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On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

机译:片从DNa微阵列经由耦合在体外转录和翻译的表面等离子体共振成像生物传感器应用蛋白质芯片的合成

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摘要

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.
机译:蛋白质微阵列是由双链DNA(dsDNA)微阵列通过单步多路酶促合成以芯片微流格式制备的,然后用于表面等离振子共振成像(SPRI)的抗体生物传感测量。编码带有His标签的绿色荧光蛋白(GFP)或带有His标签的荧光素酶蛋白的dsDNA元件的微阵列(表示为生成器元件)被用于在表面RNA聚合酶反应中创建信使RNA(mRNA)的多个副本。然后通过无细胞蛋白质合成以微流体形式将mRNA转录物翻译成蛋白质。 His标记的蛋白扩散到相邻的Cu(II)-NTA微阵列元件(称为检测器元件),并被特异性吸附。最终结果是蛋白质微阵列的芯片上无细胞合成,可立即用于SPRI蛋白质生物传感。双元素形式极大地减少了酶或蛋白质非特异性吸附的任何干扰。 SPRI测量用于检测抗GFP和抗萤光素酶抗体,以验证蛋白质微阵列的形成。这种便捷的芯片上蛋白质微阵列制造方法可在临床和研究应用中用于多重SPRI生物传感测量。

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