CN102980920A - Silicon nanowire chip simultaneously detecting miRNAs and protein markers and detection method and application of silicon nanowire chip - Google Patents

Silicon nanowire chip simultaneously detecting miRNAs and protein markers and detection method and application of silicon nanowire chip Download PDF

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CN102980920A
CN102980920A CN2012104579743A CN201210457974A CN102980920A CN 102980920 A CN102980920 A CN 102980920A CN 2012104579743 A CN2012104579743 A CN 2012104579743A CN 201210457974 A CN201210457974 A CN 201210457974A CN 102980920 A CN102980920 A CN 102980920A
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mir
silicon
layer
mirnas
silicon nanowire
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赵宇岚
朱建军
何靖
蒋宾
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华东师范大学
上海集成电路研发中心有限公司
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00Manufacture or treatment of nanostructures
    • HELECTRICITY
    • H01BASIC ELECTRIC ELEMENTS
    • H01LSEMICONDUCTOR DEVICES; ELECTRIC SOLID STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H01L29/00Semiconductor devices adapted for rectifying, amplifying, oscillating or switching, or capacitors or resistors with at least one potential-jump barrier or surface barrier, e.g. PN junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof; Multistep manufacturing processes therefor
    • H01L29/02Semiconductor bodies ; Multistep manufacturing processes therefor
    • H01L29/06Semiconductor bodies ; Multistep manufacturing processes therefor characterised by their shape; characterised by the shapes, relative sizes, or dispositions of the semiconductor regions ; characterised by the concentration or distribution of impurities within semiconductor regions
    • H01L29/0657Semiconductor bodies ; Multistep manufacturing processes therefor characterised by their shape; characterised by the shapes, relative sizes, or dispositions of the semiconductor regions ; characterised by the concentration or distribution of impurities within semiconductor regions characterised by the shape of the body
    • H01L29/0665Semiconductor bodies ; Multistep manufacturing processes therefor characterised by their shape; characterised by the shapes, relative sizes, or dispositions of the semiconductor regions ; characterised by the concentration or distribution of impurities within semiconductor regions characterised by the shape of the body the shape of the body defining a nanostructure
    • H01L29/0669Nanowires or nanotubes
    • H01L29/0673Nanowires or nanotubes oriented parallel to a substrate
    • HELECTRICITY
    • H01BASIC ELECTRIC ELEMENTS
    • H01LSEMICONDUCTOR DEVICES; ELECTRIC SOLID STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H01L29/00Semiconductor devices adapted for rectifying, amplifying, oscillating or switching, or capacitors or resistors with at least one potential-jump barrier or surface barrier, e.g. PN junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof; Multistep manufacturing processes therefor
    • H01L29/02Semiconductor bodies ; Multistep manufacturing processes therefor
    • H01L29/12Semiconductor bodies ; Multistep manufacturing processes therefor characterised by the materials of which they are formed
    • H01L29/16Semiconductor bodies ; Multistep manufacturing processes therefor characterised by the materials of which they are formed including, apart from doping materials or other impurities, only elements of Group IV of the Periodic System

Abstract

The invention discloses a silicon nanowire chip simultaneously detecting miRNAs and protein markers. The silicon nanowire chip comprises at least one of integrated configuration of single-chain deoxyribonucleic acid (DNA) probe in a silicon nanowire array, and integrated configuration of protein marker antibody in the silicon nanowire array. The invention further discloses a structure and a preparation method of the silicon nanowire chip, a detection method used for simultaneously detecting the miRNAs and the protein markers, and application in detection of the acute myocardial infarction miRNAs. The silicon nanowire chip simultaneously detecting miRNAs and protein markers and the detection method and the application of the silicon nanowire chip achieve rapid and simultaneous detection of the acute myocardial infarction miRNA and the protein markers, and have the advantages of being high in sensitivity, high in detection speed, easy to integrate, high in flux, high in stability, resistant to pollution, and the like.

Description

同时检测miRNAs与蛋白标记物的硅纳米线芯片及其检测方法和应用技术领域[0001] 本发明涉及生物分子检测领域,特别涉及一种同时检测miRNAs与蛋白标记物的硅纳米线芯片及其制备方法、检测方法及应用。 While silicon nanowire chip and a detection method and detection of miRNAs TECHNICAL FIELD protein marker [0001] The present invention relates to the field of biomolecular detection, particularly to a silicon nanowire chips simultaneously detecting miRNAs and protein markers and their preparation method, the detection method and application. 背景技术[0002]目前,心血管病是威胁人类健康的常见病高发病,而急性心肌梗塞则因为发病迅猛、致死率及致残率高,给社会及患者家庭造成极大负担。 [0002] Currently, the high incidence of cardiovascular disease is a common threat to human health, and acute myocardial infarction is because of the rapid onset, high mortality and morbidity, and the patient's family to cause great social burden. 如果要降低急性心肌梗塞的危害,早期诊断、针对性治疗以及根据病程进展调整用药等是必不可少的环节,而检测及监控某些生物标记物显然有助于这几方面的实施。 If you want to reduce the hazard of acute myocardial infarction, early diagnosis, targeted treatment and medication and other adjustments in accordance with the progression is an essential part, to detect and monitor certain biological markers clearly contributed to the implementation of these areas. 除了目前临床上常用的几种蛋白标记物cTnT、cTnI、CK-MM和CK-MB,近年来微小核糖核酸(microRNAs,miRNAs)被认为是很有应用前景的生物标记物。 In addition to several commonly used on current clinical protein markers cTnT, cTnI, CK-MM and CK-MB, in recent years, microRNAs (microRNAs, miRNAs) are considered very promising biomarkers. [0003] 按2000年欧洲心脏病学会和美国心脏病学会对心机梗死的定义,急性、演变中或新近心肌梗死诊断条件具备下列任何条件之一:心肌生化标志的典型升高和逐渐下降(cTnT或cTnl)或较快增高和下降(CK-MB),至少伴有下列情况之一者:心肌缺血症状;心电图出现病理性Q波;心电图示心肌缺血(ST段抬高或压低);冠心动脉介入术(如冠状动脉成形术)。 [0003] according to the 2000 European Society of Cardiology and the American Heart Association's definition of effort infarction, acute, evolving or recent myocardial infarction diagnosis of any one of the conditions of the following conditions are met: Typical elevated cardiac biomarkers and decreased (cTnT or of cTnl) and increased or decreased faster (CK-MB), with at least the following circumstances: symptoms of myocardial ischemia; ECG pathological Q waves; ECG showed myocardial ischemia (ST segment elevation or depression); coronary artery intervention (e.g. coronary angioplasty). 但即使是目前国际上通行诊断金标准cTn通常在心肌损伤4-8小时后升高,因而建议如果在入院时(有心肌缺血症状约I小时)检测cTn水平未升高,则需在6-9小时后再次抽血检测,甚至需要在12-24小时后第三次检测。 But even the currently accepted gold standard for the diagnosis of elevated cTn usually 4-8 hours after myocardial injury on international, and recommended (approximately I hour myocardial ischemic symptoms) did not detect elevated levels of cTn if at the time of admission, it would take six -9 hours after the blood is detected again, even be detected in 12 to 24 hours after the third time. 因而如果要早期诊断急性心梗,需要检测一些更早期即出现显著性变化的分子标记物。 Therefore, if you want early diagnosis of acute myocardial infarction, need for early detection of some of the more significant change appears molecular markers. 近年未有研究显示某些miRNAs可在急性心梗后早期出现变化,提示了miRNAs作为诊断指标的可能性。 No recent studies have shown that changes in certain miRNAs may appear early after acute myocardial infarction, suggesting the possibility of miRNAs as diagnostic indicators. 例如D' Alessandra等人采集了33例急性心肌梗死患者的血衆,TaqMan Human MicroRNA A and B Arrays筛选急性心梗后8倍以上变化的miRNAs并用实时定量PCR验证,最后选择了6个miRNAs进一步研究。 E.g. D 'Alessandra et al collected 33 cases of blood all patients with acute myocardial infarction, TaqMan Human MicroRNA A and B Arrays Screening more than 8 times after acute myocardial infarction change miRNAs and real-time quantitative PCR demonstrated that the final choice of six miRNAs further study . 结果发现急性心梗后3小时内即有miRNAs升高,其中miR-1,miR-133a, miR-133b, miR-122和miR-375的峰值比目前诊断用的“金标准”心肌肌I丐蛋白(cardiac troponin, cTn)更早出现。 It was found 3 hours after acute myocardial infarction that is elevated miRNAs, wherein miR-1, peak miR-133a, miR-133b, miR-122 and miR-375 than the current diagnostic "gold standard" Hack cardiac muscle I protein (cardiac troponin, cTn) occur earlier. Wang等人则针对性选择了肌肉富含的miR-1、miR-133a、miR_499和心肌特异性的miR-208a进行研究。 Wang and others targeted selection of the muscle rich in miR-1, miR-133a, miR_499 and cardiac-specific miR-208a were studied. 在结扎法大鼠AMI模型中,miR_208a在术后I小时即显著升高。 In the rat model of ligation of AMI, miR_208a i.e. significantly elevated after I hour. 而在临床病例中,AMI (33例)患者这四种血浆miRNAs比健康对照(30例)、非AMI型冠心病(16例)以及其它心血管病(17例)都明显升高。 In clinical cases, AMI (33 is patients) plasma miRNAs four patients than healthy controls (30 cases), non-AMI coronary artery disease (16 patients) and other cardiovascular diseases (n = 17) were significantly increased. 更重要的是miR-208a在非AMI 患者中不能检测到,而在AMI病例症状发生4小时后可100%检测到。 More important is that the miR-208a in a non-AMI patients can not be detected, the onset of AMI patients and 4 hours 100% of the symptoms detected. 之后有更多研究报道miR-1、miR-133、miR-328、miR-499_5p等对于AMI的诊断价值。 After more diagnostic value reported miR-1, miR-133, miR-328, miR-499_5p etc. for the AMI. 因为miRNAs变化出现更早,而且miRNAs本身也比较稳定,因而用miRNAs作为AMI诊断指标是相当有前景的。 Because miRNAs changes occur earlier, and miRNAs is also relatively stable, and therefore with AMI miRNAs as diagnostic indicators are quite promising. [0004] 微小RNAs (microRNAs,简称miRNAs)是一类非编码的RNA分子,长度在18-25核苷酸。 [0004] The micro RNAs (microRNAs, referred miRNAs) are a class of non-coding RNA molecules, 18-25 nucleotides in length. 近年来miRNAs对各种疾病的诊断价值越来越受到重视。 In recent years, miRNAs in the diagnosis of various diseases, more and more attention. 虽然随着现代芯片技术的应用,可以灵敏地检测出在疾病中miRNAs表达谱的变化。 Although the chip with the application of modern technology, can sensitively detect a change in the expression profiles of miRNAs disease. 然而其检测仍然存在以下缺陷:检测样品需经繁琐的步骤处理,包括细胞、组织或血液样品中总RNA的富集抽提及纯化,而后经反转录成cDNA,某些情况下还需PCR扩增,方可上样检测。 However the following shortcomings are still detects which: subject to the test sample cumbersome step process, including enrichment and purification of the extracted cell, tissue or blood samples of total RNA, followed by reverse transcription into cDNA, PCR in some cases need amplification, before the sample test. 而究其根本原因,则在于细胞、 组织或血样中miRNAs含量很低,经常在pM-fM级,超出目前的检测方法的极限低值。 And study the fundamental reason lies in the cell, tissue or blood samples miRNAs content is very low, often in the pM-fM level, beyond the limits of low current detection method. 而且生物样品中的其它复杂成分,例如糖、脂质、金属离子、大分子蛋白质等,也会干扰目标miRNA 与探针的结合,从而对检测的灵敏度和特异性产生重大影响。 Components and other complex biological sample, such as sugars, lipids, metal ions, proteins and other macromolecules, and also interfere with the binding target miRNA probe, thereby producing a significant influence on the detection sensitivity and specificity. 繁琐的前期处理过程是目前制约生物芯片在临床大规模使用的瓶颈之一。 Cumbersome pre-treatment process is currently one of biochips in clinical restrict large-scale use of bottlenecks. [0005] 最近的基于纳米技术的场效应FET的硅纳米线阵列(SiNW)芯片则有望通过提高检测灵敏度解决这个难题。 [0005] Recently chip is expected to solve this problem by improving the detection sensitivity of nanowire arrays based on silicon nano-FET field effect technology (SiNW). 以SiNW结构为核心,采用场效应晶体管实现信号采集和放大,能够更有效检测目标信号。 SiNW to the core structure, using a field effect transistor signal acquisition and amplification, can be more effectively detect the target signal. 此种芯片具有灵敏度高、检测速度快、易于集成与高通量检测的优点。 Such a chip with high sensitivity, speed, and advantages of easy integration with high-throughput testing. 因此,本发明不仅解决硅纳米线阵列保存应用中存在的容易受污染问题,且使其在生物检测中,即使待测组份本体液的呈现多样性的情况下,也同样可以使芯片面对Na、K、 Fe、Cu和Ca等离子的扩散污染的考验以及PH值等多种化学因素的影响,即实现了检测的高稳定性。 Accordingly, the present invention not only solves the problem of easily contaminated silicon nanowire arrays stored in the present application, and is allowed to bioassay, even if parts of the body fluid to be tested exhibits the diversity component of the present case, the chip can also face Effect of diffusion of contamination na, K, Fe, Cu and Ca in plasma and a variety of chemical factors PH value test, i.e., to achieve a high stability of the detection. 发明内容[0006] 本发明克服现有技术的以上缺陷,提出一种用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,可用于同时检测复杂生物样品中miRNAs和蛋白标记物,从而早期诊断急性心梗相关的miRNAs标记物。 SUMMARY OF THE INVENTION [0006] The present invention overcomes the above drawbacks of the prior art, is proposed for the simultaneous detection of miRNAs and silicon nanowires chip marker protein, can be used for simultaneous detection of miRNAs in complex biological samples and marker protein, so that early diagnosis acute myocardial infarction associated miRNAs markers. [0007] 本发明提供一种同时检测miRNAs与蛋白标记物的硅纳米线芯片,所述芯片包括: 至少一种单链DNA探针在硅纳米线阵列中的集成排布,以及蛋白标记物抗体在硅纳米线阵列中的集成排布。 [0007] The present invention provides a silicon nanowire miRNAs simultaneously detect marker protein chip, said chip comprising: at least one single-stranded DNA probes arranged in integrated silicon nanowire arrays, and protein marker antibodies in the silicon nanowire array integrated arrangement. [0008] 本发明中,所述单链DNA探针包括针对特定miRNAs的完全匹配探针、单碱基不匹配探针、阴性对照探针、线虫cel-39探针。 [0008] In the present invention, comprising a single-stranded DNA probe specific for the perfect match probe of miRNAs, single-base mismatch probes, negative control probe, nematode cel-39 probe. [0009]本发明中,所述针对特定 miRNAs 探针包括miR-1、miR_133a、miR-145、miR_146a、 miR-206、miR_208a、miR-21、miR_29a、miR-499 探针。 [0009] In the present invention, the probe comprising miR-1, miR_133a, miR-145, miR_146a, miR-206, miR_208a, miR-21, miR_29a, miR-499 probe specific miRNAs. [0010] 本发明中,所述蛋白标记物抗体包括针对cTnT、cTnl、CK-MM、CK-MB的特异性抗体、阴性对照抗体、牛血清白蛋白抗体。 [0010] In the present invention, the protein comprises a labeled antibody specific for antibodies cTnT, cTnl, CK-MM, CK-MB, and negative control antibody, bovine serum albumin antibodies. [0011] 本发明硅纳米线生物检测芯片包括半导体衬底、生长在半导体衬底上的二氧化硅隔离层、生长在二氧化硅隔离层上的多晶硅层、和生长在多晶硅层上的钝化层;其中,多晶硅层中包括图形化形成的硅纳米线阵列;钝化层的结构为从下至上依次包括SiON层、TaN 层和Ta2O5层,且TaN/Ta205层仅覆盖于硅纳米线阵列中各硅纳米线的表面和侧壁。 [0011] The present invention is a silicon nanowire biological detection chip includes a semiconductor substrate, a semiconductor grown on a silicon dioxide substrate isolation layer, the isolation layer is grown on a silica polysilicon layer, and a passivation layer is grown on the polysilicon layer; wherein the silicon nanowire array comprising a patterned polysilicon layer is formed; the structure is a passivation layer includes, in order from bottom to SiON layer, TaN layer, and a Ta2O5 layer, and TaN / Ta205 layer covers only silicon nanowire arrays surface and the sidewalls of each silicon nanowires. [0012] 本发明硅纳米线生物检测芯片中,所述二氧化硅隔离层的厚度为1000 A- 5000 Ac 所述多晶硅层厚度为5<) Α-Ι000 A。 [0012] The silicon nanowire biological detection chip of the present invention, the thickness of the silica spacer layer 1000 A- 5000 Ac said polysilicon layer having a thickness of 5 <) Α-Ι000 A. 所述硅纳米线的线宽范围为5nm〜130nm;其厚度为5nm〜IOOnm。 The line width of the silicon nanowire is 5nm~130nm; thickness of 5nm~IOOnm. 所述SiON层的厚度为10 A- 50 A;所述TaN层的厚度为10 A〜50 A;所述Ta205 层的厚度为10人〜50 A。 The thickness of the SiON layer 10 A- 50 A; the thickness of the TaN layer 10 A~50 A; Ta205 said layer has a thickness of 10 ~50 A. [0013] 本发明还提供一种用于同时检测miRNAs与蛋白标记物的硅纳米线芯片的制备方法,包括如下步骤:[0014] 步骤SOl :提供半导体衬底;[0015] 步骤S02 :在所述半导体衬底上生长二氧化硅隔离层;[0016] 步骤S03 :在所述二氧化硅隔离层上生长多晶硅层;[0017] 步骤S04 :图形化所述多晶硅层以形成硅纳米线阵列;[0018] 步骤S05 :在所述硅纳米线阵列上生长一定厚度的钝化层,其中,所述钝化层结构从上至下依次包括SiON层和TaN/Ta205层;[0019] 步骤S06 :去除硅纳米线阵列中各硅纳米线之间的TaN/Ta205层。 [0013] The present invention also provides a method for simultaneously preparing a silicon nanowire chip miRNAs detected marker protein, comprising the steps of: [0014] Step SOl: providing a semiconductor substrate; [0015] Step S02 is: in the grown silicon dioxide on a semiconductor substrate of said isolation layer; [0016] step S03: growing a polysilicon layer on the silicon dioxide isolation layer; [0017] step S04: patterning said polysilicon layer to form a silicon nanowire arrays; [0018] step S05: growing in certain thickness on the silicon nanowire arrays passivation layer, wherein the passivation layer structure includes, in order from top to bottom and SiON layer TaN / Ta205 layer; [0019] step S06: TaN removal between the silicon nanowire arrays each silicon nanowire / Ta205 layer. [0020] 本发明制备方法中,所述步骤S02中的所述二氧化硅隔离层生长工艺为湿氧氧化工艺。 [0020] The production method of the present invention, the step S02 of the silicon dioxide isolation oxide layer growth process is a wet oxidation process. [0021] 本发明制备方法中,所述步骤S04中的形成硅纳米线阵列是通过等离子干法刻蚀工艺完成的。 [0021] The production method of the present invention, a silicon nanowire arrays in the step S04 is performed by plasma dry etching process and the like to complete. [0022] 本发明制备方法中,所述步骤S05中的SiON层是通过热氧化法在硅纳米线阵列表面生长形成,所述TaN/Ta205层是通过原子层淀积工艺生长形成的。 [0022] The production method of the present invention, the step S05, the SiON layer is formed on nanowire array grown silicon surface by thermal oxidation, the TaN / Ta205 layer is grown by atomic layer deposition process is formed. [0023] 本发明制备方法中,所述步骤S06中的去除工艺是采用等离子干法刻蚀工艺。 [0023] The preparation process of the invention, the removal process in step S06 is the use of plasma dry etching process. [0024] 本发明还提供了一种同时检测miRNAs与蛋白标记物的方法,利用如权利要求I所述的用于同时检测miRNAs与蛋白标记物的娃纳米线芯片,对急性心肌梗塞相关miRNAs与蛋白标记物进行检测,包括如下步骤:[0025] I)每一条硅纳米线在上样前检测0-5V (梯度O. IV)电压下的电流值;[0026] 2)每一条硅纳米线在上样后检测0-5V (梯度O. IV)电压下的电流值;[0027] 3)根据电压电流比计算每一条硅纳米线在上样前后的电阻值;[0028] 4)每一条硅纳米线在上样前后的电阻值比作为有效参数;[0029] 5)预先获得在同批硅纳米线芯片上多种miRNAs及蛋白标记物的不同浓度标准曲线,即取O-IM浓度范围内多个点与电阻值比作图;[0030] 6)每次测定的电阻值比与特定miRNA及蛋白标记物的标准曲线比对,得到待测样品的特定miRNA及蛋白标记物的浓度。 [0024] The present invention also provides a method for the simultaneous detection of miRNAs and protein markers, using as claimed in claim I for the simultaneous detection of miRNAs and baby nanowire chip protein markers associated with acute myocardial infarction miRNAs and detecting marker protein, comprising the steps of: [0025] I) the sample prior to detection 0-5V (gradient O. IV) under the current value of voltage of each of the silicon nanowire; [0026] 2) each silicon nanowires after loading detection 0-5V (gradient O. IV) under the current value of the voltage; [0027] 3) according to each of the voltage-current ratio calculating a resistance value of the silicon nanowire before and after loading; [0028] 4) each silicon nanowires on the resistance value ratio of the sample before and after as a valid parameter; [0029] 5) previously obtained calibration curve of different concentrations of more miRNAs and protein markers on the same batch of silicon nanowire chip, i.e., taking the concentration range O-IM a plurality of points and the resistance value ratio is plotted; [0030] 6) the ratio of the resistance value of each measured and a standard curve with a particular miRNA marker protein ratio, a concentration and specific miRNA marker protein sample to be tested. [0031] 本发明还提供了一种所述同时检测miRNAs与蛋白标记物的硅纳米线芯片在检测急性心肌梗塞相关miRNAs与蛋白标记物中的应用。 [0031] The present invention further provides a method detecting the simultaneous application of silicon nanowires chip miRNAs and protein markers in the detection of acute myocardial infarction-related miRNAs and protein markers of. [0032] 本发明应用的一具体例中,取血浆样品(例如,200uL)与修饰在不同硅纳米线表面的单链DNA探针或抗体结合,导致上样前后每条硅纳米线的电阻发生变化。 [0032] The application of a particular embodiment of the present invention, plasma samples were taken (e.g., 200 uL) to a modified single-stranded DNA probes or antibodies of different binding surface of the silicon nanowire, the resistance from each of the front and rear of the silicon nanowire sample occurs Variety. 由于每一条硅纳米线修饰了针对不同miRNA的探针或针对不同蛋白标记物的抗体,通过分析电阻的变化,对比标准曲线,可以得到每一种特定miRNA或蛋白标记物的浓度绝对值。 Since each of the silicon nanowire modified with a probe or an antibody directed against different proteins of different miRNA markers, by analyzing the change in resistance, compared to the standard curve, the concentration of each obtained protein markers specific miRNA or an absolute value. 每一种指标超出正常范围即被视为异常。 Each index outside the normal range is considered abnormal. [0033] 本发明涉及一种微小核糖核酸(microRNAs, miRNAs)与蛋白标记物联合诊断急性心肌梗塞(acute myocardial infarction, AMI)相关标志物的娃纳米线芯片,其目的在于利用一张硅纳米线集成芯片同时快速检测血浆样品中某些特定miRNAs(例如,miR-1、 miR-133a、miR-145、miR_146a、miR-206、miR_208a、miR-21、miR_29a、miR-499 等)以及目前临床上常用的蛋白标记物(包括心肌肌I丐蛋白T(cardiac troponin T简称cTnT)、心肌肌隹丐蛋白I (cardiactroponin I简称cTnl)、肌酸激酶肌肉型(creatine kinase MM简称CK-MM)、肌酸激酶肌肉型杂化型(creatine kinase MB简称CK-MB))的浓度,从而判断某些特定miRNAs和蛋白标记物的量是否异常,进一步为诊断病人是否发生急性心肌梗塞提供参考。 [0033] The present invention relates to a picornavirus (microRNAs, miRNAs) protein markers in diagnosing acute myocardial infarction (acute myocardial infarction, AMI) baby nanowire chip associated markers, it is an object using a silicon nanowires the integrated chip rapid detection of miRNAs certain plasma samples (e.g., miR-1, miR-133a, miR-145, miR_146a, miR-206, miR_208a, miR-21, miR_29a, miR-499, etc.) and the current clinical commonly used protein markers (including cardiac muscle protein hack I T (cardiac troponin T referred cTnT), cardiac muscle short-tailed hack protein I (cardiactroponin I referred cTnl), muscular creatine kinase (creatine kinase MM referred to as CK-MM), muscle kinase muscular hybrid type (creatine kinase MB abbreviated CK-MB)) concentration, thereby determining the amount of certain miRNAs and protein markers are abnormal, further provides a reference for the diagnosis of whether a patient with acute myocardial infarction. [0034] 本发明用于同时检测miRNAs与蛋白标记物的娃纳米线芯片,是多种单链DNA探针与特异性抗体在硅纳米线阵列中的集成排布。 [0034] The present invention is for the simultaneous detection of miRNAs and protein markers baby nanowire chip, a plurality of single-stranded DNA probe and specific antibody arranged in integrated silicon nanowire arrays. 其中,DNA探针包含三大类:1)用于做阴性对照的完全不匹配探针;2)用于检测样品内参的线虫cel-39探针;以及3)针对目标miRNAs 的探针。 Wherein, DNA probe comprises three categories: 1) was used as a negative control probe does not match exactly; 2) for detecting the internal control nematode sample probe cel-39; and 3) target probe of miRNAs. 每类探针包含三组探针:包括完全匹配探针以及2条单碱基不匹配探针,以去除非特异性结合的影响。 Each probe type probe comprises three groups: a perfect match probe and the two single-base mismatch probes, to remove non-specifically bound impact. 蛋白标记物特异性抗体包含针对cTnT、cTnl、CK-MM和CK-MB的特异性抗体、空白对照、用于阴性对照的兔抗鼠抗体、以及用于检测样品内参的牛血清白蛋白(BSA)抗体。 Marker protein-specific antibodies comprise antibodies specific cTnT, cTnl, CK-MM and CK-MB, and the control, rabbit anti-mouse antibody for the negative control, and bovine serum albumin (BSA internal reference for the test sample )antibody. [0035] 本发明硅纳米线生物检测芯片用于同时检测复杂生物样品中miRNAs和蛋白标记物。 [0035] The silicon nanowire biological detection chip of the present invention for simultaneously detecting miRNAs in complex biological samples and protein markers. 本发明硅纳米线芯片包括:多种单链DNA探针在硅纳米线阵列中的集成排布;以及四种蛋白标记物cTnT、cTnl、CK-MM和CK-MB抗体在硅纳米线阵列中的集成排布。 Silicon nanowire chip of the present invention comprising: a plurality of single-stranded DNA probes arranged in integrated silicon nanowire arrays; four proteins and marker cTnT, cTnl, CK-MM and CK-MB antibody silicon nanowire arrays integration arrangement. 所选miRNAs 和蛋白标记物皆在急性心肌梗塞时相比较正常人的血浆有显著的增高,而不同的miRNAs 和蛋白标记物最早出现变化的时间段各不相同,通过联合检测,达到在较宽的时间窗内诊断急性心梗的目的。 Selected miRNAs and protein markers of acute myocardial infarction during both normal human plasma compared significantly increased, and the time period different miRNAs and protein markers earliest changes vary by joint detection, to achieve a wide the purpose of the diagnosis of acute myocardial infarction in a time window. 更重要的是,由于通常特定miRNAs的变化出现比蛋白标记物更早,本发明本发明硅纳米线生物检测芯片可用于实现早期(临床症状出现1-2小时后)诊断急性心梗的目的。 More importantly, due to changes in specific miRNAs typically occurs earlier than the marker protein, according to the present invention, a silicon nanowire biological detection chip of the present invention may be used for the purpose of early (1-2 hours after the onset of clinical symptoms) diagnosis of acute myocardial infarction. 附图说明[0036] 图I表明本发明硅纳米线芯片中探针及抗体的集成排布示意图。 BRIEF DESCRIPTION [0036] FIG integrated exhaust schematic cloth inventive silicon nanowire chips and probe I show that antibodies. [0037] 图2表明以miR-1区为例的不同类探针的排布图。 [0037] FIG. 2 shows that miR-1 in an Example of different types of probes are arranged in FIG. [0038] 图3表明以cTnT区为例的抗体的排布图。 [0038] Figure 3 shows the arrangement of FIG region cTnT antibody to Case. [0039] 图4表明本发明硅纳米线芯片的制备方法的示意图。 [0039] Figure 4 shows a schematic method of preparing a silicon nano wire according to the present invention the chip. [0040] 图5表明一条硅纳米线上样前后电压电流曲线图,显示上miRNA样后电阻增大。 [0040] Figure 5 shows a graph showing the voltage-current silicon nanoparticles before and after the sample line, an increase in resistance after display miRNA-like. 具体实施方式[0041] 结合以下具体实施例和附图,对本发明作进一步的详细说明,本发明的保护内容不局限于以下实施例。 DETAILED DESCRIPTION [0041] The following specific embodiments and in conjunction with the accompanying drawings, the present invention will be described in further detail, protection of the present invention is not limited to the following examples. 在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点部被包括在本发明中,并且以所附的权利要求书为保护范围。 Without departing from the spirit and scope of the inventive concept, those skilled in the art can conceive of variations and advantages of the present invention are included in the portion, and to the appended claims for the scope of protection. 实施本发明的过程、 条件、试剂、实验方法等,除以下专门提及的内容之外,均为本领域的普遍知识和公知常识, 本发明没有特别限制内容。 The process embodiment of the present invention, conditions, reagents, experimental methods, etc., in addition to content specifically mentioned in the following, are common knowledge and common knowledge in the art, the present invention is not particularly restricted content. [0042] 图I-图3对本发明用于同时检测急性心梗miRNAs与蛋白标记物的硅纳米线芯片中的探针及抗体集成排布进行详细说明。 [0042] FIG I- FIG. 3 of the present invention for the simultaneous detection of acute myocardial infarction miRNAs and protein markers silicon nanowire chip integrated arrangement and antibody probes described in detail. 图4对本发明硅纳米线芯片的制备方法进行详细说明。 FIG method for preparing a silicon nanowire chip of the present invention will be described in detail. [0043] 本发明用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,包括半导体衬底;生长在所述半导体衬底上的二氧化硅隔离层,其厚度为1000 A〜5000 A;生长在所述二氧化硅隔离层上的多晶硅层,其厚度为50 A〜1000 A;生长在所述多晶硅层上的钝化层,该钝化层结构从下至上依次包括SiON层、TaN/Ta205层。 [0043] The present invention is for the simultaneous detection of miRNAs and protein markers silicon nanowire chip comprising a semiconductor substrate; grown silicon dioxide on the semiconductor substrate an isolation layer with a thickness of 1000 A~5000 A; Growth a polysilicon layer on the silicon dioxide isolation layer having a thickness of 50 A~1000 a; polysilicon layer grown on the passivation layer, the passivation layer structure includes, in order from bottom to top SiON layer, TaN / Ta205 Floor. SiON层的厚度为10 A〜50 A; TaN/Ta205层仅覆盖于所述硅纳米线阵列中各硅纳米线的表面和侧壁。 The thickness of SiON layer 10 A~50 A; TaN / Ta205 only layer covering the silicon surface of each sidewall and a silicon nanowire in the nanowire array. TaN层的厚度为10 A〜50 A; Ta2O5层的厚度为10人〜50 Ai多晶硅层中包括图形化形成的硅纳米线阵列;硅纳米线的线宽范围为5nm〜130nm ;娃纳米线的厚度为5nm〜lOOnm。 The thickness of the TaN layer 10 A~50 A; Ta2O5 layer thickness of a silicon nanowire array 10 ~50 Ai comprises a patterned polysilicon layer is formed; the line width of the silicon nanowire is 5nm~130nm; baby nanowires thickness 5nm~lOOnm. [0044] 本发明实施例中,优选地,二氧化硅隔离层的厚度为2000A,多晶硅层的厚度为500A;Si()N层的厚度为10 A5TaN层的厚度为20 A; Ta2O5层的厚度为20 A;硅纳米线的线宽范围为20nm ;硅纳米线的厚度为50nm。 [0044] The thickness of the embodiment of the present invention, preferably, the thickness of the silica spacer layer 2000A, a polysilicon layer 500A; thickness Si () N layer 10 A5TaN layer thickness is 20 A; Ta2O5 layer thickness It is 20 a; the line width of the silicon nanowire is 20 nm; thickness of the silicon nanowire is 50nm. [0045] 图I所示为本发明用于同时检测急性心梗miRNAs与蛋白标记物的硅纳米线芯片一具体实施例的结构示意图。 [0045] As shown in Figure I of the present invention for the simultaneous detection of acute myocardial infarction miRNAs and protein markers silicon nanowires chip a schematic structural diagram of a specific embodiment. 本发明硅纳米线芯片包括一种或多种单链DNA探针在硅纳米线阵列中的集成排布,以及蛋白标记物抗体在硅纳米线阵列中的集成排布,如图I所示,其包括:[0046] I)目标miRNA 区:包含miR-1 区、miR_133a 区、miR-145 区、miR_146a 区、miR-206 区、miR-208a 区、miR-21 区、miR_29a 区、miR-499 区等;[0047] 2)miRNA内参区:即线虫Cel-39区;[0048] 3)miRNA阴性对照区:即与所有miRNAs都有10个以上碱基不匹配的完全不匹配区;[0049] 4)目标蛋白标记物区:包含cTnT区、cTnl区、CK-MM区和CK-MB区;[0050] 6)蛋白内参区:为BSA区;[0051] 7)蛋白阴性对照区:即与所有人源抗原都不能特异性结合的兔抗鼠抗体;[0052] 5)空白区:此区的硅纳米线既不修饰探针也不修饰抗体,目的在于去除背景值的影响。 Silicon nanowire chip of the present invention comprises one or more single-stranded DNA probe in the silicon nanowire array integrated arrangement, a marker protein antibody and silicon nanowire array integrated arrangement, as shown in FIG. I, comprising: [0046] I) miRNA target zone: comprising miR-1 region, miR_133a region, miR-145 region, miR_146a region, miR-206 region, miR-208a region, miR-21 region, miR_29a region, miR-499 zones; [0047] 2) miRNA internal reference area: the area nematode Cel-39; [0048] 3) miRNA negative control zone: i.e. all miRNAs have more than 10 bases that do not match completely matching area; [0049 ] 4) a protein marker region: containing cTnT region, cTnl region, CK-MM region and CK-MB region; [0050] 6) protein internal reference zone: as BSA region; [0051] 7) protein negative control area: i.e. rabbit anti-mouse antibodies to all antigens are not specifically bound; [0052] 5) the blank area: this area silicon nanowire modified neither probe nor modified antibodies, aims to remove the influence of background values. [0053] 本发明用于同时检测急性心梗miRNAs与蛋白标记物的硅纳米线芯片结构与现有的芯片结构显著不同的是,现有的芯片仅在硅纳米线修饰抗体来检测常用的cTnT、CK-MM、 CK-MB指标,从未联合修饰多种miRNA的探针来检测在急性心梗早期即发生显著变化的miRNAs指标。 [0053] The present invention is for the simultaneous detection of acute myocardial infarction miRNAs and protein markers silicon nanowires chip structure and the structure of a conventional chip is significantly different from existing chip only modified antibodies of the silicon nanowire to detect common cTnT , CK-MM, CK-MB index, has never been combined modified miRNA probes to detect various miRNAs an index changes significantly in the early acute myocardial infarction. 本发明中,由于miRNAs指标变化发生的时相较早(例如,本发明中选取的是2小时即有显著升高的miRNAs),使得本发明能达到检测出与早期急性心梗有关的miRNAs, 有助于作为早期诊断的判断指标。 In the present invention, miRNAs index changes due to an earlier phase (e.g., the present invention is selected for 2 hours to have a significant increase miRNAs), so that the present invention can achieve the miRNAs detected early associated with acute myocardial infarction, as early indicators help determine the diagnosis. 同时,在较晚的时相则能通过本发明检测到cTnT等蛋白指标的变化,有助于作为进一步确诊急性心梗的判断指标。 Meanwhile, the present invention at a later time by detecting a change in the phase cTnT proteins such indicators, as a further contributes to the diagnosis of acute myocardial infarction determination index. [0054] 本发明所述硅纳米线芯片中,单链DNA探针可以是针对特定miRNAs的完全匹配探针,或单碱基不匹配探针、或阴性对照探针、或线虫cel-39探针。 [0054] The present invention is a silicon nanowire chip, single-stranded DNA probes can be specific for a perfect match probe of miRNAs, or single-base mismatched probes or negative control probes, or nematode cel-39 probe needle. 优选地,所述针对特定miRNAs 探针包括miR-1、miR_133a、miR-145、miR_146a、miR-206、miR_208a、miR-21、 miR-29a、miR-499探针。 Preferably, the probe comprising miR-1, miR_133a, miR-145, miR_146a, miR-206, miR_208a, miR-21, miR-29a, miR-499 probe specific miRNAs. 本发明一具体实施例的miRNAs区不同类探针的排布图,如图2所示。 A particular embodiment of the present invention, the arrangement of FIG different classes of probes region miRNAs embodiment, as shown in FIG. 在本发明的一个实施例miR-1区,不同类探针的排布如下:1)完全匹配探针:每一个碱基都与目标miRNA互相匹配,此类探针重复修饰3-10条硅纳米线;2)单碱基不匹配探针: 除一个碱基外,其余碱基都与目标miRNA互相匹配,此类探针2条,重复修饰3-10条硅纳米线。 In a region of miR-1 Example embodiment of the present invention, different types of probes are arranged as follows: 1) the perfect match probe: each of the target base matches each miRNA, such probes 3-10 repeating silicone-modified nanowires; 2) single-base mismatch probes: In addition to a base, bases are matched to each other and the rest of the target miRNA, such probes 2, 3-10 repeating modified silicon nanowire. [0055] 本发明中,根据实验结果,在急性心梗2小时显著变化的miRNAs包括以下:[0056] 升高:mir_872、mir-136、mir-122、mir_l、mir-150、mir-434、mir-322、mir-335、 mir-484、mir-378、mir_144、mir_206、mir-145、mir_133a、mir-361、mir_106b、mir_18a、 mir_208a、mir-28、mir_29a、no-mir-16、mir-374、mir-320、mir_15b、mir_99b、mir-330、 mir-350、mir-342、mir-146a、mir-152、mir-24-l、mir-24-2、mir-451、mir-146b、mir-505、 mir-21、mir-93、mir_450a、mir-101b、mir-10b、mir_200c、mir-24-2、mir_34b、mir_34c、 mir-96、mir-425、mir-101a、mir_98、mir_130a、mir_23b、mir_92b、mir_210、mir_140、 mir_133b、let_7d、mir_30e、mir_23a、mir—203、mir—429、mir_27a、mir_199a、mir_130b、mir-217、mir-652、mir_29c、mir_27b、mir_30a、mir-126、mir_30d、mir_200b、mir_148b、 mir-127、mir-127、mir_133a、mir_216a、mir-221、mir_28、mir_379、mir-455、mir_487b、 mir-499、mir-7a_l、mir-872 ;[0057] 降低:mir-10a [0055] In the present invention, according to experimental results, in acute myocardial infarction 2 hours miRNAs significant changes include the following: [0056] increased: mir_872, mir-136, mir-122, mir_l, mir-150, mir-434, mir-322, mir-335, mir-484, mir-378, mir_144, mir_206, mir-145, mir_133a, mir-361, mir_106b, mir_18a, mir_208a, mir-28, mir_29a, no-mir-16, mir- 374, mir-320, mir_15b, mir_99b, mir-330, mir-350, mir-342, mir-146a, mir-152, mir-24-l, mir-24-2, mir-451, mir-146b, mir-505, mir-21, mir-93, mir_450a, mir-101b, mir-10b, mir_200c, mir-24-2, mir_34b, mir_34c, mir-96, mir-425, mir-101a, mir_98, mir_130a, mir_23b, mir_92b, mir_210, mir_140, mir_133b, let_7d, mir_30e, mir_23a, mir-203, mir-429, mir_27a, mir_199a, mir_130b, mir-217, mir-652, mir_29c, mir_27b, mir_30a, mir-126, mir_30d, mir_200b, mir_148b, mir-127, mir-127, mir_133a, mir_216a, mir-221, mir_28, mir_379, mir-455, mir_487b, mir-499, mir-7a_l, mir-872; [0057] reduction: mir-10a mir-139、mir_328b、mir-365、let_7f_l、mir_135a、let_7f_2、 let_7a_2、mir-145、let_7a_l、mir-221、let_7c_2、let_7c_l、mir-132、let_7e、mir-212、 let_7b、let_7i、mir_135b、mir_181c、mir-187、mir_23a、mir-30c_l、mir-324、mir-338、 mir-351、mir-3545、mir-377、mir-421、mir-451、mir_708o[0058] 针对上述部分变化最显著的miRNAs,本发明设计的检测探针及序列如表I所示:[0059] 表I :miRNAs的检测探针序列[0060] mir-139, mir_328b, mir-365, let_7f_l, mir_135a, let_7f_2, let_7a_2, mir-145, let_7a_l, mir-221, let_7c_2, let_7c_l, mir-132, let_7e, mir-212, let_7b, let_7i, mir_135b, mir_181c, mir-187, mir_23a, mir-30c_l, mir-324, mir-338, mir-351, mir-3545, mir-377, mir-421, mir-451, mir_708o [0058] for the partial change of the most significant miRNAs the present invention is designed and detection probe sequences as shown in table I: [0059] table I: detection of the probe sequence miRNAs [0060]

Figure CN102980920AD00101
Figure CN102980920AD00111

[0062] 本发明所述娃纳米线芯片中,所述蛋白标记物抗体是针对cTnT、cTnl、CK-MM> CK-MB的特异性抗体、或阴性对照抗体、或牛血清白蛋白抗体。 [0062] The present invention doll nanowire chip, the protein is an antibody against the marker cTnT, cTnl, CK-MM> CK-MB-specific antibody or negative control antibody, antibody or bovine serum albumin. 优选地,如图3所示的本发明一具体实施例的抗体的排布图。 Preferably, the present invention is shown in FIG. 3, a specific antibody embodiment of the arrangement of FIG. 在本发明的一个实施例cTnT区,特异性的兔抗人cTnT抗体重复修饰3-10条硅纳米线。 In one embodiment of the present invention, region cTnT embodiment, specific rabbit anti-cTnT antibody 3-10 repeating modified silicon nanowire. [0063] 实施例I制备用于同时检测miRNAs与蛋白标记物的硅纳米线芯片[0064] 本发明用于同时检测miRNAs与蛋白标记物的硅纳米线芯片的制备方法及过程, 如图4所示,如下:[0065] 步骤SOl :提供半导体衬底I ;[0066] 步骤S02 :采用湿氧氧化工艺,在半导体衬底上生长二氧化硅隔离层2 ;[0067] 步骤S03 :在二氧化硅隔离层上生长多晶硅层3 ;[0068] 步骤S04 :采用等离子干法刻蚀工艺,图形化多晶硅层3以形成硅纳米线阵列4 ;[0069] 步骤S05 :采用热氧化法,在娃纳米线阵列4上生长一定厚度(例如:3nm〜15nm) 的钝化层,通过原子层淀积工艺生长形成TaN/Ta205层,其中,钝化层结构从上至下依次包括SiON 层5 和TaN/Ta205 层6 ;[0070] 步骤S06 :采用等离子干法刻蚀工艺,去除硅纳米线阵列4中各硅纳米线之间的TaN/Ta205 层6。 [0063] Example I is prepared for simultaneously detecting miRNAs silicon nanowires chip with the protein marker [0064] The present invention is a method and process for the simultaneous preparation miRNAs detected marker protein chip silicon nanowires, as shown in FIG 4 shown as follows: [0065] step sOl: providing a semiconductor substrate I; [0066] step S02 is: wet oxygen oxidation process, grown silicon dioxide isolation layer on a semiconductor substrate 2; [0067] step S03: in dioxide growing a polysilicon layer 3 on the silicon separation layer; [0068] step S04: using plasma dry etching process, patterning the polycrystalline silicon layer 3 to form a silicon nanowire array 4; [0069] step S05: thermal oxidation method, the baby nano growth on a thickness line array 4 (e.g.: 3nm~15nm) passivation layer grown by atomic layer deposition process is formed TaN / Ta205 layer, wherein the passivation layer structure includes, in order from top to bottom SiON layer 5 and TaN / Ta205 layer 6; [0070] step S06: using plasma dry etching process, the TaN removal between the silicon nanowire array 4 each silicon nanowires / Ta205 layer 6. [0071] 然后,点样机在硅纳米线阵列中分别点上探针和抗体,其排布见图1。 [0071] Then, the point prototype silicon nanowire array probes and antibodies, respectively, the point that the arrangement shown in Figure 1. [0072] 实施例2同时检测急性心梗miRNAs与蛋白标记物[0073] 利用可同时检测急性心梗miRNAs与蛋白标记物的硅纳米线芯片,通过在硅纳米线修饰不同的探针或抗体,达到在血浆样品中直接同时检测急性心肌梗塞相关的miRNAs 和蛋白标记物的研究目的。 [0072] Example 2 miRNAs simultaneously detect AMI marker protein [0073] miRNAs acute myocardial infarction can be detected using the marker protein while silicon nanowires chip, by modifying different probes or antibodies silicon nanowires, plasma samples reached directly simultaneous detection of acute myocardial infarction object related miRNAs and protein markers. [0074] 其检测方法如下:[0075] I)每一条硅纳米线在上样前检测0-5V (梯度O.1V)电压下的电流值;[0076] 2)每一条硅纳米线在上样后检测0-5V (梯度O.1V)电压下的电流值;[0077] 3)根据电压电流比计算每一条硅纳米线在上样前后的电阻值;[0078] 4)每一条硅纳米线在上样前后的电阻值比作为有效参数;[0079] 5)预先获得在同批硅纳米线芯片上多种miRNAs及蛋白标记物的不同浓度标准曲线,即取O-1M浓度范围内多个点与电阻值比作图;[0080] 6)每次测定的电阻值比与特定miRNA及蛋白标记物的标准曲线比对,推算待测样品的特定miRNA及蛋白标记物的浓度。 [0074] The detection method is as follows: [0075] I) the current value of each of the silicon nanowires at a sample prior to detection 0-5V (gradient O.1V) voltage; [0076] 2) Each silicon nanowires after the sample is detected 0-5V (gradient O.1V) at a current value of voltage; [0077] 3) a resistance value per silicon nanowires on the voltage and current before and after the sample is calculated according to the ratio; [0078] 4) each silicon nano the resistance value of the line than in the sample before and after as a valid parameter; [0079] 5) previously obtained calibration curve of different concentrations of more miRNAs and protein markers on the same batch of silicon nanowire chip, i.e., taking the multi-O-1M concentration range and the resistance value ratio points plotted; [0080] 6) the ratio of the resistance value of each assay a standard curve and specific miRNA marker protein comparison, and estimate the concentration of a particular miRNA marker protein sample to be tested. [0081] 图5为其中一条娃纳米线上样前后电压电流曲线图,显示上miRNA(本例为U6完全匹配IpM)样后电阻增大。 [0081] FIG. 5 is a graph in which the voltage and current before and after a nanowire-like doll, the display miRNAs (U6 exact match to the present embodiment IPM) increase in resistance after the sample. [0082] 实施例3本发明硅纳米线芯片的检测应用[0083] 由于miRNAs和蛋白标记物在心梗患者血浆样品中出现变化的时相不同,本发明加长了诊断心梗的时间窗,例如,1-72小时,尤其是增加了早期(2小时内)诊断的可能性。 [0082] Example 3 of the present invention, the silicon nanowire chip detection applications [0083] Since the marker proteins and miRNAs when changes occur in the patient plasma samples with different MI, the present invention is a diagnosis of myocardial infarction longer time window, e.g. , 1-72 hours, in particular increasing the likelihood of early diagnosis (2 hours). 取200uL血浆样品与修饰在不同硅纳米线表面的单链DNA探针或抗体结合,导致上样前后每条硅纳米线的电阻发生变化。 200uL plasma samples taken to a modified single-stranded DNA probe or antibody silicon nanowires different binding surface, resulting in the resistance of each of the silicon nanowire changes before and after loading. 由于每一条硅纳米线修饰了针对不同miRNA的探针或针对不同蛋白标记物的抗体,通过分析电阻的变化,对比标准曲线,可以得到每一种特定miRNA 或蛋白标记物的浓度绝对值。 Since each of the silicon nanowire modified with a probe or an antibody directed against different proteins of different miRNA markers, by analyzing the change in resistance, compared to the standard curve, the concentration of each obtained protein markers specific miRNA or an absolute value. 步骤如下:[0084] I)获取待测血浆样品。 The following steps: [0084] I) obtaining test plasma sample. [0085] 收集急性心肌梗塞患者出现胸痛症状后I小时、2小时、4小时、8小时、24小时时提取的血浆样品各200ul,上样于实施例I所制备的硅纳米线芯片;[0086] 2)在37度摇床中杂交半小时;[0087] 3) IXSSC 清洗四遍;[0088] 4)芯片吹干;[0089] 5)每一条硅纳米线在上样后检测0-5V (梯度O. IV)电压下的电流值;[0090] 6)根据电压电流比计算每一条硅纳米线在上样后的电阻值;[0091] 7)每一条硅纳米线在上样后的电阻值与预先测定的上样前的电阻值比作为有效参数;[0092] 8)对照miRNA及蛋白标记物的标准曲线,计算待测样品的特定miRNA及蛋白标记物的浓度。 [0085] in patients with acute myocardial infarction were collected after chest pain I hour, 2 hours, 4 hours, 8 hours, 24 hours extraction in plasma samples of each 200 ul of, loaded on the silicon nanowire chip prepared as described in Example I; [0086 ] 2) hybridization and a half hours at 37 ° shaker; [0087] 3) IXSSC washed four times; [0088] 4) dry chip; [0089] 5) each silicon nanowires after loading detection 0-5V (gradient O. IV) under the current value of the voltage; [0090] 6) a voltage-current ratio calculated for each of the silicon nanowire resistance value after loading according; [0091] 7) each silicon nanowires after loading the the resistance value of the resistance value measured in advance before loading ratio as an effective parameter; [0092] 8) and control miRNA marker protein standard curve and calculate the concentration of a specific miRNA marker protein sample to be tested. [0093]实验结果显示:miR-l、miR_133a、miR_208a、miR_29a、miR-499 在胸痛症状后I 小时取得的血浆样品中即相对正常血浆有显著增高,在胸痛症状后2-8小时取得的样品中达到峰值,在胸痛症状后24小时取得的样品中基本回落。 [0093] The results show: miR-l, miR_133a, miR_208a, miR_29a, miR-499 plasma samples taken after I h symptoms of chest pain that is relatively normal plasma was significantly increased in samples 2-8 hours after chest pain obtained reached the peak, the sample 24 hours after chest pain fall substantially achieved. 而cTnT、cTnl在在胸痛症状后4 小时取得的血浆样品中相对正常血浆有显著增高,在胸痛症状后8-24小时取得的样品中持续升高。 And cTnT, cTnl plasma samples at 4 hours after chest pain relative to normal plasma obtained in significantly increased, continues to rise in a sample taken after 8-24 hours of chest pain. [0094] 以上所述仅为本发明的较佳实施例,并非用来限定本发明的实施范围。 [0094] The foregoing is only preferred embodiments of the present invention, not intended to limit the scope of embodiments of the present invention. 任何所属技术领域中具有通常知识者,在不脱离本发明的精神和范围内,当可作各种变动与润饰,本发明保护范围应以权利要求书所界定的保护范围为准。 Any skilled in the art having ordinary knowledge in the present invention without departing from the spirit and scope, as various changes and modifications may be made, the scope of the present invention should be the scope of the appended claims and their equivalents.

Claims (16)

1. 一种用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,其特征在于,所述芯片包括:至少一种单链DNA探针在硅纳米线阵列中的集成排布,以及蛋白标记物抗体在硅纳米线阵列中的集成排布。 1. A method for the simultaneous detection of miRNAs and silicon nanowires chip marker protein, wherein said chip comprising: at least one single-stranded DNA probes arranged in integrated silicon nanowire arrays, and protein labeling antibody arranged in integrated silicon nanowire arrays.
2.如权利要求I所述的用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,其特征在于,所述单链DNA探针包括针对miRNAs的完全匹配探针、单碱基不匹配探针、阴性对照探针、线虫cel-39探针。 I as claimed in claim 2 for the simultaneous detection of miRNAs and silicon nanowires chip marker protein, wherein said probe comprises a single stranded DNA probe for the exact match of miRNAs, single-base mismatch probe needle, negative control probe, nematode cel-39 probe.
3.如权利要求2所述的用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,其特征在于,所述针对miRNAs 探针包括miR-1、miR_133a、miR-145、miR_146a、miR-206、miR_208a、miR-21、miR-29a、miR-499 探针。 As claimed in claim 3 for the simultaneous detection of miRNAs and silicon nanowires chip marker protein of claim 2, wherein, for the miR-1, miR_133a, miR-145, miR_146a, miR-206 miRNAs probe comprising , miR_208a, miR-21, miR-29a, miR-499 probe.
4.如权利要求I所述的用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,其特征在于,所述蛋白标记物抗体包括针对cTnT、cTnl、CK-MM、CK-MB的特异性抗体、阴性对照抗体、牛血清白蛋白抗体。 As claimed in claim I for the simultaneous detection of miRNAs and silicon nanowires chip marker protein, wherein said protein comprises a labeled antibody specific for cTnT, cTnl, CK-MM, CK-MB of antibody, negative control antibody, bovine serum albumin antibodies.
5.如权利要求I所述的用于同时检测miRNAs与蛋白标记物的硅纳米线芯片,其特征在于,所述芯片结构包括: 半导体衬底; 二氧化硅隔离层,其生长在所述半导体衬底上; 多晶硅层,其生长在所述二氧化硅隔离层上;所述多晶硅层中包括图形化形成的硅纳米线阵列; 钝化层,其生长在所述多晶硅层上;所述钝化层结构从下至上依次包括SiON层、TaN/Ta2O5层;其中,所述TaN/Ta205层仅覆盖于所述硅纳米线阵列中各硅纳米线的表面和侧壁。 As claimed in claim I for the simultaneous detection of miRNAs and silicon nanowires chip marker protein, wherein said chip structure comprising: a semiconductor substrate; silicon dioxide spacer layer grown on said semiconductor a substrate; a polysilicon layer, which is grown on the silicon dioxide isolation layer; the patterned polysilicon layer comprises forming a silicon nanowire arrays; a passivation layer, which is grown on the polysilicon layer; the blunt layered structure from bottom to top comprises SiON layer, TaN / Ta2O5 layer; wherein the TaN / Ta205 only layer covering the silicon surface of each sidewall and a silicon nanowire in the nanowire array.
6.如权利要求5所述的硅纳米线芯片,其特征在于,所述二氧化硅隔离层的厚度为I000A〜5000Ao 6. The silicon nanowire chip according to claim 5, wherein the thickness of the silicon dioxide spacer layer is I000A~5000Ao
7.如权利要求5所述的硅纳米线芯片,其特征在于,所述多晶硅层厚度为50 A-1000 Ad 7. The silicon nanowire chip according to claim 5, wherein said polysilicon layer having a thickness of 50 A-1000 Ad
8.如权利要求5所述的娃纳米线芯片,其特征在于,所述娃纳米线的线宽范围为5nm〜130nm ;其厚度为5nm〜lOOnm。 8. The baby nanowire chip according to claim 5, wherein the line width of the baby nanowires 5nm~130nm; thickness of 5nm~lOOnm.
9.如权利要求5所述的硅纳米线芯片,其特征在于,所述SiON层的厚度为10 A〜50 A;所述TaN层的厚度为10 A〜50 A;所述Ta2O5层的厚度为10 A〜50 A0 9. The silicon nanowire chip of claim 5, wherein the thickness of SiON layer 10 A~50 A; the thickness of the TaN layer 10 A~50 A; the thickness of the Ta2O5 layer to 10 A~50 A0
10. 一种用于同时检测miRNAs与蛋白标记物的硅纳米线芯片的制备方法,其特征在于,包括如下步骤: 步骤SOI:提供半导体衬底; 步骤S02 :在所述半导体衬底上生长二氧化硅隔离层; 步骤S03 :在所述二氧化硅隔离层上生长多晶硅层; 步骤S04 :图形化所述多晶硅层以形成硅纳米线阵列; 步骤S05 :在所述硅纳米线阵列上生长钝化层,其中,所述钝化层结构从下至上依次包括SiON 层和TaN/Ta205 层; 步骤S06 :去除硅纳米线阵列中各硅纳米线之间的TaN层和Ta2O5层。 10. A method for simultaneously detecting method for producing silicon nanowires miRNAs marker protein chip, characterized in that it comprises the following steps: the SOI steps of: providing a semiconductor substrate; Step S02 is: two grown on the semiconductor substrate, a silicon oxide spacer layer; step S03: silicon dioxide grown on the spacer layer, the polysilicon layer; step S04: patterning said polysilicon layer to form a silicon nanowire array; step S05: blunt grown on the silicon nanowire arrays layer, wherein the passivation layer structure comprising successively from bottom to top and SiON layer TaN / Ta205 layer; step S06: removing the TaN layer between the silicon nanowire arrays each layer of silicon nanowires and Ta2O5.
11.如权利要求10所述的制备方法,其特征在于,所述步骤S02中的所述二氧化硅隔离层生长工艺为湿氧氧化工艺。 11. The method as recited in claim 10, wherein, in the step S02 of the silicon dioxide isolation oxide layer growth process is a wet oxidation process.
12.如权利要求10所述的制备方法,其特征在于,所述步骤S04中的形成硅纳米线阵列是通过等离子干法刻蚀工艺完成的。 12. The method as recited in claim 10, wherein said forming a silicon nanowire arrays step S04 is performed by plasma dry etching process and the like to complete.
13.如权利要求10所述的制备方法,其特征在于,所述步骤S05中的SiON层是通过热氧化法在硅纳米线阵列表面生长形成,所述TaN/Ta205层是通过原子层淀积工艺生长形成的。 13. The method as recited in claim 10, wherein, SiON layer in the step S05 is grown nanowire array formed on a silicon surface by thermal oxidation, the TaN / Ta205 layer by atomic layer deposition process of growth formation.
14.如权利要求10所述的制备方法,其特征在于,所述步骤S06中的去除工艺是采用等尚子干法刻蚀工艺。 14. The method as recited in claim 10, wherein said removal process in step S06 is employed Naoko like dry etching process.
15. 一种利用如权利要求I所述的硅纳米线芯片同时检测miRNAs与蛋白标记物的检测方法,其特征在于: 1)每一条硅纳米线在上样前检测0-5V (梯度O. IV)电压下的电流值; 2)每一条硅纳米线在上样后检测0-5V(梯度O. IV)电压下的电流值; 3)根据电压电流比计算每一条硅纳米线在上样前后的电阻值; 4)每一条硅纳米线在上样前后的电阻值比作为有效参数; 5)预先获得在同批硅纳米线芯片上多种miRNAs及蛋白标记物的不同浓度标准曲线,取O-IM浓度范围内多个点与电阻值比作图; 6)每次测定的电阻值比与特定miRNA及蛋白标记物的标准曲线比对,得到待测样品的特定miRNA及蛋白标记物的浓度。 15. A method as claimed in claim I using the silicon nanowires chip miRNAs simultaneously detecting method for detecting marker protein, wherein: 1) each of silicon nanowires in the sample prior to detection 0-5V (gradient O. IV) under the current value of voltage; 2) a current value of each of the silicon nanowire at a detection 0-5V (gradient O. IV) the voltage on the sample; 3) according to each of the voltage-current ratio is calculated on a sample of silicon nanowires the resistance values ​​before and after; 4) each in a silicon nanowire on a resistance value ratio of the sample before and after as a valid parameter; 5) previously obtained calibration curve of different concentrations of more miRNAs and protein markers on the same batch of silicon nanowire chip, taken O-IM plurality of points over the range of concentrations was plotted and the resistance value; 6) the ratio of the resistance value of each assay a standard curve and specific miRNA marker protein alignment give specific miRNA marker protein and the sample to be tested concentration.
16.如权利要求I所述的硅纳米线芯片在检测急性心肌梗塞miRNAs中的应用,其特征在于,所述硅纳米线芯片在急性心肌梗塞症状出现的I小时-72小时内检测到急性心肌梗塞miRNAs的变化。 I as claimed in claim 16. Use of the silicon nanowires chip miRNAs detected in acute myocardial infarction, characterized in that said silicon nanowire chips detected within 72 hours of acute myocardial I hour in onset of symptoms of acute myocardial infarction infarct changes of miRNAs.
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