生物芯片技术 刘超 李世燕 谢宏林 2008.11.3

什么是生物芯片 生物芯片（Biochips）是90年代中期发展起来的一项尖端技术。一种微型多参数生物传感器。它通过在一微小的基片表面固定大量的分子识别探针，或构建微分析单元和系统，实现对化合物、蛋白质、核酸、细胞或其它生物组分准确、快速、大信息量的筛选或检测。 他是继大规模集成电路之后的又一次具有深远意义的科学技术革命。

生物芯片种类 ——按芯片上固定的生物材料分 生物芯片种类 ——按芯片上固定的生物材料分

Gene chips 是最早出现的一种生物芯片。 是指通过微阵列(Microarray)技术将高密度DNA片段阵列通过高速机器人或原位合成方式以一定的顺序或排列方式使其附着固相表面，以荧光标记的DNA探针，借助碱基互补杂交原理，进行大量的基因表达及监测等方面研究的最新革命性技术。

Protein chips 蛋白质芯片是一种高通量的蛋白功能分析技术，可用于蛋白质表达谱分析，研究蛋白质与蛋白质的相互作用，甚至DNA－蛋白质、RNA－蛋白质的相互作用，筛选药物作用的蛋白靶点等。

Lab chips 芯片实验室是以芯片为平台的微全分析系统，它是把生物和化学等领域所涉及的样品制备、生物与化学反应、分离与检测等基本操作单元集成到一块几平方厘米的芯片上，用以完成不同的生物或化学反应过程，并对其产物进行分析的一种技术。通俗言之，就是把实验室搬到芯片上。

优点：芯片上集成了各种不同的实验室单元技术，能够在短时间内分析大量的生物分子，准确获取样品中的大量信息，信息量是传统检测手段的成百上千倍。

生物芯片技术的优点 与传统的仪器检测方法相比： 高通量 微型化 自动化 高灵敏 成本低 防污染

生物芯片技术的应用 1、基因表达水平的检测 2、基因诊断 目前应用较多的领域，用基因芯片进行的表达水平检测可自动、快速地检测出成千上万个基因的表达情况，通过分析那些有表达差异的基因来达到研究目的。 2、基因诊断 从正常人的基因组中分离出DNA与DNA芯片杂交就可以得出标准图谱。从病人的基因组中分离出DNA与DNA芯片杂交就可以得出病变图谱。通过比较、分析这两种图谱，就可以得出病变的DNA信息。

3、药物筛选     目前,药物开发中的重大障碍是----如何分离和鉴定药的有效成份进而达到最佳治疗效果，基因芯片技术是解决这一障碍的有效手段。它能够大规模地筛选、通用性强，能够从基因水平解释药物的作用机理。 4、个体化医疗     临床上，不同的人对相同剂量的药物的反应不一样，主要是由于病人遗传学上存在差异。如果将这些基因突变部位的全部序列构建为DNA芯片，则可快速地检测病人是这一个或那一个或多个基因发生突变，从而可对症下药。

5、测序     用基因芯片进行的表达水平检测可自动、快速地检测出成千_卜万个基因的表达情况。 Mark chee等用含135000个寡核苷酸探针的阵列测定了全长为16.6kb的人线粒体基因组序列，准确率达99%。 6、生物信息学研究 使用基因芯片技术可以同时测定成千上万个基因的作用方式，几周内获得用其它方法需要几年才能得到的信息。它成为基因组信息学研究的主要技术支撑。

　生物芯片的发展趋势 Greater density Accelerated automation Cost reduction

Literature

Abstract A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signalswere consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.

　　HPV的传统检测方法

Principle Fig. 1. Experiment cycle. (a) The oligonucleotide probe was immobilized on the GMR sensor surface. (b) Target sample containing the HPV DNAs was applied to the sensor. Complementary DNAs were hybridized and stayed on the sensor surface. Non-complementary DNAs were washed away. (c) Solution containing magnetic particles was applied to the sensor. The streptavidin molecules on the magnetic particles bound to the biotin group on the free end of the double stranded DNA on the sensor surface.

Experimental Procedure 传感器的制备 聚核苷酸探针的制备及固定于传感器 目标DNA的生物素酰基化 磁性颗粒的功能化 加样 目标DNA与磁性颗粒结合 检测

Fig. 2. A typical measurement cycle and signal curve Fig. 2. A typical measurement cycle and signal curve. (A) The chip was placed in the measurement setup and signal was recorded to establish a base signal level. (B) Phosphate buffer solution (PBS, pH 7.2) was applied to measure the “water level” of the sensor. (C) PBS was removed and the chip was left in dry condition. (D) The MACS particles were added to the chip. (Interim between D and E) The chip was rinsed with DI water and dried. (E) The chip was left in dry condition to measure the final signal level.

　Results

Fig. 3. Signal curves of four assays in experiment phase I Fig. 3. Signal curves of four assays in experiment phase I. The phase I experiment used four chips to detect HPV DNA in the same sample. There are four probes on each chip, corresponding to HPV16, 18, 33 and 45 strains. The target sample contains HPV16 and 45, confirmed by pyrosequencing. The concentration of each type of DNA was ∼10 nM. The purpose of phase I was to test the stability and signal consistency of GMR sensors.

Conlusion GMR生物芯片用于HPV的基因定型的准确率达到90%以上,并且可以运用于各种涉及到基因检测的医学诊断。 相对于其他类型的生物芯片,这种新型GMR 生物芯片具有磁标记稳定,灵敏度高,与IC 工艺兼容,适用于自动化分析,不依赖于昂贵、高精度的光学测量系统、易于小型化等优势,同时利于减小甚至消除背景噪声,而且可以进行单片多通量检测。

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