On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications.
On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications.
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DOI：
10.1021/ja304187r
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发表时间：
2012-08-01
影响因子：
15
通讯作者：
Corn RM
中科院分区：
文献类型：
--
作者：
Seefeld TH;Halpern AR;Corn RM
蛋白质微阵列是由双链DNA(DsDNA)微阵列通过一步、多路酶合成以芯片上微流控的形式制备的，然后用于表面等离子体共振成像(SPRI)的抗体生物传感测量。编码His标记的绿色荧光蛋白(GFP)或His标记的荧光素酶蛋白的dsDNA元件(表示为生成器元件)的微阵列被用于在表面RNA聚合反应中产生多个拷贝的信使RNA(MRNA)；然后，通过以微流控形式的无细胞蛋白质合成将所述信使RNA转录物翻译成蛋白质。组氨酸标记的蛋白质扩散到相邻的铜(II)-NTA微阵列元件(表示为检测器元件)，并被特异性地吸附。最终结果是芯片上、无细胞合成的蛋白质微阵列可以立即用于SPRI蛋白质生物传感。双元素格式大大减少了酶或蛋白质的非特异性吸附造成的任何干扰。用SPRI检测抗体、抗GFP和抗荧光素酶来验证蛋白质芯片的形成。这种方便的芯片上蛋白质微阵列制作方法可以在临床和研究应用中实现多路SPRI生物传感测量。
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.