CN100424186C - High sensitivity biosensor gene chip and clinical diagnosis technology - Google Patents

High sensitivity biosensor gene chip and clinical diagnosis technology Download PDF

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CN100424186C
CN100424186C CN 200610024197 CN200610024197A CN100424186C CN 100424186 C CN100424186 C CN 100424186C CN 200610024197 CN200610024197 CN 200610024197 CN 200610024197 A CN200610024197 A CN 200610024197A CN 100424186 C CN100424186 C CN 100424186C
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nucleic acid
probe
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CN1850989A (en
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宋家武
杨又春
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宋家武
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Abstract

本发明涉及一种高灵敏度生物传感器基因芯片及临床诊断技术。 The present invention relates to a high-sensitivity biosensor microarray and clinical diagnostics. 应用纳米材料探针技术和光学传感器技术,实现了检测信号的放大,达到肉眼判读的水平,从而突破传统基因芯片技术在检测中需要使用激光扫描仪等大型设备的瓶颈限制,基因芯片无需专用设备而可广泛使用。 Nanomaterials probe technology and optical sensor technology, the amplification of the detection signal, to the naked eye interpretation level to break the bottleneck of the conventional microarray techniques require the use of large equipment like laser scanner in the detection, the microarray without special equipment and it can be widely used.

Description

一种高灵敏度生物传感器基因芯片及临床诊断技术 A high-sensitivity biosensor clinical diagnostics and gene chip

技术领域 FIELD

本发明属于生物基因技术和生物传感器领域,涉及-一种基因芯片技术及临床疾病诊断技术及其衍生的各种基因诊断产品。 The present invention belongs to the field of gene technology and biosensors biological relates to - one gene chip technology and clinical diagnosis techniques and diagnostic products derived from various genes.

背景技术 Background technique

基因芯片技术是二十世纪九十年代发展起来的一项高新生物技术(可参见 Gene chip technology is developed in the nineties of the twentieth century, a high-tech biotechnology (see

Collins, FS,」ZiefK/ o/jcAei/w/e gaw/ wwder 6wc/g"., fAe Gewo附e /Vq/ecf /?。wes ,卿6/,M勿.Proc Natl Acad Sci USA, 1995. 92(24): p. 10821-3; Pease, AC,等, i/g/7Ngewe;Y//ed o//gowwc/eo"We flAr。 Collins, FS, "ZiefK / o / jcAei / w / e gaw / wwder 6wc / g"., FAe Gewo attached e / Vq / ecf /?.wes, Qing 6 /, M do .Proc Natl Acad Sci USA, 1995 .. 92 (24): p 10821-3; Pease, AC, etc., i / g / 7Ngewe; Y // ed o // gowwc / eo "We flAr. ys井r napiW sequence awfl/yw's. Proc Natl Acad Sci USA, 1994. 91(11): p. 5022-6;和Shalon, D" SJ Smith和PO Brown, ys well r napiW sequence awfl / yw's Proc Natl Acad Sci USA, 1994. 91 (11): p 5022-6; and Shalon, D "SJ Smith and PO Brown,..

'/7worejce/^/?a"o6e /iy6r/<i/za"ow. Genome Res, 1996. 6(7): p. 639-45), 其基本原理是将大量的靶基因片段有序地,较高密度地排列在特定的载体基质(如硅、 玻璃、纤维或尼龙膜上),称为基因芯片(微矩阵法,Microarray)或在玻片, 或应用半导体蚀刻技术,直接原位合成寡核苷酸探针。 '/ 7worejce / ^ / a "o6e / iy6r / <i / za" ow Genome Res, 1996. 6 (7):?.. P 639-45), the basic principle is the large amount of target gene fragment orderly high density arranged in the particular carrier matrix (such as silicon, glass, or nylon membranes), called gene chip (microarray method, the microarray) or slide, or a semiconductor etching technique application directly in situ synthesis oligonucleotide probes. 前者最早由美国Stanford 大学P.Brown发明U995年),后者由美国Affymetrix公司拥有其专利。 The former was first proposed by Stanford University P.Brown invention U995年), which owns the patent by the US company Affymetrix. 由于其具有通量高(按需要检测数十至数万条基因),平行分析(在同一条件下进行)及核酸杂交所具有的高度特异性(可鉴别单个核苷酸的改变)等优点, 一经发现即为世界各国广泛应用。 Because of its high flux (as required to detect several tens of thousands of bar gene), parallel analysis (carried out under the same conditions) and highly specific nucleic acid hybridization has (single nucleotide change can be identified), etc., upon discovery that is widely used around the world. 基因芯片技术已成为基因组学和后基因组学研究中的基本技术。 Microarray technology has become a basic technical school and post-genomics research in the genome.

早期的基因芯片技术主要用于高通量,大规模研究基因功能的需求而产生,但随着基因芯片技术的进步,人们惊喜发现,除原有的作用外,基因芯片在各种基因相关性疾病,如传染病和遗传性疾病的诊断、基因多态性分析等具 Early gene chip technology is mainly used for high-throughput, large-scale studies of gene function and generate demand, but with the advances in gene chip technology, people are pleasantly surprised to find, in addition to the existing role of gene chips in a variety of genetic relatedness diseases, such as diagnosis of infectious and genetic diseases, analysis of gene polymorphism and the like with

有独特的应用前景: Unique prospects:

l.其检测片段为各类致病基因片段,由于探针量大,通量高,显著提高检测效率;可一次完成多种疾病和同一疾病状态多种不同状态或/和遗传背景的检测: . L which detects all kinds of pathogenic gene fragment is a fragment, since the probe volume, high throughput, significantly improve the detection efficiency; can be a complete detection of various diseases and disease states of the same plurality of different states and / or genetic background:

2. 检测中应用具有高度特异性的核酸杂交技术,其特异性可以分辨出一个核酸的改变,因此,具有最高精度的特异性。 2. Application of detecting highly specific nucleic acid hybridization techniques which change the specificity of a nucleic acid can be distinguished, and therefore, with the most precision specificity.

3. 检测直接应用核酸,只需痕量的标本即可完成诊断,如对病原体检测, 无需免疫反应阶段,在感染初期即可完成诊断; 3. The direct application of nucleic acid detection, only a trace amount of samples to complete the diagnosis, as the detection of pathogens, without stages of the immune response, can be completed in the early diagnosis of infection;

4. 自动化程度高,可重复性好,可适应于大规模检测及应用。 4. High degree of automation, repeatability, and can be adapted to large-scale testing and application.

然而,传统的基因芯片技术的检测,是基于荧光标记探针的荧光发光来进行相应的检测的。 However, the conventional detection of gene chip technology, and is based on the fluorescent light-emitting fluorescently labeled probes corresponding to detection. 其信号弱,必须经过激发,才能发光,需要昂贵的激光扫描设备。 Its signal is weak, must be excited to emit light, it requires an expensive laser scanning devices. 在临床检测应用过程中,由于检测单位需要投资购置大型的激光扫描设备, 一方面,显著增加了进行相关检测的投资成本,另一方面,亦直接增加了应用的成本,显著地限制了该技术的应用范围,并成为该技术在临床推广中的瓶颈问题。 In the clinical application of the detection process, since the detection units need to invest in large-scale laser scanning device, on the one hand, a significant increase in investment costs related to detection, on the other hand, also directly increase the cost of applications, significantly limits the technology range of applications, and become a bottleneck in the technology in the clinic. 使这-对疾病诊断具有高度特异性和高通量的检测技术,受检测成本的限制,无法在临床实践中得到广泛应用。 Make this - with a highly specific detection technology and high-throughput diagnosis of disease, detection is limited by cost, not widely used in clinical practice. 但其潜在的社会进步和巨大产业价值,促使人们不断地发明新的技术和方法。 But its potential social progress and great industrial value, prompting people are constantly inventing new techniques and methods.

发明内容 SUMMARY

本发明的目的之--是,应用纳米材料标记技术取代传统的荧光探针,改变探针的物理光学特性,使探针在可见光谱范围内进行检测,解决荧光检测需要使用大型设备的不足,从而在基因芯片应用过程中无需大型激光扫描设备, 便于基因芯片技术广泛应用。 The object of the present invention - is substituted nanomaterials labeling techniques conventional fluorescent probes, changes in the physical properties of the optical probe, the probe is detected in the visible spectral range, solve the problem of fluorescence detection requires the use of large equipment, eliminating the need for large-scale gene chip laser scanning apparatus in the application process, to facilitate gene chip technology is widely used.

本发明的目的之二是,应用生物传感器技术,将较弱的可见光检测信号放大,实现芯片检测信号肉眼或借助简单的光学设备判读,并可借助简单的数值化设备如数码相机实现芯片的计算机自动化处理。 Second object of the present invention is applied biosensor technology, the less visible light detection signal amplifying, signal detection chip to achieve the naked eye or by a simple interpretation of the optical device, and by means of simple numerical devices such as digital cameras implemented in computer chips automated processing.

本发明的目的之三是,应用特殊的阳性信号处理过程,获得高特异性的检验信号.. Another object of the present invention, the positive application of special signal processing, to obtain a high specificity test signal ..

本发明的目的之四是,应用特定的基因探针及相应的微矩阵,实现疾病的 A fourth object of the present invention is the application of specific gene probes and microarray corresponding to achieve disease

特异性诊断。 Specific diagnosis.

为此,本发明第一方面涉及一种生物芯片,该芯片包括:二氧化硅基片; 存在于该二氧化硅基片上的光学涂层;和存在于该光学涂层上的多聚苯丙氨酸-赖試酸涂层。 For this purpose, a first aspect of the present invention relates to a bio-chip, the chip comprising: a substrate of silica; optical coatings present on the silica substrate; and poly-phenylpropyl present on the optical coating acid - LAI acid test coating.

在本发明一个实施方式中,本发明生物芯片的二氧化硅基片的厚度为 In one embodiment of the present invention, the thickness of the silica-based biochip sheet of the present invention

2-4mm,光学涂层包含350-550埃的氮化硅和80-200埃的T结构聚氨二甲基硅烷烃。 2-4mm, optical coating comprising silicon nitride and 80-200 Å 350-550 Å T dimethyl silicone alkane polyurethane structure.

在本发明另-实施方式中,该芯片还包括通过化学键与其光学涂层相连从而固定于芯片表面的核酸探针。 In the present invention, another - embodiment, the chip further comprising a connected and fixed to the chip surface by chemical nucleic acid probe and an optical coating.

在本发明一个实施方式中,该芯片的多聚苯丙氨酸-赖氨酸涂层的厚度为20-200埃。 In one embodiment of the present invention, the chip polyphenylalanine - lysine coated thickness of 20 to 200 angstroms.

本发明另一方面还涉及一种制备本发明所述生物芯片的方法,该方法包 The present invention further aspect the present invention relates to a process for preparing a biochip, the method

括: include:

(1) 在二氧化硅基片上镀以光学涂层; (1) on the silica substrate is coated with an optical coating;

(2) 在步骤(1)所得的涂层上再镀以多聚苯丙氨酸-赖氨酸涂层,获得镀了光学涂层和多聚苯丙氨酸-赖氨酸涂层的二氧化硅基片;(3)用盐酸琥珀酰亚胺基肼烟酸盐处理步骤(2)所得的二氧化硅基片,洗净,得到所述芯片。 (2) in step (1) and then coated on the resultant coating to polyphenylalanine - lysine coated to give a coated optical coating and polyphenylalanine - lysine coated titanium oxidizing the silicon substrate; (3) with a hydrazine hydrochloride succinimidyl nicotinate obtained in process step (2) a silica substrate, and washed to obtain a chip.

在本发明一-个实施方式中,该盐酸琥珀酰亚胺基肼烟酸盐的浓度为l-10pM,处理时间为10-30分钟。 In the present invention, one - embodiment, the concentration of the hydrochloric acid succinimidyl nicotinate hydrazine is l-10pM, processing time is 10-30 minutes.

在本发明另一实施方式中,该方法还包括步骤(4):点样固定探针,使其经室温与芯片表面的化学涂层进行氨醛综合反应,形成共价结合的化合物, 固定于芯片的表面上。 In another embodiment of the invention, the method further comprises the step of (4): spotting a probe fixed, so that the aldehyde ammonia synthesis reaction was room temperature and the chemical coating of the chip surface, covalently bonded compound formed, is fixed to the upper surface of the chip.

本发明另一方面还涉及一种检测样品中的待检核酸的方法,该方法包括: Another aspect of the invention relates to a method for detecting a nucleic acid to be detected in a sample, the method comprising:

(1)提供本发明的芯片,在其表面上固定与待检核酸特异性结合的核酸 (1) providing a chip according to the present invention, on the surface thereof to be fixed to the subject that specifically binds to the nucleic acid

探针: Probe:

(2.)使歩骤U)所述的芯片与样品、与待检核酸特异性杂交的标记探针接触,其中,该标记探针标记有生物素; (2) reacting ho step U) of the chip and the sample, to be examined in contact with the labeled probe specifically hybridizes nucleic acid, wherein the labeled probe is labeled with biotin;

(3) 用NaOH溶液进行洗脱,获得未被洗脱的产物; (3) eluting with a NaOH solution to obtain a non-eluting product;

(4) 用纳米金探针标记的链亲蛋白与步骤(3)获得的未被洗脱的产物接制!,形成核酸探针-标记探针-生物素-链亲蛋白-纳米金复合物;和 (4) a streptavidin protein in step (3) Gold Nanoparticle Probes labeled with non-eluted product obtained by the above system, the nucleic acid probe is formed! - labeled probe - biotin - streptavidin protein - composite nano-gold ;with

(5) 检测芯片信号,确定待检核酸的存在。 (5) detecting a signal chip, the presence of the test nucleic acid to be determined.

在本发明一个实施方式中,上述方法步骤(3)中NaOH的浓度为0.001-0.1M。 In one embodiment of the present invention, the above-described step (3) the concentration of NaOH is 0.001-0.1M. 本发明的技术方案具有如下优点: Aspect of the present invention has the following advantages:

(1) 应用纳米材料技术,将传统基因芯片检测信号,由荧光探针技术, 转换为可见光探针技术,实现了基因信号的可见光判读,从而从技术上实现了不需耍荧光扫描仪等大型设备的难题。 (1) Application of nano-materials technology, the traditional microarray detection signal from the fluorescent probe technology, into visible light probe technology, gene interpretation visible light signal, thereby achieving a fluorescence scanner without playing technically other large equipment problems.

(2) 常规的纳米材料可见光探针技术在临床应用过程中,存在着灵敏度偏低,普通光学设各判读时,存在着灵敏度不够和获得信号欠稳定,为此,我们引进了生物传感器技术,再次将纳米材料探针信号特异性放大,显著提高基因芯片的检测信号特异性和检测灵敏度,较常规芯片检测技术检测灵敏度提高1000至百万倍以上,完全实现基因芯片信号的特异性检测,直至达到肉眼判读的灵敏度水平。 (2) a conventional probe nanomaterials visible in clinical application process, there is the low sensitivity, the respective interpretation of ordinary optical design, there is insufficient sensitivity and less stable signal is obtained, Therefore, we introduced a biosensor technology, again nanomaterials probes specific amplification signal, the detection signal significantly improved the specificity and sensitivity of detection of gene chip, compared with conventional chip by detection sensitivity can be improved from 1000 to 1,000,000 or more times, fully achieve specific detection of gene chip signal until reach the level of sensitivity visually interpreted.

(3) 能实现基因芯片信号的简单解读。 (3) to achieve a simple interpretation of microarray signals. 应用范围更广,便于基层临床检测和适宜简陋的的野战条件等是其最大特点之一。 Wider range of applications, for clinical detection of the crude base and a suitable field conditions is one of its most important feature. 由于应用可见光谱及高灵敏的生物传感器技术,使芯片信号达到肉眼判读水平,并可借助简单的光学设备, 实现自动化和批量化检测。 Since the application of the visible spectrum and highly sensitive biosensor technology, the chip enable signal reaches a macroscopic level interpretation, and by a simple optical device, automation and mass detection. 因此,在应用中无需大型设备,极易推广应用。 Therefore, in the application without the need of large equipment, easy application.

(4) 采用特殊高温连接技术,显著提高杂交信号的特异性,降低背景信号, 信号信噪比显著提高,实现了芯片单个核酸分子差异的信号有和无的清晰准确判读。 (4) The temperature signal obtained by special connection technology, significantly increase the specificity of the hybridization signal, reduce the background signal, the signal to noise ratio significantly increased, to achieve a single nucleic acid molecule chip differences with and without a clear and accurate interpretation.

(5) 成本低廉。 (5) low cost. 由于检测时无需大型设备,使用成本显著下降和易于推广,必将促进基因芯片这--高新技术的普及和推广.并随着技术的普及及推广, 必将更进一步降低芯片成本。 Since the detection without the need of large equipment, the cost decreased significantly and easy to spread, it will promote gene chips - popularization and promotion of high-tech and with the popularity and promotion of technology, will further reduce the cost of the chip.

(6) 操作过程简单。 (6) The operation is simple. 简化应用,使临床检测过程时间显著縮短,通过的独特的信号处理过程,实现特异性信号的检测;检测过程时间短(除体外PCR扩增外, 杂交检测过程只需要20分钟)。 Simplify the application, causing the clinical detection process significantly shortened, by the unique signal processing, to realize detection of a specific signal; detecting short process times (except in vitro PCR amplification, the hybridization process takes only 20 minutes).

附图说明 BRIEF DESCRIPTION

图1显示阴阳性信号。 Figure 1 shows the negative and positive signal. 图1A为阴性,1B为阳性。 FIG negative 1A, 1B positive. 图2显示实际芯片及其检测信号。 Figure 2 shows an actual chip and signal detection.

图3昆示芯片结果的肉眼判读。 Figure 3 shows microarray results Kun Macroscopic interpretation. 图3A为实际结果的数码照片,3B为芯片信号设计示意图,按其阳性信号出现在芯片中的定位,判读其阳性信号代表的突变类型。 3A is a result of the actual digital photos, 3B is a schematic diagram of a chip design signal, a positive signal is positioned in its chip occurs, the interpretation of the type of mutation positive signals representative. 阁4显示薄膜生物传感器芯片上特异可见光信号的实现原理。 4 shows a specific implementation principle Court visible light signals on the film biosensor chip. 其中,图4A 捕获探针通过-NH-键固定于制备的生物传感器芯片上;图4B通过捕获探针, 形成目标片段-捕获探针-标记探针双链DNA,并在目标片段为桥的基础上,捕获探针与生物紫标记探针进行链接:图4C洗脱后能完成链接反应而形成的标记探针与捕获探针链接形成特异单链,其一端与生物传感器通过共价键连接固定,另一端带有生物素标记:图4D含有纳米金的通用探针与牛:物素结合,完成核酸与纳米金的连接,后者形成可见光信号,并经生物传感器放大而为肉眼或简单光学设备所判读。 Wherein FIG. 4A -NH- bond by the capture probe immobilized on the biosensor chip produced; FIG. 4B by capturing probe, target segment formed - capture probe - the DNA double-stranded labeled probe, and target segment of the bridge based on the capture probe and labeled probe to link biological violet: forming Figure 4C to complete the elution of the reaction link labeled probe specific to the capture probe forming a single chain link, having one end connected to the biosensor via a covalent bond fixed and the other end with biotin-labeled: FIG. 4D universal probe containing nano gold and bovine: biotin-binding gold nanoparticles to complete nucleic connection, which forms a visible light signal, and enlarged by the naked eye biosensor or simple the interpretation of the optical device.

具体实施方式 Detailed ways

--•、检测用薄膜生物传感器芯片的制备 - •, prepared film biosensor chip for detecting

1. 薄膜生物传感器的表面处理: 1. biosensor surface treatment film:

在直径约10-20cm硅片(S02)上镀以一定厚度的氮化硅(Si4N3)按真空气相沉积法镀膜,及旋转真空镀膜机将TSPS (T结构聚氨二甲基硅烷烃,UCT, Bristol, PA)的薄膜(Jenison, R., La, H., Haeberli, A" Ostroff, R. & Polisky, B. (2001) Clin. Chem.(Washington, DC) 47, 1894- l卯O: Jenison, R., Yang, S., Haeberli, A. & Polisky,B. (2001) Nat. Biotechnol. 19 , 62- 65),制备出相应的牛物传感器并再按常规的方法镀以多聚苯丙氨酸-赖氨酸涂层,最后经1-lOnM 盐酸琥珀酰亚胺基肼烟酸盐(succinimidyl hydrazinium nicotinate hydrochloride) 处理约10-30分钟(如15-25分钟,较佳约20分钟),清水洗净供芯片制作。 About 10-20cm in diameter silicon wafer (S02 is) coated with a predetermined thickness of silicon nitride (Si4N3) film by vacuum vapor deposition method, vacuum coating and the rotating machine TSPS (T dimethyl silicone alkane polyurethane structure, the UCT, bristol, PA) film (Jenison, R., La, H., Haeberli, a "Ostroff, R. & Polisky, B. (2001) Clin Chem (Washington, DC) 47, 1894- l d O..: Jenison, R., Yang, S., Haeberli, A. & Polisky, B. (2001) Nat. Biotechnol. 19, 62- 65), the preparation of the corresponding bovine sensor and then plated with a conventional method multimeric phenylalanine - lysine coated, finally processed (succinimidyl hydrazinium nicotinate hydrochloride) was 1-lOnM succinic acid imide group, hydrazino nicotinate about 10 to 30 minutes (e.g., 15-25 minutes, preferably about 20 minutes ), rinse with water for chip production.

该硅片的厚度可以是l-5毫米,优选是2-4毫米;该氮化硅的厚度可以是如350-550埃、优选400-500埃:该TSPS的厚度可以是80-200埃,如100-150 埃:该多聚苯闪弒酸-赖氨酸涂层的厚度可以是20-200埃,如可以是50-150埃、 80-120埃。 The thickness of the silicon wafer may be l-5 mm, preferably 2-4 mm; the thickness of the silicon nitride may be as 350-550 Angstroms, preferably 400-500 Angstroms: the thickness may be 80-200 angstroms TSPS, such as 100-150 Å: the killing polyphenyl flash acid - lysine coating thickness may be 20-200 angstroms, as can be 50-150 Å 80-120 Å.

2. 基因芯片微矩阵的设计及实现 2. Design and Implementation of a microarray gene chip

游离的待检核酸,经过核苷酸的碱基互补原则(AT, CG),通过与固定于芯片表面的特异性捕获探针结合而被固定于芯片表面。 Free nucleic acid to be detected, through the principle of complementary nucleotide bases (AT, CG), it is fixed to the surface of the chip by the chip is fixed to the surface of a specific capture probe binding. 非特异性的核酸,因未捕获固定,经洗脱过程而被洗脱,从而实现待检核酸与其它核酸分离。 Non-specific nucleic acids, failing to trap a fixed, but was eluted after the elution process, thereby achieving the test nucleic acid to be separated from the other nucleic acids.

基因捕获探针C1的设计:经检索分析现有美国国家医学图书馆基因库中所有乙型肝炎病毒(Hepatitis B virus, HBV)基因序列,本发明选择高度保守的HBV片段,设计并制备合成相应的探针,经标准的DNA合成过程(Applied Biosystems)或商业化合成,高效液相色谱纯化,获得色谱纯的DNA探针。 C1 gene capture probe design: Analysis All retrieved hepatitis B virus genes existing National Library of Medicine database (Hepatitis B virus, HBV) gene sequence, the present invention is highly conserved HBV fragment selection, design and synthesis of the corresponding preparation probe, by standard DNA synthesis (Applied Biosystems) or a commercially available synthetic, purified by HPLC chromatography to obtain a DNA probe. 并以().IM PBS pH 7.8缓冲液稀释为1 M浓度的工作液,并按疾病诊断的需求, 设计相应的矩阵进行点样和制作。 And diluted with () .IM PBS pH 7.8 buffer was 1 M concentration of the working fluid, and demand-driven disease diagnosis, corresponding matrix design and production spotting.

通过不同的捕获探针组成的矩阵,将不同的目的核酸分离于特定的区域, 并通过检测固定在不同物理部位的核酸分子,实现特定的核酸的分子的判读。 Matrix composed of different capture probes, the nucleic acid of different isolated to a particular area, and is fixed by detecting different physical parts of the nucleic acid molecule, to achieve a particular interpretation of the nucleic acid molecule.

3.薄膜生物传感器芯片表面的基因探针点样及化学交联点样固定后的捕获探针经室温下与薄膜生物传感器芯片表面的化学涂层进行氨醛综合反应,形成共价结合的化合物,稳定地固定于芯片的表面,并保持良好的生物学活性;再经清水漂洗,干燥;可供室温下保存一年或以上。 Gene probes spotted chemical crosslinking points 3 and a thin film biosensor chip surface of the integrated samples of aminal was reacted at room temperature the capture probe is fixed with a chemical coating film biosensor chip surface, forming a compound covalently bound stably fixed to the surface of the chip, and good biological activity; and then by water rinse, and dried; stored for a year or more at room temperature. 经过以上工艺,完成了薄膜生物传感器芯片的制作过程。 After the above processes, the complete production process of the thin-film biosensor chip.

二、薄膜生物传感器芯片上特异可见光信号的实现及读取1.特异性检测探针制备(1)通用金纳米材料探针及其制备 Second, to achieve specific detection and 1. Preparation of a probe (1) Material Gold universal probe specific for the visible light signal read on the film biosensor chip and its preparation

传统的基因芯片的标记探针系统采用的是cy3 cy5荧光素标记,而cy3,cy5 必须经过激发,才能发生可见光,亦是在基因芯片中检测需要昂贵的激光扫描仪的根本原因。 Traditional microarray system uses a labeled probe fluorescein labeled Cy3 Cy5, and Cy3, Cy5 must be excited to visible light is generated, is also detected in the microarray need root cause expensive laser scanner. 因此,检测中如要获得可见光信号,必须改变探针的标记分子, 为适宜高灵敏度和高分辩率的检测信号,金纳米材料作为标记分子,是现有最成熟的标记分子,其制备工艺简单,纳米材料直径易控。 Thus, the visible light detection signal to be obtained as necessary to change the labeled probe molecule, detection signal suitable for high sensitivity and high resolution, a material as gold marker molecule, most existing mature labeled molecule, simple preparation process , easy to control the diameter of the nanomaterial. 为此,本申请选用该分子作为标记分子。 To this end, the present application as a selection marker molecule of the molecule.

专用纳米金探针制备方法很多,有些发明使用了核酸直接标记纳米金技术,特异性较好,但由f链上标记金纳米分子较少,灵敏度相对较低;基因芯片中,检测探针数量庞大,•一一标记纳米金探针,工程量大,通用性低。 Many methods for nano gold probes prepared, some invention uses a technique gold nanoparticles direct labeling nucleic acid, better specificity, but less marked by the f gold chain molecules, relatively low sensitivity; gene chip, the number of detection probes huge, • eleven labeled gold nanoparticle probes, large projects, general low. 本发明采用了将金纳米材料标记链亲蛋白(streptaviditi)的方法,制备了链亲蛋白纳米金通用探针,该探针具有与任一标记有生物素分子的特异性核酸链高结合力亲合,从而获得通用性极高的通用检测探针。 The method of the present invention employs the gold labeled streptavidin protein material (streptaviditi) of the gold nano-chain pro-protein preparation universal probe, the probe having any one of the labeled nucleic acid strand with a high specificity binding force affinity biotin molecules together, thereby obtaining a highly versatile universal detection probe. 通过利用通用纳米材料探针, 提高纳米探针的通用性,减少纳米探针的制备工艺,降低芯片系统成本。 By using a universal probe nanomaterials, improving versatility nanoprobes, reducing nanoprobes preparation, to reduce the chip cost of the system. 由此制得的链亲蛋白纳米金通用探针,能与任一标记有生物素分子的特异性核酸链高结合力亲合,从而通过与带有生物素标记分子的任一特异的核酸探针结合,形成探针-生物素-链亲蛋白-纳米金复合物,迅速批量制备数量庞大的特异性检测核酸变异的特异的核酸纳米金探针- Streptavidin thus obtained protein nano-gold universal probe can be labeled with any one of the specific nucleic acid chain biotin molecule a high affinity binding force, so that by a specific nucleic acid probe with any biotinylated molecule with needle, forming probe - biotin - streptavidin protein - nano-gold complex, a huge number of rapidly detecting specific nucleic acid variations of specific nucleic acid prepared in bulk Nanoparticle probes -

(2)特异性标记探针及其制备通用性纳米探针具有良好的通用性和适宜批量制备,但不具备芯片检测所需要的特异性,为此,实现芯片特异性信号的检测,还需要制备大量特异性的标记探针。 (2) specific for a labeled probe prepared by the general nanoprobe and having excellent versatility and a suitable bulk preparation, but do not have the required specificity microarray, therefore, a chip enable detection of the specific signal, but also preparation of a large number of specific labeled probe. 为此,我们还需要依据所检测的目的基因的不同,利用核酸AT CG互补的特性,应用通用的化学合成方法(可委托国内外商业化公司合成), 合成特异性的互补性探针,并在其3,端标记上生物素分子(biotin),以利于其与通用型的纳米探针的结合连接。 To this end, we need different purposes based on the detected gene, using a nucleic acid complementary to AT CG characteristics, application of a common chemical synthesis methods (commercially available domestic and foreign companies commissioned synthesis), synthesis of specific complementary probe, and at its 3, end labeled biotin molecule (biotin), which is connected to facilitate binding universal nano probe.

2.薄膜生物传感器芯片上肉眼判读信号的实现 Achieve visually signal interpretation film biosensor chip 2.

(1) 待检基因片段的PCR扩增及产物的单链化处理待检的核酸分子,经PCR扩增,获得待检的核酸DNA片段,后者变性后 Single-stranded nucleic acid molecule of the subject to be treated (. 1) and the PCR amplification products to be subject gene fragments amplified by PCR, DNA fragments to obtain the nucleic acid to be detected, which denaturation

(例如在95'C处理2分钟),迅速置于冰上,形成单链的核酸片段。 (E.g. treatment 95'C 2 min), placed on ice for rapid, single-stranded nucleic acid fragments formed.

(2) 与待检核酸完整的双链DNA的形成及特异性生物素化探针的标记单链化后的待检核酸分子,通过核酸A-GC-T互补的原理,在待检核酸为 The nucleic acid molecule to be detected after the labeled single stranded form (2) with the nucleic acid to be detected and the complete double-stranded DNA-specific biotinylated probe, through the principle of A-GC-T nucleic acid complementary to, the nucleic acid is to be detected

桥杂交形成双链DNA分子,并在高温下(55-65'C,较佳60'C),捕获探针的3' 端-OH基与含有生物素的标记探针进行链接反应,形成60-100bp长度的完整的DNA双链,并完成对该双链上标记上生物素分子。 Bridge hybridize to form double stranded DNA molecule, and at elevated temperature (55-65'C, preferably 60'C), the capture probe the 3 'end of the link -OH groups react with the labeled probe containing biotin, 60 are formed full length DNA duplexes -100bp, and the completion flag of the double-stranded molecule of biotin.

(3) 含生物素特异性探针在芯片上固定及非特异的生物素特异探针的清 (3) specific biotin-containing probe is immobilized and non-specific biotinylated probes on the chip specific for clear

except

标记有生物素的完整双链的待检核酸,在严谨的NaOH溶液中((圆誦0.1M)或高温条件下(如65-80。C,通常为70-75。C,如70'C )再次解4连,形成争链,与捕获探针完全链接的标记有生物素的标记探针,因为有捕获探针与芯片形成的化学连接,冈此在此严谨洗涤条件下不被洗脱。而在非目的探针的待检核酸为桥的条件下,标记探针不能与芯片上的捕获探针完全链接, 因此不能形成长链的标记有生物素的核酸,因而在此条件下被洗脱。从而完成芯片对H的片段的特异性分离,并将特异性的待检核酸保留并固定在基因芯片衷面。 Full duplex is labeled with biotin to the nucleic acid to be examined, under stringent NaOH solution ((ROUND recite 0.1M) or high temperatures (e.g. 65-80.C, usually 70-75.C, as 70'C ) solution 4 is connected again, contention chains are formed, completely linked to the capture probe labeled with biotin labeled probe, the capture probe because chip formed chemically linked, Gang will not be eluted at this stringency wash conditions while under conditions of non-target nucleic acid probes to be examined for the bridge, the labeled probe can not be completely linked to the capture probes on the chip, and therefore can not form a nucleic acid labeled with long-chain biotin, thus this condition is eluted thereby completing the specific isolation of the fragment H of the chip, and fixed and retained co microarray surface to be specific for the subject nucleic acid.

3. 薄膜生物传感器上特异性待检信号的实现 Achieve specific signal to be detected on the film 3. The biosensor

前述洗脱和分离后的芯片,在特定的位置上,保留有带有生物素标记的特异性核酸,而其它位点上,因被洗脱而不再含有生物素分子的DNA链。 After elution of the chip and separated at a specific location, a specific nucleic acid retains biotin-labeled, while other sites, because no longer is eluted DNA strand containing the biotin molecule. 前者再与通用的金纳米探针结合,实现了对特异性待检探针的金纳米探针标记。 Recombined with the former common gold probe, to achieve the gold labeled probe specific for the probe to be detected.

通用金纳米探针与未洗脱的带有生物素分子的标记探针-捕获探针链发生亲合反应,形成捕获探针-标记探针-生物素-链亲蛋白-纳米金复合物,并形成沉淀=常规的玻璃芯片,由于光学信号的丢失,本实验条件下所形成的金纳米光学沉淀,既使是经过银离子的进一步放大(约10000倍),仍难以实现稳定的肉眼判读的信号强度或灵敏度。 General non-gold probe is eluted with a biotin labeled probe molecules - affinity capture probe strand reactions occur, to form a capture probe - labeled probe - biotin - streptavidin protein - nano-gold composite, = precipitate formed and visually conventional glass chips, of gold since the lower the optical loss of the optical signal, the experimental conditions of the formed precipitate, even after a further enlarged silver ions (about 10,000 times), still difficult to achieve a stable interpreted signal strength or sensitivity. 而薄膜生物传感器芯片所具备的光学特性,可将此可见光学沉淀信号显著放大,达到肉眼可见光信号的判读。 And the optical properties of the films included in the biosensor chip, this may be visible precipitate optical signal amplified significantly, to interpret visually visible light signals.

4. 薄膜生物传感器芯片上肉眼判读信号的读取 Read visually judging signal from the thin-film biosensor chip 4

信号的肉眼判读或简单数字成像设备(数码像机、CCD成像装置或扫描仪) 成像后的计算机批量处理。 Visually judging signal or simply a digital imaging device (digital camera, CCD imaging device or scanner) the imaging computer after batch processing. 具体的检测步骤如下: •将薄膜生物传感器芯片置60'C预热; Detecting specific steps are: • film biosensor chip set 60'C preheating;

•连接液复合物50-10(Hil置于薄膜生物传感器芯片上,60'C反应IO分钟; • connecting the liquid complex 50-10 (Hil biosensor chip was placed on the film, 60'C the reaction IO min;

'在60"C用预热的O.OlNNaOH洗涤3次,每次5-10秒; 'In 60 "C and washed three times with prewarmed O.OlNNaOH, every 5-10 seconds;

•再将芯片置0.1XSSC中室温下洗涤3次,每次l-2分钟; • then washed at room temperature 3 0.1XSSC chip set times l-2 min;

•链亲蛋白-Au溶液按1:1000在1X杂交缓冲液(5XSSC)稀释,并取50-100 • -Au streptavidin protein solution was diluted 1: in 1X hybridization buffer (5XSSC) 1000, and takes 50-100

Hl加入到芯片表面; Hl was added to the chip surface;

•室温下反应5分钟; • at room temperature for 5 minutes;

'用0.1XSSC洗涤芯片3次,每次l-2分钟;并在最后用净空气吹千芯片表面; 'Chip was washed three times with 0.1XSSC, each l-2 min; and the chip surface in the last one thousand blowing with clean air;

肉服或借助放大镜判读:或应用数码相机摄像、计算机判读信号。 Meat served with a magnifying glass or interpretation: or digital camera to camera, computer interpretation of signals.

下文将以具体实施例的方式阐述本发明。 Example manner hereinafter set forth specific embodiments of the present invention will. 应理解,这些实施例仅仅是阐述 It should be understood that these embodiments are merely illustrative

性的而非限制性的,本发明并不受到这些具体实施例的限制。 And not restrictive, and the invention is not to be limited to these specific embodiments. 本发明的范围由权利要求书的内容所限定。 Contents of the book scope of the invention as defined by the appended claims.

实施例l:薄膜生物传感器的制备 Biosensor film preparation: Example l embodiment

按真空气相沉积法镀膜,使用旋转真空镀膜机在厚度约为卜3mm、直径约15cm硅片(SO。上镀以475埃的氮化硅(Si3N4)及135埃的TSPS的薄膜,制备出相应的生物传感器,并覆盖150埃的多聚苯丙氨酸-赖氨酸涂层,最后经卜10uM盐酸琥珀酰亚胺基肼烟酸盐处理约20分钟,清水洗净供芯片制作。 Vacuum vapor deposition method by coating, using a rotary vacuum coater in a thickness of about Bu 3mm, 15cm diameter silicon wafer (SO. Coated with 475 Angstroms of silicon nitride (Si3N4) TSPS 135 Å and a film on to prepare the corresponding biosensor, and covers 150 angstroms polyphenylalanine - lysine coated, the final succinic acid imide group Bu 10uM hydrazino nicotinate for about 20 minutes and rinse for chip production.

实施例2:捕获探针的制备 Preparation of the capture probe: Example 2

采用本领域常规的核酸化学合成方法制得以下捕获探针(可委托国内或国际上专业公司,按以下化学结构进行制备): Using conventional in the art of nucleic acid chemistry synthetic methods of the following capture probes (national or international can delegate specialist, prepared according to the following chemical structure):

L528M P1-T SEQ ID NO: 1 ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCT L528M P1-T SEQ ID NO: 1 ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCT

L528M P1-A SEQ ID NO: 2 ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCA L528M P1-A SEQ ID NO: 2 ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCA

M539V P1-A SEQ ID NO: 3 ALD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATA M539V P1-A SEQ ID NO: 3 ALD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATA

M539V Pl-G SEG ID NO: 4 AIjD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATG M539V Pl-G SEG ID NO: 4 AIjD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATG

M539I Pl-G SEQ ID NO: 5 AIjD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATG M539I Pl-G SEQ ID NO: 5 AIjD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATG

M539I Pl-T SEQ ID NO: 6 AIjD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATT M539I Pl-T SEQ ID NO: 6 AIjD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATT

(ALD二醛基) (ALD dialdehydo)

实施例3:薄膜生物传感器芯片的制备 Thin-film biosensor chip was prepared: Example 3

在实施例1的芯片上点样固定实施例2制得的捕获探针,使其经室温与芯片表面的化学涂层进行氨醛综合反应,形成共价结合的化合物,固定于芯片的表面上,制得带有罔定的薄膜生物传感器芯片。 On the chip of Example 1 prepared in Example 2 was spotted capture probe embodiment is fixed, so that the aldehyde ammonia synthesis reaction was room temperature and the chemical coating of the chip surface, covalently bound compound formed, is fixed to the upper surface of the chip to prepare a film with a given indiscriminately biosensor chip.

实施例4:通用型金纳米材料标记探针的制备配置用子25批次的如下溶液: Example 4: Preparation of gold material arranged universal probes labeled with 25 sub-batch was as follows:

1.0 mM氯金酸溶液:将0.1 gHAuCl溶解于500 mL蒸馏水中即可。 1.0 mM chloroauric acid solution: 0.1 gHAuCl was dissolved in 500 mL of distilled water to. 可置棕色瓶中长期保存。 Brown bottle can set long-term preservation. 1%枸椽酸三钠(Na3C6H507*2H20):将0.5 g Na3C6H507'2H20溶入50 mL 1% trisodium citrate (Na3C6H507 * 2H20): The 0.5 g Na3C6H507'2H20 dissolved in 50 mL

蒸馏水中。 Distilled water.

1 M NaCI:将0.5 g of NaCl溶入10 mL蒸馏水中。 1 M NaCI: the 0.5 g of NaCl dissolved in 10 mL of distilled water.

将20 mL 1.0 mM HAuCU加入到50 mL三角烧瓶中,置入电磁搅拌棒并置千电磁搅拌器上加热,搅拌至沸腾。 The 20 mL 1.0 mM HAuCU added to 50 mL Erlenmeyer flask, and placed a magnetic stirring bar on a magnetic stirrer set one thousand heated, with stirring to boiling. 缓慢加入2mL的1%枸椽酸三钠,可见金纳米逐渐形成(约10分钟),当转为深红色时,停止搅拌加热。 Was slowly added 2mL of 1% trisodium citrate, visible, gold gradually formed (about 10 minutes), when turned dark red, stirring was stopped heating.

将纳米金pH调节至8.0,磁性搅拌下加入少量双蒸水溶解的链亲蛋白(Sigma-Eldrich, USA),作用10分钟,即可得到标记有纳米金的链亲蛋白;再加入5%牛血淸白蛋白(BSA: Boehringer Co., Gereman),使其终浓度至1%。 The gold nanoparticles is adjusted to pH 8.0, under magnetic stirring was added a small amount of double distilled water to dissolve the protein streptavidin (Sigma-Eldrich, USA), for 10 min, to obtain gold nano-labeled streptavidin protein; was added with 5% bovine Qing serum albumin (BSA: Boehringer Co., Gereman), to give a final concentration of 1%. 反应后生成的链亲蛋白-Au以15000g离心,吸取上清,过Sephacryl S-400柱(Pharmacia),收集中间段深红纯化部分,4'C保存备用。 Generated after the reaction to streptavidin protein -Au 15000g centrifugation, the supernatant was drawn, through Sephacryl S-400 column (Pharmacia), purified fractions were collected magenta intermediate section, 4'c for use.

实施例5:乙型肝炎病毒YMDD突变的检测1.样品准备 YMDD motif mutation Detection 1. Sample preparation: 5 Example

(1) 抗凝或不抗凝血2ml,常温下1000rpm离心5分钟,用微量吸液器取血淸或血浆100^1; (1) no anticoagulant or anticoagulant 2ml, centrifuged at 1000rpm at room temperature for 5 minutes, micro pipette blood or plasma Qing 100 ^ 1;

(2) 加等体积的具有如下组成的试剂1, 100'C煮沸5rnin: (2) adding an equal volume of a reagent having the following composition 1, 100'C boil 5rnin:

HBV正向引物(SEQ ID NO: 7) TGCACCTGTATTCCCATCC HBV forward primer (SEQ ID NO: 7) TGCACCTGTATTCCCATCC

HBV反向引物(SEQ ID NO: 8) GCGGTATAAAGGGACTCACG HBV reverse primer (SEQ ID NO: 8) GCGGTATAAAGGGACTCACG

(3) 5000g离心5分钟备用 (3) 5000g centrifugation for 5 minutes standby

2.目的片段的PCR扩增 2. PCR amplification of fragment

(1) 室温下取PCR扩增管编号,按顺序放置; PCR amplification number taken tube (1) at room temperature, placed in sequence;

(2) 取上述制备的样品上清5pl; Sample (2) take the above prepared supernatant 5pl;

(3) 加去离子水19nl; (3) deionized water 19nl;

(4) 加Taq嗨(珠海赛乐奇生物技术有限公司)lpl; (4) plus Taq Hi (Zhuhai race Lucky Biotechnology Co., Ltd.) lpl;

(5) 4。 (5) 4. C离心1分钟; C and centrifuged for 1 min;

(6) 按以下条件进行扩增:94。 (6) was amplified under the following conditions: 94. C变性5分钟;再按94。 C denaturation for 5 min; then 94. C, 30秒一一56'C, 3()秒——72°C, 30秒进行30轮循环,72'C延伸10分钟后4t密封保存备用。 C, 30 seconds eleven 56'C, 3 () seconds --72 ° C, 30 seconds 30 cycles, 72'C extending 4t sealed standby after 10 minutes. 3.采用本领域常规的核酸化学合成方法制得以下捕获探针(可委托国内或国际上专业公司,按以下化学结构进行制备): 3. The nucleic acid according to the present art conventional chemical synthetic methods of the following capture probes (national or international can delegate specialist, prepared according to the following chemical structure):

L,528M P2-生物素(SEQ ID NO: 9) Phosphate-TGGCTCAGTTTACTAGTGCC-biotin L, 528M P2- biotin (SEQ ID NO: 9) Phosphate-TGGCTCAGTTTACTAGTGCC-biotin

M539V P2-生物素(SEQ ID NO: 10) Phosphate-TGGATGATGTGGTATTGGGG-biotin M539V P2- Biotin (SEQ ID NO: 10) Phosphate-TGGATGATGTGGTATTGGGG-biotin

M539I P2-生物紫(SEQ ID NO: 11) Phosphate-GATGATGTGGTATTGGGGGC-biotin M539I P2- biological violet (SEQ ID NO: 11) Phosphate-GATGATGTGGTATTGGGGGC-biotin

(其中,"Phosphate"指磷酸根,而"biotin"指生物素) (Wherein, "Phosphate" refers to a phosphate, and "biotin" refers to biotin)

4. 基因芯片检测 4. Microarray

(1) 将PCR扩增产物95。 (1) The PCR amplification product 95. C加热3分钟,迅速置于冰上; C for 3 minutes, rapidly and placed on ice;

(2) 取10mlPCR扩增产物至实施例3制得的光学传感器基因芯片上; (2) to take 10mlPCR embodiment the amplification products obtained in Example 3 of the optical sensor microarray;

(3) 取1^1连接酶(珠海赛乐奇生物技术有限公司)加入芯片上; (3) 1 ^ 1 taken ligase (Zhuhai race Leqi Biotechnology Co., Ltd.) was added to the chip;

(4) 取60'C预热连接液3(链接酶5u,链接酶缓冲液,5mg/mlATC,酸化) (珠海赛乐奇生物技术有限公司)滴于芯片上,在60'C反应10分钟: (4) 3 was connected to the preheating takes 60'C (5U ligase, ligase buffer, 5mg / mlATC, acidification) (Zhuhai race Leqi Biotechnology Co., Ltd.) was dropped on the chip, the reaction 60'C 10 minutes :

(5) 在用60。 (5) 60. C预热的0.01-0.1 NNaOH洗涤3次,每次5-10秒; 0.01-0.1 NNaOH C preheated washed 3 times for 5-10 seconds;

(6) 室温下再将芯片置0.1XSSC中洗涤3次,每次l-2分钟;然后空气中干燥; (6) set at room temperature and then the chip was washed three times with 0.1XSSC, each l-2 min; then air-dried;

(7) 将实施例4制得的金纳米材料探针每片3滴滴于芯片表面,并在常温下反应5分钟; (7) to the chip surface prepared in Example 4 was gold probe material drops per tablet of embodiment 3, and reacted at room temperature for 5 minutes;

(8) 用0.1XSSC洗涤芯片3次,每次l-2分钟;并在最后用干净空气吹干芯片表面: (8) the chip was washed three times with 0.1XSSC, each l-2 min; and finally blown dry with clean air at the surface of the chip:

(9) 迅速地用水冲洗芯片3次,干净空气吹干芯片表面,进行芯片判读。 (9) the chip quickly rinsed three times with water, air dry and clean surface of the chip, the chip for interpretation.

5. 结果判定 The result of the determination

用普通数码相机照相或扫描输入电脑,依信号出现的部位,确实突变的类型.,结果显示在图3中。 Entered into the computer with a general digital camera or camera scan, by the signal parts appear, indeed mutant types, results are shown in FIG. 图3显示芯片结果的肉眼判读。 FIG 3 the interpretation of microarray results visually displayed. 图3A为实际结果的数码照片,3B为芯片信号设计示意图,按其阳性信号出现在芯片中的定位,判读其阳性信号代表的突变类型。 3A is a result of the actual digital photos, 3B is a schematic diagram of a chip design signal, a positive signal is positioned in its chip occurs, the interpretation of the type of mutation positive signals representative.

结果证明,采用PCR技术扩增乙型肝炎病毒(HBV)的P基因,再利用核酸杂交技术和基因芯片技术,检测HBV的P基因的单个核苷酸的变异,并应用纳米材料探针技术和光学传感器技术,实现了检测信号的放大,达到肉眼判读的水平,从而突破传统基因芯片技术在检测中需要使用激光扫描仪等大型设备的瓶颈限制,基因芯片无需专用设备而可广泛使用。 The results demonstrate, P gene was amplified by PCR of hepatitis B virus (HBV), and then utilizes nucleic acid hybridization techniques and gene chip technology to detect single nucleotide mutation of HBV P gene, and probe technology and nanomaterials optical sensor technology, the amplification of the detection signal, to the naked eye interpretation level to break the bottleneck of the conventional microarray techniques require the use of large equipment like laser scanner in the detection, the microarray without special equipment can be widely used.

本款芯片主要针对核苷类抗HBV治疗过程中,诱导HBV产生YMDD区突变,从而导致病毒对拉米夫定的耐药的遗传学特性,应用基因芯片技术,检测HBV的YMDD区的基因突变类型,从而为临床抗病毒治疗提供科学的用药依据。 This paragraph chip targeted nucleoside anti-HBV treatment, the induction YMDD HBV generating region mutations, resulting in genetic properties of the virus resistant to lamivudine, gene chip technology to detect mutations in the YMDD HBV region type, thus providing a scientific basis for clinical drug antiretroviral therapy. 50 50

50 50

50 50

50 50

序列表 Sequence Listing

':11()> 宋家武 ': 11 ()> Song Jiawu

.120〉--种^灵敏度生物传感器基闵芯片及临床诊断技术 .120> - ^ Species Sensitivity Min yl biosensor chip and clinical diagnostics

'1:〗0: 059H20 '1:〗 0: 059H20

'.,11 '. 11

•:17()> fJaiL>mln version 3.3 •: 17 ()> fJaiL> mln version 3.3

'■>】 '■>]

aaaaaiiaiiiifi cgcaaaatac ctatgggagt gggcctcagt ccgtttctct aaaaaiiaiiiifi cgcaaaatac ctatgggagt gggcctcagt ccgtttctct

<■〉 2 <■> 2

aaaaaaaaaa cgcaaaatac ctatgggagt gggcctcagt ccgtttctca aaaaaaaaaa cgcaaaatac ctatgggagt gggcctcagt ccgtttctca

:■> 3 : ■> 3

artaaaaaaau gttcgtaggg ctttccccca ctgtttggct ttcagctatet artaaaaaaau gttcgtaggg ctttccccca ctgtttggct ttcagctatet

.'210:' .1 ,21l> 50 .'210: '.1, 21l> 50

213> 'jl物 213> 'jl thereof

:.iO()> .1 : .IO ()> .1

;化緣uiaiia" gttcgtaggg ctttccccca ctgtUggct ttcagctatg ; Alms uiaiia "gttcgtaggg ctttccccca ctgtUggct ttcagctatg

、:21()::' 5 :211> 50 .212: l)NA ,: 21 () :: '5: 211> 50 .212: l) NA

16 16

:2i()〉 : 2i ()>

〈212〉 <212>

:213〉 : 213>

<211> <213〉 <211> <213>

:2io> : 2io>

<211> 〈212〉 <213> <211> <212> <213>

1 5 ci JIJ 1 5 ci JIJ

2 2

A物 A matter

D弓 D bow

A:物 A: was

g引50 50 g lead

50 50

19 19

20 20

20 20

20 20

•'213> 引物 • '213> Primer

"aaaaai.ui;ui lcgtagggct ttcccccact gtttggcttt cagctatatg "Aaaaai.ui; ui lcgtagggct ttcccccact gtttggcttt cagctatatg

■■211... 5() '212卩D、A 引物 ■■ 211 ... 5 () '212 Jie D, A primer

iuifumiuiaafi u'guigggct ttcccccact gtttggcUt cagctatatt iuifumiuiaafi u'guigggct ttcccccact gtttggcUt cagctatatt

<4()0〉 7 <4 () 0> 7

tgcacctgta ttcccatcc tgcacctgta ttcccatcc

<210〉 8 <210> 8

<211〉 20 <211> 20

<212〉 腿 <212> leg

:213〉 引物 : 213> Primer

<■〉 K <■> K

gcggtataaa gggactcacg gcggtataaa gggactcacg

<210〉 9 <210> 9

:211> 20 : 211> 20

<212〉 隠(21:!〉引物 <212> Yin (21:!> Primer

'、顿):'9 'Dayton):' 9

l.Kgcu'agtl lactagtgc(: l.Kgcu'agtl lactagtgc (:

10 10

211!/ 20 211! / 20

t附u则gi ggtaugggg t u is attached gi ggtaugggg

、:21())' :211〉 ,: 21 ()) ': 211>

A啦 A friends

.y N — .y N -

,■40u:- 11 , ■ 40u: - 11

糾gatgtgg tattgggggc 20 Correct gatgtgg tattgggggc 20

:210〉 : 210>

:2ii〉' :212〉、 : 2ii> ': 212>,

A华 A China

1 o \.;;;; 1 o \. ;;;;

1 2 D lo 1 2 D lo

Claims (9)

1. 一种生物芯片,其特征在于,该芯片包括: 二氧化硅基片; 存在于该二氧化硅基片上的光学涂层,其中,该光学涂层包含厚度为350-550埃的氮化硅和厚度为80-200埃的T结构聚氨二甲基硅烷烃;和存在于该光学涂层上的多聚苯丙氨酸-赖氨酸涂层。 A bio-chip, wherein the chip comprises: a substrate of silica; optical coatings present on the silica substrate, wherein the optical coating comprises a nitride thickness of 350-550 Å silicon and having a thickness of 80-200 angstroms T dimethyl silicone polyurethane structure alkanes; and present on the optical coating polyphenylalanine - lysine coated.
2. 如权利要求1所述的生物芯片,其特征在于,所述二氧化硅基片的厚度为2-4mm。 2. The biochip according to claim 1, wherein the thickness of the silica substrate is 2-4mm.
3. 如权利要求1所述的生物芯片,其特征在于,该芯片还包括通过化学键与其光学涂层相连从而固定于芯片表面的核酸探针。 The biochip as claimed in claim 1, wherein the chip further comprises an optical coating by a chemical bond therewith and fixed to the surface of the chip is connected to a nucleic acid probe.
4. 如权利要求1所述的生物芯片,其特征在于,所述多聚苯丙氨酸-赖氨酸涂层的厚度为20-200埃。 4. The biochip according to claim 1, wherein said polyphenylalanine - lysine coated thickness of 20 to 200 angstroms.
5. —种制备权利要求1所述的生物芯片的方法,其特征在于,该方法包括:(1) 在二氧化硅基片上镀以光学涂层,其中该光学涂层包含厚度为350-550埃的氮化硅和厚度为80-200埃的T结构聚氨二甲基硅烷烃:(2) 在步骤(1)所得的涂层上再镀以多聚苯丙氨酸-赖氨酸涂层,获得镀了光学涂层和多聚苯丙氨酸-赖氨酸涂层的二氧化硅基片:(3) 用盐酸琥珀酰亚胺基肼烟酸盐处理步骤(2)所得的二氧化硅基片, 洗净,得到所述芯片。 5. - Method according to claim 1 for preparing a biochip species claims, characterized in that, the method comprising: (1) coated with an optical coating, wherein the optical coating comprises a thickness on the silica substrate is 350-550 angstroms of silicon nitride and having a thickness of 80-200 angstroms T butyldimethylsilyl alkanes polyurethane structure: (2) in step (1) on the resulting coating and then plated with polyphenylalanine - lysine coated layer, the optical coating is obtained and coating polyphenylalanine - lysine coated silica substrates: the resulting (3) nicotinate hydrochloride succinimidyl with hydrazine in step (2) two oxidizing the silicon substrate, and washed to obtain a chip.
6. 如权利要求5所述的方法,其特征在于,所述盐酸琥珀酰亚胺基肼烟酸盐的浓度为ll(HiM,处理时间为10-30分钟。 6. The method according to claim 5, characterized in that the concentration of succinimidyl nicotinate hydrazine hydrochloride is the ll (HiM, the processing time is 10-30 minutes.
7. 如权利要求5所述的方法,其特征在于,该方法还包括:(4)点样固定探针,使其经室温与芯片表面的化学涂层进行氨醛综合反应,形成共价结合的化合物,固定于芯片的表面上。 7. The method according to claim 5, characterized in that the method further comprises: (4) immobilized probes spotted so aldehyde ammonia synthesis reaction was room temperature and the chemical coating of the chip surface, to form a covalent binding compound immobilized on the surface of the chip.
8. —种检测样品中的待检核酸的方法,其特征在于,该方法包括:(1) 提供权利要求1所述的芯片,在其表面上固定与待检核酸特异性结合的核酸探针:(2) 使步骤(1)所述的芯片与样品、与待检核酸特异性杂交的标记探针接触,其中,该标记探针标记有生物素;(3) 用NaOH溶液进行洗脱,获得未被洗脱的产物;(4) 用纳米金探针标记的链亲蛋白与步骤(3)获得的未被洗脱的产物接触,形成核酸探针-标记探针-生物素-链亲蛋白-纳米金复合物;和(5) 检测芯片信号,确定待检核酸的存在。 8. - The method of nucleic acid species to be detected in the test sample, wherein the method comprises: (1) providing a chip according to claim 1, the nucleic acid probe is fixed on the surface thereof that specifically binds to the nucleic acid to be detected and : (2) sample chips of step (1), the contact labeled probe that specifically hybridizes with the nucleic acid to be detected, wherein the labeled probe is labeled with biotin; (3) eluting with NaOH solution, eluted product is not obtained; not eluted product (4) gold Nanoparticle probes labeled with streptavidin protein in step (3) obtained by contacting the nucleic acid probe is formed - labeled probe - biotin - streptavidin protein - nano-gold compound; and (5) detection chip signal, determining the presence of the nucleic acid to be detected.
9. 如权利要求8所述的方法,特征在于,步骤(3)中NaOH的浓度为0,001-0.1M。 9. The method as claimed in claim 8, wherein the concentration in step (3) of NaOH was 0,001-0.1M.
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