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目的:在传统基因芯片技术基础上,应用生物传感器技术,研制一种能将基因芯片信号原位放大后达肉眼判读灵敏度,无需专用基因芯片检测仪器就可使用,易于在基层医疗单位推广的薄膜生物传感器基因芯片诊断系统.方法:在薄膜生物传感器基片基础上,经过表面化学处理,使特定的基因捕获探针在传感器表面固定,形成特定检测目的生物传感器基因芯片.并以乙肝病毒YMDD区的特异序列设计为例,通过特定的YMDD区的捕获探针,以矩阵的形式点样于传感器芯片表面来实现本系统.同时,纳米金标记的检测探针取代了传统芯片中的荧光标记探针.扩增后的目的PCR片段与捕获探针、生物素标记探针、链亲蛋白纳米金探针进行反应,最后得到探针-生物素-链亲蛋白-纳米金复合物,并在芯片表面经生物传感器芯片将信号原位放大,获得肉眼观的芯片信号并进行分析,完成芯片诊断.以乙型肝炎病毒YMDD突变为例,观察该芯片系统对临床诊断标本诊断的可靠性.结果:基因芯片的检测信号经生物传感器原位放大后能肉眼判读或借助普通数码照像机或计算机扫描,根据信号出现的特定位置即可确定突变的类型.且该生物传感器基因芯片系统信噪比高,在人工合成的寡核苷酸及临床血清的检测中,均可实现生物芯片阴阳性信号完全的有或无的判读;临床血清标本检测证实,使用该传感器芯片系统对前期经过测序确定为YMDD突变的23份临床血清结果与测序结果完全一致.结论:薄膜生物传感器基因芯片集纳米材料、生物传感器技术及原位放大技术为一体,实现了信号的肉眼判读,具有通用性高,准确可靠,无需大型设备,易于在基层医疗单位推广使用.
OBJECTIVE: On the basis of traditional gene chip technology, the application of biosensor technology to develop a can amplify the signal of the gene chip in situ amplification of the sensitivity of the naked eye interpretation, without special gene chip detection equipment can be used easily spread in the primary medical units of the film Biosensor gene chip diagnostic system.Methods: Based on the thin film biosensor substrate, the specific gene capture probe was immobilized on the surface of the sensor through the surface chemical treatment to form a specific biosensor gene chip for detection purpose, and the hepatitis B virus YMDD region For example, the capture probe in a specific YMDD region, dotted on the sensor chip surface in the form of a matrix to realize the system.At the same time, the gold-labeled detection probe replaced the fluorescent labeled probe in the traditional chip The amplified target PCR fragment is reacted with a capture probe, a biotin-labeled probe and a chain-on protein nanogold probe to finally obtain a probe-biotin-streptavidin-nanogold complex, The surface of the biosensor chip amplifies the signal in situ to obtain a macroscopic view of the chip signal and analyze to complete the chip diagnosis. Hepatitis B virus YMDD mutation was used as an example to observe the reliability of the chip system in the diagnosis of clinical diagnostic specimens.Results: The detection signal of gene chip can be read by the naked eye or scanned by ordinary digital camera or computer through the biosensor in situ amplification, According to the specific position of the signal, the type of mutation can be determined, and the biosensor gene chip system has high signal-to-noise ratio, which can achieve both positive and negative signal of the biochip in the detection of synthetic oligonucleotide and clinical serum With or without interpretation of the clinical serum samples confirmed that the use of the sensor chip system on the pre-sequencing identified as YMDD mutations in 23 clinical serum results and sequencing consistent with the results.Conclusion: The film biosensor gene chip set of nano-materials, biosensors Technology and in situ amplification technology as a whole, to achieve the naked eye signal interpretation, with high versatility, accurate and reliable, without large equipment, easy to promote the use of primary health care units.