的多重面板中，以在三个对数范围内测量蛋白质靶标，浓度低至 1 pg ml(-1)。
