WO2014075373A1 - Silicon nanowire chip for simultaneously detecting mirnas and protein markers, detecting method and application thereof - Google Patents

Silicon nanowire chip for simultaneously detecting mirnas and protein markers, detecting method and application thereof Download PDF

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WO2014075373A1
WO2014075373A1 PCT/CN2012/087861 CN2012087861W WO2014075373A1 WO 2014075373 A1 WO2014075373 A1 WO 2014075373A1 CN 2012087861 W CN2012087861 W CN 2012087861W WO 2014075373 A1 WO2014075373 A1 WO 2014075373A1
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silicon nanowire
mir
layer
silicon
mirnas
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PCT/CN2012/087861
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French (fr)
Chinese (zh)
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朱建军
赵宇岚
蒋宾
何靖
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上海集成电路研发中心有限公司
华东师范大学
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Publication of WO2014075373A1 publication Critical patent/WO2014075373A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L29/00—Semiconductor 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
    • H01L29/02—Semiconductor bodies ; Multistep manufacturing processes therefor
    • H01L29/06—Semiconductor 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
    • H01L29/0657—Semiconductor 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
    • H01L29/0665—Semiconductor 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
    • H01L29/0669—Nanowires or nanotubes
    • H01L29/0673—Nanowires or nanotubes oriented parallel to a substrate
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L29/00—Semiconductor 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
    • H01L29/02—Semiconductor bodies ; Multistep manufacturing processes therefor
    • H01L29/12—Semiconductor bodies ; Multistep manufacturing processes therefor characterised by the materials of which they are formed
    • H01L29/16—Semiconductor 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

Definitions

  • Silicon nanowire chip for simultaneously detecting miRNAs and proteins, and method and application thereof
  • the invention relates to the field of biomolecule detection, in particular to a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, a preparation method thereof, a detection method and an application thereof.
  • cardiovascular disease is a common disease that threatens human health, and acute myocardial infarction is extremely burdensome for society and patients due to rapid onset, mortality and disability. If the risk of acute myocardial infarction is to be reduced, early diagnosis, targeted treatment, and adjustment of medication based on progression of the disease are essential, and the detection and monitoring of certain biomarkers clearly contributes to these aspects.
  • cTnT, cTnI, CK-MM and CK-MB, microRNAs (miRNAs) have been considered as promising biomarkers in recent years.
  • Typical elevation and gradual decline in myocardial biochemical markers cTnT or cTnl
  • CK-MB faster increase and decrease
  • miR-208a was significantly elevated 1 hour after surgery.
  • the four plasma miRNAs in patients with AMI 33 patients were significantly higher than those in healthy controls (30 patients, non-AMI coronary heart disease (16 patients), and other cardiovascular diseases (17 patients). More important MiR-208a is not detectable in non-AMI patients, but 100% after 4 hours of AMI symptoms.
  • More studies have reported miR-l, miR-133, miR-328, miR-499- The diagnostic value of 5p et al for AMI. Because miRNAs change earlier, and miRNAs themselves are relatively stable, the use of miRNAs as diagnostic indicators for AMI is quite promising.
  • MicroRNAs are a class of non-coding RNA molecules that are 18-25 nucleotides in length.
  • the diagnostic value of miRNAs for various diseases has received increasing attention.
  • changes in the expression profile of miRNAs in diseases can be sensitively detected.
  • the detection still has the following defects:
  • the detection of samples requires cumbersome steps, including enrichment of total RNA in cells, tissues or blood samples, followed by purification, and then reverse transcription into cDNA, and in some cases PCR Amplification before testing.
  • the root cause is The amount of miRNAs in cells, tissues or blood samples is very low, often at the pM-fM level, which is outside the limits of current assays.
  • the recent nanotechnology-based field effect F E T silicon nanowire array (SiNW) chip is expected to solve this problem by improving detection sensitivity.
  • the field effect transistor is used for signal acquisition and amplification, which can detect the target signal more effectively.
  • This chip has the advantages of high sensitivity, fast detection, easy integration and high-throughput detection. Therefore, the present invention not only solves the problem of easy contamination in the storage application of the silicon nanowire array, but also enables the chip to face in the biodetection even if the composition of the component body to be tested is diverse.
  • the test of diffusion contamination of Na, K, Fe, Cu and Ca plasmas and the influence of various chemical factors such as enthalpy value achieve high detection stability. Summary of invention
  • the present invention overcomes the above drawbacks of the prior art, and proposes a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, which can be used for simultaneously detecting miRNAs and protein markers in complex biological samples, thereby early diagnosis of acute myocardial infarction. miRNAs markers.
  • the present invention provides a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, the chip comprising: an integrated arrangement of at least one single-stranded DNA probe in a silicon nanowire array, and a protein marker antibody in silicon nanometer The integrated arrangement in the line array.
  • the single-stranded DNA probe includes a perfectly matched probe for a specific miRNA, Single base mismatched probe, negative control probe, nematode cel-39 probe.
  • the probes for specific miRNAs include miR-1, miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499 probes.
  • the protein marker antibody includes a specific antibody against cTnT, cTnI, CK-MM, CK-MB, a negative control antibody, and a bovine serum albumin antibody.
  • the silicon nanowire biodetection chip of the present invention comprises a semiconductor substrate, a silicon dioxide isolation layer grown on the semiconductor substrate, a polysilicon layer grown on the silicon dioxide isolation layer, and a passivation layer grown on the polysilicon layer;
  • the polysilicon layer includes a patterned silicon nanowire array;
  • the passivation layer has a structure including a SiON layer, a TaN layer, and a Ta 2 0 5 layer from bottom to top, and the TaN/Ta 2 0 5 layer covers only the silicon nanometer. The surface and sidewalls of each silicon nanowire in the line array.
  • the thickness of the silicon dioxide isolation layer is 1000 A to 5000 A.
  • the polysilicon layer has a thickness of 50 A to 1000 A.
  • the silicon nanowires have a line width ranging from 5 nm to 130 nm; and a thickness of 5 nm to 100 nm.
  • the thickness of the SiON layer is 10 A to 50 A; the thickness of the TaN layer is 10 A to 50 A; and the thickness of the Ta205 layer is 10 A to 50 A.
  • the invention also provides a preparation method of a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, comprising the following steps:
  • Step S01 providing a semiconductor substrate
  • Step S02 growing a silicon dioxide isolation layer on the semiconductor substrate
  • Step S03 growing a polysilicon layer on the silicon dioxide isolation layer
  • Step S04 patterning the polysilicon layer to form a silicon nanowire array
  • Step S05 growing a passivation layer of a certain thickness on the silicon nanowire array, wherein The passivation layer structure includes a SiON layer and a TaN/Ta 2 0 5 layer in order from top to bottom;
  • Step S06 removing the TaN/Ta 2 0 5 layer between the silicon nanowires in the silicon nanowire array.
  • the growth process of the silicon dioxide isolation layer in the step S02 is a wet oxidation oxidation process.
  • the forming of the silicon nanowire array in the step S04 is performed by a plasma dry etching process.
  • the SiON layer in the step S05 is formed by surface growth on the surface of the silicon nanowire array by thermal oxidation, and the TaN/Ta 2 0 5 layer is formed by an atomic layer deposition process.
  • the removal process in the step S06 is a plasma dry etching process.
  • the present invention also provides a method for simultaneously detecting miRNAs and protein markers, using the silicon nanowire chip for simultaneously detecting miRNAs and protein markers according to claim 1, for miRNAs and protein markers related to acute myocardial infarction To perform the test, the following steps are included:
  • Each silicon nanowire detects the current value at 0-5V (gradient 0.1V) voltage before loading
  • the resistance value of each measurement is compared with a standard curve of a specific miRNA and a protein marker, and the concentration of a specific miRNA and a protein marker of the sample to be tested is obtained.
  • the invention also provides a silicon nanowire chip for simultaneously detecting miRNAs and protein markers for detecting acute myocardial infarction-related miRNAs and protein markers.
  • a plasma sample (e.g., 200 uL) is combined with a single-stranded DNA probe or antibody modified on the surface of different silicon nanowires, resulting in a change in the electrical resistance of each of the silicon nanowires before and after loading. Since each of the silicon nanowires modifies probes for different miRNAs or antibodies against different protein markers, by analyzing the change in resistance, the absolute value of each specific miRNA or protein marker can be obtained by comparing the standard curve. Each indicator is considered abnormal if it is outside the normal range.
  • the invention relates to a silicon nanowire chip for synthesizing acute myocardial infarction (AMI) related markers in combination with microRNAs (miRNAs) and protein markers, the purpose of which is to utilize a silicon nanowire integrated chip simultaneously Rapid detection of certain specific miRNAs in plasma samples (eg, miR-K miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499, etc.) and current Clinically used protein markers (including cardiac troponin T (cTnT), cardiac troponin I (cTnI), creatine kinase MM (CK-MM), The concentration of creatine kinase MB (CK-MB) is used to determine whether the amount of certain specific miRNAs and protein markers is abnormal, and further provides a reference for diagnosing whether patients have acute myocardial infarction.
  • AMI acute myocardial infarction
  • miRNAs microRNAs
  • protein markers e
  • the invention relates to a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, and is an integrated arrangement of a plurality of single-stranded DNA probes and specific antibodies in a silicon nanowire array.
  • DNA probes include three major categories: 1) completely mismatched probes for negative control; 2) nematode cel-39 probes for detecting sample internal references; and 3) probes for target miRNAs.
  • Each type of probe contains three groups Probes: Includes perfectly matched probes and 2 single base mismatched probes to remove the effects of non-specific binding.
  • Protein marker-specific antibodies comprise specific antibodies against cTnT, cTnI, CK-MM and CK-MB, blank controls, rabbit anti-mouse antibodies for negative controls, and bovine serum albumin (BSA) for the detection of sample internal reference ) antibodies.
  • BSA bovine serum albumin
  • the silicon nanowire biodetection chip of the invention is used for simultaneously detecting miRNAs and protein markers in complex biological samples.
  • the silicon nanowire chip of the present invention comprises: an integrated arrangement of a plurality of single-stranded DNA probes in a silicon nanowire array; and four protein markers cTnT, cTnI, CK-MM and CK-MB antibodies in a silicon nanowire array Integrated layout.
  • the selected miRNAs and protein markers were significantly increased in the acute myocardial infarction compared with the normal human plasma, while the different miRNAs and protein markers showed different changes in the earliest time period, which was achieved by joint detection. The purpose of diagnosing acute myocardial infarction in the time window.
  • the silicon nanowire biodetection chip of the present invention can be used for the purpose of early diagnosis (after 1-2 hours of clinical symptoms) for the diagnosis of acute myocardial infarction.
  • Fig. 1 is a view showing the integrated arrangement of probes and antibodies in the silicon nanowire chip of the present invention.
  • Figure 2 shows the layout of different types of probes using the miR-1 region as an example.
  • Figure 3 shows the arrangement of antibodies using the cTnT region as an example.
  • Fig. 4 is a view showing the preparation method of the silicon nanowire chip of the present invention.
  • Figure 5 shows a graph of voltage and current before and after a sample on a silicon nanowire, showing an increase in resistance after miRNA-like.
  • Fig. 1 - Fig. 3 are a detailed description of the probe and antibody integrated arrangement in the silicon nanowire chip of the present invention for simultaneously detecting acute myocardial infarction miRNAs and protein markers.
  • Fig. 4 is a detailed description of the preparation method of the silicon nanowire chip of the present invention.
  • the invention relates to a silicon nanowire chip for simultaneously detecting miRNAs and a protein label, comprising a semiconductor substrate; a silicon dioxide isolation layer grown on the semiconductor substrate, having a thickness of 1000 A ⁇ 5000 A; growing in the a polysilicon layer on the silicon dioxide isolation layer having a thickness of 50 A to 1000 A; a passivation layer grown on the polysilicon layer, the passivation layer structure including a SiON layer, TaN/Ta 2 0 5 in this order from bottom to top Floor.
  • the thickness of the SiON layer is 10 A to 50 A; the TaN/Ta 2 0 5 layer covers only the surface and sidewalls of the respective silicon nanowires in the silicon nanowire array.
  • the thickness of the TaN layer is 10 A to 50 A; the thickness of the Ta 2 0 5 layer is 10 A to 50 A.
  • the polysilicon layer includes a patterned silicon nanowire array; the silicon nanowire has a line width ranging from 5 nm to 130 nm; and the silicon nanowire has a thickness of 5 nm to 100 nm.
  • the thickness of the silicon dioxide isolation layer is 2000A
  • the thickness of the polysilicon layer is 500A
  • the thickness of the SiON layer is 10 A
  • the thickness of the TaN layer is 20 A
  • the thickness of the Ta 2 0 5 layer is 20 A
  • the line width of the silicon nanowires is 20 nm
  • the thickness of the silicon nanowires is 50 nm.
  • Figure 1 shows the silicon of the present invention for simultaneous detection of acute myocardial infarction miRNAs and protein markers.
  • the silicon nanowire chip of the present invention comprises an integrated arrangement of one or more single-stranded DNA probes in a silicon nanowire array, and an integrated arrangement of protein label antibodies in a silicon nanowire array, as shown in FIG. It includes:
  • miRNA region including 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 District, etc.
  • miRNA internal reference area nematode Cel-39 area
  • miRNA negative control region a completely mismatched region with 10 or more base mismatches with all miRNAs
  • Target protein marker region including cTnT region, cTnl region, CK-MM region and CK-MB region;
  • Protein negative control region a rabbit anti-mouse antibody that does not specifically bind to all human antigens
  • Blank area The silicon nanowires in this area neither modify the probe nor modify the antibody, in order to remove the influence of the background value.
  • the silicon nanowire chip structure for simultaneously detecting acute myocardial infarction miRNAs and protein markers is significantly different from the existing chip structure, and the existing chip only detects the commonly used cTnT, CK- in the silicon nanowire modified antibody.
  • MM, CK-MB index probes that have never been combined with multiple miRNAs to detect miRNAs that are significantly altered early in acute myocardial infarction.
  • the present invention can detect miRNAs associated with early acute myocardial infarction, Helps to serve as an indicator of early diagnosis.
  • the later phase it can pass The invention detects changes in protein indexes such as cTnT, and is helpful as a judgment index for further diagnosis of acute myocardial infarction.
  • the single-stranded DNA probe may be a perfectly matched probe for a specific miRNA, or a single base mismatched probe, or a negative control probe, or a nematode cel-39 probe.
  • the probes for specific miRNAs include miR-1, miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499 probes.
  • An arrangement of different types of probes in the miRNAs region of one embodiment of the present invention is shown in FIG.
  • the miR-1 region the different types of probes are arranged as follows: 1) perfectly matched probes: each base matches the target miRNA, and the probe repeats modification of 3-10 silicon Nanowires; 2) Single-base mismatched probes: In addition to one base, the remaining bases are matched with the target miRNA. Two such probes are repeatedly modified to modify 3-10 silicon nanowires.
  • miRNAs which significantly change in acute myocardial infarction for 2 hours include the following:
  • 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- 23 a , mir-30c-l, mir-324, mir-338, mir-351, mir-3545, mir-377, mir-421 > mir-451, mir-708.
  • miR-133a UUUGGUCCCCUUCAACCAGCUG Perfect match CAGCTGGTTGAAGGGGACCAAA
  • miR-208a AUAAGACGAGCAAAAAGCUUGU exact match: ACAAGCTTTTTGCTCGTCTTAT
  • miR-29a UAGCACCAUCUGAAAUCGGUUA exact match: TAACCGATTTCAGATGGTGCTA
  • the protein marker antibody is a specific antibody against cTnT, cTnK CK-MM, CK-MB, or a negative control antibody, or a bovine serum albumin antibody.
  • a specific rabbit anti-human cTnT antibody is repeatedly modified to modify 3-10 silicon nanowires.
  • Step S01 providing a semiconductor substrate 1 ;
  • Step S02 using a wet oxygen oxidation process, growing a silicon dioxide isolation layer 2 on the semiconductor substrate; step S03: growing a polysilicon layer 3 on the silicon dioxide isolation layer; Step S04: using a plasma dry etching process, patterning the polysilicon layer 3 to form a silicon nanowire array 4;
  • Step S05 using a thermal oxidation method, a passivation layer of a certain thickness (for example, 3 nm to 15 nm) is grown on the silicon nanowire array 4, and a TaN/Ta 2 0 5 layer is formed by an atomic layer deposition process, wherein, passivation is performed.
  • the layer structure includes a SiON layer 5 and a TaN/Ta 2 0 5 layer 6 in order from top to bottom;
  • Step S06 removing the TaN/Ta 2 0 5 layer 6 between the silicon nanowires in the silicon nanowire array 4 by a plasma dry etching process.
  • the detection method is as follows:
  • Each silicon nanowire detects the current value at 0-5V (gradient 0.1V) voltage before loading
  • Each silicon nanowire detects the current value at a voltage of 0-5V (gradient 0.1V) after loading; 3) calculates the resistance value of each silicon nanowire before and after loading according to the voltage-current ratio;
  • Figure 5 is a graph of voltage and current before and after sample on a silicon nanowire, showing the upper miRNA (this For example, U6 is perfectly matched to ⁇ ) and the resistance increases.
  • the present invention lengthens the time window for diagnosing myocardial infarction, for example, 1-72 hours, especially increased early (within 2 hours) diagnosis. possibility.
  • a 200 uL plasma sample was bound to a single-stranded DNA probe or antibody modified on the surface of different silicon nanowires, resulting in a change in the resistance of each silicon nanowire before and after loading. Since each of the silicon nanowires modifies probes against different miRNAs or antibodies against different protein markers, the absolute value of each specific miRNA or protein marker can be obtained by analyzing the change in resistance and comparing the standard curve. Proceed as follows:
  • Plasma samples extracted at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after the symptoms of chest pain occurred in patients with acute myocardial infarction were collected, and the silicon nanowire chips prepared in Example 1 were loaded;
  • the experimental results show: miR- miR-133a, miR-208a, miR-29a, miR-499
  • the plasma samples obtained 1 hour after the symptoms of chest pain showed a significant increase from normal plasma, peaked in the samples taken 2-8 hours after the chest pain symptoms, and basically fell in the samples taken 24 hours after the chest pain symptoms.
  • c TnT and cTnl showed a significant increase in plasma samples obtained 4 hours after the symptoms of chest pain compared with normal plasma, and continued to increase in samples taken 8-24 hours after chest pain symptoms.

Abstract

A silicon nanowire chip for simultaneous detection of miRNAs and protein markers, comprises at least a single-strand DNA probe and protein markers integrated arranged on a silicon nanowire array. The silicon nanowire chip includes: a semiconductor substrate, a silicon dioxide isolating layer, a polycrystalline silicon layer and a passivation layer. The preparation method of the silicon nanowire chip comprises the following steps: providing a semiconductor substrate, growing a silicon dioxide isolating layer on the semiconductor substrate, growing a polycrystalline silicon layer on the silicon dioxide isolating layer, patterning the polycrystalline silicon layer to form the silicon nanowire array, then forming the passivation layer on the formed nanowire array, and finally removing the passivation layer among nanowires in the silicon nanowire array. The operation method of the silicon nanowire chip comprises the steps of: detecting current values of silicon nanowires before and after sampling, accordingly calculating the resistance values before and after sampling, regarding the ratio of the resistance values as an effective parameter, obtaining standard curve of concentration of special miRNAs and protein markers, and acquiring concentration of miRNAs and protein markers in samples upon comparing the tested values with the standard curve. The silicon nanowires chip can detect miRNAs and protein markers simultaneously, and shows good sensitivity, easy integration, high throughput, high stability and antipolution.

Description

同时检测 miRNAs与蛋白^ 物的硅纳米线芯片及 测方法和应用 技术领域  Silicon nanowire chip for simultaneously detecting miRNAs and proteins, and method and application thereof
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。 技术背景  The invention relates to the field of biomolecule detection, in particular to a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, a preparation method thereof, a detection method and an application thereof. technical background
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。 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。 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。  At present, cardiovascular disease is a common disease that threatens human health, and acute myocardial infarction is extremely burdensome for society and patients due to rapid onset, mortality and disability. If the risk of acute myocardial infarction is to be reduced, early diagnosis, targeted treatment, and adjustment of medication based on progression of the disease are essential, and the detection and monitoring of certain biomarkers clearly contributes to these aspects. In addition to several protein markers commonly used in clinical practice, cTnT, cTnI, CK-MM and CK-MB, microRNAs (miRNAs) have been considered as promising biomarkers in recent years.
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）。 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 1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。 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。近年来有研究显示某些 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。 例如 D' Alessandra等人采集了 33例急性 心肌梗死患者的血浆， 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。 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"心肌肌钙蛋白 （cardiac troponin, cTn)更早出现。 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。在结扎法大鼠 AMI模型中， miR-208a在术后 1小时即显著升高。 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。 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。 之后有更多研究报道 miR-l、 miR-133、 miR-328、miR-499-5p等对于 AMI的诊断价值。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。 According to the definition of cardiac infarction in the 2000 European Society of Cardiology and the American College of Cardiology, the diagnosis of acute, evolving or recent myocardial infarction has one of the following conditions: Typical elevation and gradual decline in myocardial biochemical markers (cTnT or cTnl) Or faster increase and decrease (CK-MB), at least accompanied by one of the following conditions: myocardial ischemia symptoms; electropathogram with pathological Q wave; electrocardiogram showing myocardial ischemia (ST segment elevation or depression); coronary artery Intervention (such as coronary angioplasty). However, even the current international diagnostic gold standard cTn usually rises 4-8 hours after myocardial injury, it is recommended that if the cTn level is not increased at the time of admission (about 1 hour with myocardial ischemia), it is required to be 6 The blood test was performed again after -9 hours, and even the third test was required after 12-24 hours. Therefore, if you want to diagnose acute myocardial infarction early, you need to detect some molecular markers that show significant changes earlier. Things. In recent years, studies have shown that certain miRNAs may change early after acute myocardial infarction, suggesting the possibility of miRNAs as diagnostic indicators. For example, D'Alessandra et al. collected plasma from 33 patients with acute myocardial infarction. TaqMan Human MicroRNA A and B Arrays screened miRNAs with more than 8 fold changes after acute myocardial infarction and verified by real-time quantitative PCR. Finally, 6 miRNAs were selected for further study. The results showed that miRNAs were elevated within 3 hours after acute myocardial infarction, and the peaks of miR-1, miR-133a, miR-133b, miR-122 and miR-375 were higher than the current "gold standard" cardiac troponin. (cardiac troponin, cTn) appeared earlier. Wang et al. targeted the selection of muscle-rich miR-1, miR-133a, miR-499 and myocardial-specific miR-208a. In the AMI model of ligation, miR-208a was significantly elevated 1 hour after surgery. In clinical cases, the four plasma miRNAs in patients with AMI (33 patients) were significantly higher than those in healthy controls (30 patients, non-AMI coronary heart disease (16 patients), and other cardiovascular diseases (17 patients). More important MiR-208a is not detectable in non-AMI patients, but 100% after 4 hours of AMI symptoms. More studies have reported miR-l, miR-133, miR-328, miR-499- The diagnostic value of 5p et al for AMI. Because miRNAs change earlier, and miRNAs themselves are relatively stable, the use of miRNAs as diagnostic indicators for AMI is quite promising.
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。近年来 miRNAs对各种疾病的诊断价值越来越受到重视。 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。 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。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。 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。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。 MicroRNAs (miRNAs) are a class of non-coding RNA molecules that are 18-25 nucleotides in length. In recent years, the diagnostic value of miRNAs for various diseases has received increasing attention. Although with the application of modern chip technology, changes in the expression profile of miRNAs in diseases can be sensitively detected. However, the detection still has the following defects: The detection of samples requires cumbersome steps, including enrichment of total RNA in cells, tissues or blood samples, followed by purification, and then reverse transcription into cDNA, and in some cases PCR Amplification before testing. The root cause is The amount of miRNAs in cells, tissues or blood samples is very low, often at the pM-fM level, which is outside the limits of current assays. Moreover, other complex components in biological samples, such as sugars, lipids, metal ions, macromolecular proteins, etc., also interfere with the binding of the target miRNA to the probe, thereby having a significant impact on the sensitivity and specificity of the assay. The cumbersome pre-processing process is one of the bottlenecks that restrict the large-scale use of biochips in the clinic.
最近的基于纳米技术的场效应 F E T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。 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。 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。 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 ΙΉ值等多种化学因素的影响， 即实现了检测的高稳定性。 发明概要  The recent nanotechnology-based field effect F E T silicon nanowire array (SiNW) chip is expected to solve this problem by improving detection sensitivity. Taking the SiNW structure as the core, the field effect transistor is used for signal acquisition and amplification, which can detect the target signal more effectively. This chip has the advantages of high sensitivity, fast detection, easy integration and high-throughput detection. Therefore, the present invention not only solves the problem of easy contamination in the storage application of the silicon nanowire array, but also enables the chip to face in the biodetection even if the composition of the component body to be tested is diverse. The test of diffusion contamination of Na, K, Fe, Cu and Ca plasmas and the influence of various chemical factors such as enthalpy value achieve high detection stability. Summary of invention
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。  The present invention overcomes the above drawbacks of the prior art, and proposes a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, which can be used for simultaneously detecting miRNAs and protein markers in complex biological samples, thereby early diagnosis of acute myocardial infarction. miRNAs markers.
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。  The present invention provides a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, the chip comprising: an integrated arrangement of at least one single-stranded DNA probe in a silicon nanowire array, and a protein marker antibody in silicon nanometer The integrated arrangement in the line array.
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。 In the present invention, the single-stranded DNA probe includes a perfectly matched probe for a specific miRNA, Single base mismatched probe, negative control probe, nematode cel-39 probe.
本发明中， 所述针对特定 miRNAs 探针包括 miR-l、 miR-133a、 miR-145、 miR-146a、 miR-206、 miR-208a、 miR-21、 miR-29a、 miR-499探 针。  In the present invention, the probes for specific miRNAs include miR-1, miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499 probes.
本发明中，所述蛋白标记物抗体包括针对 cTnT、 cTnI、 CK-MM、 CK-MB 的特异性抗体、 阴性对照抗体、 牛血清白蛋白抗体。  In the present invention, the protein marker antibody includes a specific antibody against cTnT, cTnI, CK-MM, CK-MB, a negative control antibody, and a bovine serum albumin antibody.
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 Ta205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。 The silicon nanowire biodetection chip of the present invention comprises a semiconductor substrate, a silicon dioxide isolation layer grown on the semiconductor substrate, a polysilicon layer grown on the silicon dioxide isolation layer, and a passivation layer grown on the polysilicon layer; The polysilicon layer includes a patterned silicon nanowire array; the passivation layer has a structure including a SiON layer, a TaN layer, and a Ta 2 0 5 layer from bottom to top, and the TaN/Ta 2 0 5 layer covers only the silicon nanometer. The surface and sidewalls of each silicon nanowire in the line array.
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 A。所述多晶硅层厚度为 50 A〜1000 A。所述硅纳米线的线宽范围为 5nm〜130nm; 其厚度为 5nm〜100nm。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 A〜 50 A。  In the silicon nanowire biodetection chip of the present invention, the thickness of the silicon dioxide isolation layer is 1000 A to 5000 A. The polysilicon layer has a thickness of 50 A to 1000 A. The silicon nanowires have a line width ranging from 5 nm to 130 nm; and a thickness of 5 nm to 100 nm. The thickness of the SiON layer is 10 A to 50 A; the thickness of the TaN layer is 10 A to 50 A; and the thickness of the Ta205 layer is 10 A to 50 A.
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：  The invention also provides a preparation method of a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, comprising the following steps:
步骤 S01 : 提供半导体衬底；  Step S01: providing a semiconductor substrate;
步骤 S02: 在所述半导体衬底上生长二氧化硅隔离层；  Step S02: growing a silicon dioxide isolation layer on the semiconductor substrate;
步骤 S03 : 在所述二氧化硅隔离层上生长多晶硅层；  Step S03: growing a polysilicon layer on the silicon dioxide isolation layer;
步骤 S04: 图形化所述多晶硅层以形成硅纳米线阵列；  Step S04: patterning the polysilicon layer to form a silicon nanowire array;
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； Step S05: growing a passivation layer of a certain thickness on the silicon nanowire array, wherein The passivation layer structure includes a SiON layer and a TaN/Ta 2 0 5 layer in order from top to bottom;
步骤 S06: 去除硅纳米线阵列中各硅纳米线之间的 TaN/Ta205层。 Step S06: removing the TaN/Ta 2 0 5 layer between the silicon nanowires in the silicon nanowire array.
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。  In the preparation method of the present invention, the growth process of the silicon dioxide isolation layer in the step S02 is a wet oxidation oxidation process.
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。  In the preparation method of the present invention, the forming of the silicon nanowire array in the step S04 is performed by a plasma dry etching process.
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。 In the preparation method of the present invention, the SiON layer in the step S05 is formed by surface growth on the surface of the silicon nanowire array by thermal oxidation, and the TaN/Ta 2 0 5 layer is formed by an atomic layer deposition process.
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。  In the preparation method of the present invention, the removal process in the step S06 is a plasma dry etching process.
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 1所述的用于同时检测 miRNAs与蛋白标记物的硅纳米线芯片，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：  The present invention also provides a method for simultaneously detecting miRNAs and protein markers, using the silicon nanowire chip for simultaneously detecting miRNAs and protein markers according to claim 1, for miRNAs and protein markers related to acute myocardial infarction To perform the test, the following steps are included:
1 ) 每一条硅纳米线在上样前检测 0-5V (梯度 0.1V) 电压下的电流值； 1) Each silicon nanowire detects the current value at 0-5V (gradient 0.1V) voltage before loading;
2) 每一条硅纳米线在上样后检测 0-5V (梯度 0.1V) 电压下的电流值；2) The current value of each silicon nanowire is detected after 0-5V (gradient 0.1V) voltage after loading;
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； 3) calculating the resistance value of each silicon nanowire before and after loading according to the voltage-current ratio;
4) 每一条硅纳米线在上样前后的电阻值比作为有效参数；  4) The ratio of resistance values of each silicon nanowire before and after loading is taken as an effective parameter;
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 0- 1 M浓度范围内多个点与电阻值比作图；  5) Pre-acquisition of different concentration standard curves of various miRNAs and protein markers on the same batch of silicon nanowire chips, that is, taking a ratio of multiple points in the concentration range of 0-1 M to the resistance value;
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。 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。 6) The resistance value of each measurement is compared with a standard curve of a specific miRNA and a protein marker, and the concentration of a specific miRNA and a protein marker of the sample to be tested is obtained. The invention also provides a silicon nanowire chip for simultaneously detecting miRNAs and protein markers for detecting acute myocardial infarction-related miRNAs and protein markers.
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。 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。 每一种指标超出正常范围即 被视为异常。  In a specific embodiment of the application of the present invention, a plasma sample (e.g., 200 uL) is combined with a single-stranded DNA probe or antibody modified on the surface of different silicon nanowires, resulting in a change in the electrical resistance of each of the silicon nanowires before and after loading. Since each of the silicon nanowires modifies probes for different miRNAs or antibodies against different protein markers, by analyzing the change in resistance, the absolute value of each specific miRNA or protein marker can be obtained by comparing the standard curve. Each indicator is considered abnormal if it is outside the normal range.
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) 相关标志物的硅纳米 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-K 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 （包括心肌肌钙蛋白 T (cardiac troponin T 简称 cTnT)、 心肌肌钙蛋 白 I (cardiac troponin I 简称 cTnI)、 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。  The invention relates to a silicon nanowire chip for synthesizing acute myocardial infarction (AMI) related markers in combination with microRNAs (miRNAs) and protein markers, the purpose of which is to utilize a silicon nanowire integrated chip simultaneously Rapid detection of certain specific miRNAs in plasma samples (eg, miR-K miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499, etc.) and current Clinically used protein markers (including cardiac troponin T (cTnT), cardiac troponin I (cTnI), creatine kinase MM (CK-MM), The concentration of creatine kinase MB (CK-MB) is used to determine whether the amount of certain specific miRNAs and protein markers is abnormal, and further provides a reference for diagnosing whether patients have acute myocardial infarction.
本发明用于同时检测 miRNAs与蛋白标记物的硅纳米线芯片，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。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。 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。 蛋白标记物特异性抗体包含针对 cTnT、 cTnI、 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。 The invention relates to a silicon nanowire chip for simultaneously detecting miRNAs and protein markers, and is an integrated arrangement of a plurality of single-stranded DNA probes and specific antibodies in a silicon nanowire array. Among them, DNA probes include three major categories: 1) completely mismatched probes for negative control; 2) nematode cel-39 probes for detecting sample internal references; and 3) probes for target miRNAs. Each type of probe contains three groups Probes: Includes perfectly matched probes and 2 single base mismatched probes to remove the effects of non-specific binding. Protein marker-specific antibodies comprise specific antibodies against cTnT, cTnI, CK-MM and CK-MB, blank controls, rabbit anti-mouse antibodies for negative controls, and bovine serum albumin (BSA) for the detection of sample internal reference ) antibodies.
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。 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、 cTnI、 CK-MM和 CK-MB 抗体在硅纳米线阵列中的集成排布。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。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。 國删  The silicon nanowire biodetection chip of the invention is used for simultaneously detecting miRNAs and protein markers in complex biological samples. The silicon nanowire chip of the present invention comprises: an integrated arrangement of a plurality of single-stranded DNA probes in a silicon nanowire array; and four protein markers cTnT, cTnI, CK-MM and CK-MB antibodies in a silicon nanowire array Integrated layout. The selected miRNAs and protein markers were significantly increased in the acute myocardial infarction compared with the normal human plasma, while the different miRNAs and protein markers showed different changes in the earliest time period, which was achieved by joint detection. The purpose of diagnosing acute myocardial infarction in the time window. More importantly, since the change of specific miRNAs usually occurs earlier than the protein marker, the silicon nanowire biodetection chip of the present invention can be used for the purpose of early diagnosis (after 1-2 hours of clinical symptoms) for the diagnosis of acute myocardial infarction. Country deletion
图 1 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。  Fig. 1 is a view showing the integrated arrangement of probes and antibodies in the silicon nanowire chip of the present invention.
图 2 表明以 miR-1区为例的不同类探针的排布图。  Figure 2 shows the layout of different types of probes using the miR-1 region as an example.
图 3 表明以 cTnT区为例的抗体的排布图。  Figure 3 shows the arrangement of antibodies using the cTnT region as an example.
图 4表明本发明硅纳米线芯片的制备方法的示意图。  Fig. 4 is a view showing the preparation method of the silicon nanowire chip of the present invention.
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。 ^咖 Figure 5 shows a graph of voltage and current before and after a sample on a silicon nanowire, showing an increase in resistance after miRNA-like. ^Caf
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。 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都被包括在本实用新型中， 并且以所附的权 利要求书为保护范围。 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 present invention will be further described in detail in conjunction with the following specific embodiments and drawings, which are not limited to the following embodiments. Variations and advantages that may be conceived by those skilled in the art are included in the present invention, and are in the scope of the appended claims. The processes, conditions, reagents, experimental methods, and the like of the present invention are generally recognized and common knowledge in the art, and the present invention is not particularly limited.
图 1 -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。 图 4对本发明硅纳米线芯 片的制备方法进行详细说明。  Fig. 1 - Fig. 3 are a detailed description of the probe and antibody integrated arrangement in the silicon nanowire chip of the present invention for simultaneously detecting acute myocardial infarction miRNAs and protein markers. Fig. 4 is a detailed description of the preparation method of the silicon nanowire chip of the present invention.
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。 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。 TaN层的厚度为 10 A〜50 A; Ta205 层的厚度为 10 A〜50 A。 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; 硅纳米线的厚度为 5nm〜100nm。 The invention relates to a silicon nanowire chip for simultaneously detecting miRNAs and a protein label, comprising a semiconductor substrate; a silicon dioxide isolation layer grown on the semiconductor substrate, having a thickness of 1000 A~5000 A; growing in the a polysilicon layer on the silicon dioxide isolation layer having a thickness of 50 A to 1000 A; a passivation layer grown on the polysilicon layer, the passivation layer structure including a SiON layer, TaN/Ta 2 0 5 in this order from bottom to top Floor. The thickness of the SiON layer is 10 A to 50 A; the TaN/Ta 2 0 5 layer covers only the surface and sidewalls of the respective silicon nanowires in the silicon nanowire array. The thickness of the TaN layer is 10 A to 50 A; the thickness of the Ta 2 0 5 layer is 10 A to 50 A. The polysilicon layer includes a patterned silicon nanowire array; the silicon nanowire has a line width ranging from 5 nm to 130 nm; and the silicon nanowire has a thickness of 5 nm to 100 nm.
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; SiON层的厚度为 10 A; TaN层的厚度为 20 A; Ta205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。 In the embodiment of the present invention, preferably, the thickness of the silicon dioxide isolation layer is 2000A, the thickness of the polysilicon layer is 500A; the thickness of the SiON layer is 10 A; the thickness of the TaN layer is 20 A; the thickness of the Ta 2 0 5 layer is 20 A; The line width of the silicon nanowires is 20 nm; the thickness of the silicon nanowires is 50 nm.
图 1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。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 1所示， 其包括： Figure 1 shows the silicon of the present invention for simultaneous detection of acute myocardial infarction miRNAs and protein markers. A schematic diagram of the structure of a rice wire chip in a specific embodiment. The silicon nanowire chip of the present invention comprises an integrated arrangement of one or more single-stranded DNA probes in a silicon nanowire array, and an integrated arrangement of protein label antibodies in a silicon nanowire array, as shown in FIG. It includes:
1 ) 示 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； 1) miRNA region: including 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 District, etc.
2) miRNA内参区： 即线虫 Cel-39区； 2) miRNA internal reference area: nematode Cel-39 area;
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；  3) miRNA negative control region: a completely mismatched region with 10 or more base mismatches with all miRNAs;
4) 目标蛋白标记物区： 包含 cTnT区、 cTnl区、 CK-MM区和 CK-MB 区；  4) Target protein marker region: including cTnT region, cTnl region, CK-MM region and CK-MB region;
6) 蛋白内参区： 为 BSA区；  6) Intraprotein reference area: BSA area;
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；  7) Protein negative control region: a rabbit anti-mouse antibody that does not specifically bind to all human antigens;
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。  5) Blank area: The silicon nanowires in this area neither modify the probe nor modify the antibody, in order to remove the influence of the background value.
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。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。 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。 The silicon nanowire chip structure for simultaneously detecting acute myocardial infarction miRNAs and protein markers is significantly different from the existing chip structure, and the existing chip only detects the commonly used cTnT, CK- in the silicon nanowire modified antibody. MM, CK-MB index, probes that have never been combined with multiple miRNAs to detect miRNAs that are significantly altered early in acute myocardial infarction. In the present invention, since the change of the miRNAs index occurs earlier (for example, the miRNAs which are significantly increased in 2 hours in the present invention), the present invention can detect miRNAs associated with early acute myocardial infarction, Helps to serve as an indicator of early diagnosis. At the same time, in the later phase, it can pass The invention detects changes in protein indexes such as cTnT, and is helpful as a judgment index for further diagnosis of acute myocardial infarction.
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。优选地，所述针对特定 miRNAs探针包括 miR-l、 miR-133a、 miR-145、 miR-146a、 miR-206、 miR-208a、 miR-21、 miR-29a、 miR-499探针。 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。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 the silicon nanowire chip of the present invention, the single-stranded DNA probe may be a perfectly matched probe for a specific miRNA, or a single base mismatched probe, or a negative control probe, or a nematode cel-39 probe. Preferably, the probes for specific miRNAs include miR-1, miR-133a, miR-145, miR-146a, miR-206, miR-208a, miR-21, miR-29a, miR-499 probes. An arrangement of different types of probes in the miRNAs region of one embodiment of the present invention is shown in FIG. In one embodiment of the invention, the miR-1 region, the different types of probes are arranged as follows: 1) perfectly matched probes: each base matches the target miRNA, and the probe repeats modification of 3-10 silicon Nanowires; 2) Single-base mismatched probes: In addition to one base, the remaining bases are matched with the target miRNA. Two such probes are repeatedly modified to modify 3-10 silicon nanowires.
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：  In the present invention, according to the experimental results, miRNAs which significantly change in acute myocardial infarction for 2 hours include the following:
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- 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; Elevation: 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-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;
降低： 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-708。 Lower: 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- 23 a , mir-30c-l, mir-324, mir-338, mir-351, mir-3545, mir-377, mir-421 > mir-451, mir-708.
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 1所示：  For the miRNAs most prominent in the above partial changes, the detection probes and sequences designed by the present invention are shown in Table 1:
表 1 : miRNAs的检测探针序列  Table 1: Detection probe sequences for miRNAs
miRNA 序列 探针序列  miRNA sequence probe sequence
miR-1 UGGAAUGUAAAGAAGUAUGUAU 完全匹配： ATACATACTTCTTTACATTCCA  miR-1 UGGAAUGUAAAGAAGUAUGUAU exact match: ATACATACTTCTTTACATTCCA
单碱基不匹配： ATACATTCTTCTTTACATTCCA;  Single base mismatch: ATACATTCTTCTTTACATTCCA;
ATACTTACTTCTTTACATTCCA  ATACTTACTTCTTTACATTCCA
miR-133a UUUGGUCCCCUUCAACCAGCUG 完全匹配 CAGCTGGTTGAAGGGGACCAAA  miR-133a UUUGGUCCCCUUCAACCAGCUG Perfect match CAGCTGGTTGAAGGGGACCAAA
单碱基不匹配： CAGCTGATTGAAGGGGACCAAA;  Single base mismatch: CAGCTGATTGAAGGGGACCAAA;
CAGCAGGTTGAAGGGGACCAAA  CAGCAGGTTGAAGGGGACCAAA
miR-145 GGAUUCCUGGAAAUACUGUUCU 完全匹配： AGAACAGTATTTCCAGGAATCC  miR-145 GGAUUCCUGGAAAUACUGUUCU exact match: AGAACAGTATTTCCAGGAATCC
单碱基不匹配： AGAACAATATTTCCAGGAATCC;  Single base mismatch: AGAACAATATTTCCAGGAATCC;
AGAAGAGTATTTCCAGGAATCC  AGAAGAGTATTTCCAGGAATCC
miR-146a UGAGAACUGAAUUCCAUGGGUU 完全匹配： AACCCATGGAATTCAGTTCTCA  miR-146a UGAGAACUGAAUUCCAUGGGUU exact match: AACCCATGGAATTCAGTTCTCA
单碱基不匹配： AACCCACGGAATTCAGTTCTCA;  Single base mismatch: AACCCACGGAATTCAGTTCTCA;
AACCAATGGAATTCAGTTCTCA miR-206 UGGAAUGUAAGGAAGUGUGUGG 完全匹配： CCACACACTTCCTTACATTCCA  AACCAATGGAATTCAGTTCTCA miR-206 UGGAAUGUAAGGAAGUGUGUGG Exact match: CCACACACTTCCTTACATTCCA
单碱基不匹配： CCACACGCTTCCTTACATTCCA;  Single base mismatch: CCACACGCTTCCTTACATTCCA;
CCACTCACTTCCTTACATTCCA  CCACTCACTTCCTTACATTCCA
miR-208a AUAAGACGAGCAAAAAGCUUGU 完全匹配： ACAAGCTTTTTGCTCGTCTTAT  miR-208a AUAAGACGAGCAAAAAGCUUGU exact match: ACAAGCTTTTTGCTCGTCTTAT
单碱基不匹配： ACAAGCCTTTTGCTCGTCTTAT; ACAATCTTTTTGCTCGTCTTAT miR-21 UAGCUUAUCAGACUGAUGUUGA 完全匹配： TCAACATCAGTCTGATAAGCTA Single base mismatch: ACAAGCCTTTTGCTCGTCTTAT; ACAATCTTTTTGCTCGTCTTAT miR-21 UAGCUUAUCAGACUGAUGUUGA Exact match: TCAACATCAGTCTGATAAGCTA
单碱基不匹配： TCAACACCAGTCTGATAAGCTA;  Single base mismatch: TCAACACCAGTCTGATAAGCTA;
TCAATATCAGTCTGATAAGCTA  TCAATATCAGTCTGATAAGCTA
miR-29a UAGCACCAUCUGAAAUCGGUUA 完全匹配： TAACCGATTTCAGATGGTGCTA  miR-29a UAGCACCAUCUGAAAUCGGUUA exact match: TAACCGATTTCAGATGGTGCTA
单碱基不匹配： TAACCGGTTTCAGATGGTGCTA;  Single base mismatch: TAACCGGTTTCAGATGGTGCTA;
TAACAGATTTCAGATGGTGCTA  TAACAGATTTCAGATGGTGCTA
miR-499 UUAAGACUUGCAGUGAUGUUU 完全匹配： AAACATCACTGCAAGTCTTAA  miR-499 UUAAGACUUGCAGUGAUGUUU exact match: AAACATCACTGCAAGTCTTAA
单碱基不匹配： AAACATAACTGCAAGTCTTAA;  Single base mismatch: AAACATAACTGCAAGTCTTAA;
AAACGTCACTGCAAGTCTTAA  AAACGTCACTGCAAGTCTTAA
Cel-39 UCACCGGGUGUAAAUCAGCUUG 完全匹配： CAAGCTGATTTACACCCGGTGA  Cel-39 UCACCGGGUGUAAAUCAGCUUG exact match: CAAGCTGATTTACACCCGGTGA
单碱基不匹配： CAAGCTAATTTACACCCGGTGA;  Single base mismatch: CAAGCTAATTTACACCCGGTGA;
CAAGATGATTTACACCCGGTGA  CAAGATGATTTACACCCGGTGA
阴性对照 完全不匹配： GATCCACTACTCAGTCCTTCGC  Negative control completely mismatched: GATCCACTACTCAGTCCTTCGC
TATACATTCACCATATCCTGCA GATCCACTACTCAGTCCTTCGC  TATACATTCACCATATCCTGCA GATCCACTACTCAGTCCTTCGC
本发明所述硅纳米线芯片中， 所述蛋白标记物抗体是针对 cTnT、 cTnK 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。 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。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 the silicon nanowire chip of the present invention, the protein marker antibody is a specific antibody against cTnT, cTnK CK-MM, CK-MB, or a negative control antibody, or a bovine serum albumin antibody. Preferably, an arrangement of antibodies of a particular embodiment of the invention as shown in Figure 3. In a cTnT region of one embodiment of the invention, a specific rabbit anti-human cTnT antibody is repeatedly modified to modify 3-10 silicon nanowires.
实施例 1 制备用于同时检测 miRNAs与蛋白标记物的硅纳米线芯片 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：  EXAMPLE 1 Preparation of Silicon Nanowire Chip for Simultaneous Detection of miRNAs and Protein Labels The method and process for preparing silicon nanowire chips for simultaneous detection of miRNAs and protein markers are shown in Figure 4, as follows:
步骤 S01 : 提供半导体衬底 1 ;  Step S01 : providing a semiconductor substrate 1 ;
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; 步骤 S03： 在二氧化硅隔离层上生长多晶硅层 3； 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; Step S02: using a wet oxygen oxidation process, growing a silicon dioxide isolation layer 2 on the semiconductor substrate; step S03: growing a polysilicon layer 3 on the silicon dioxide isolation layer; Step S04: using a plasma dry etching process, patterning the polysilicon layer 3 to form a silicon nanowire array 4;
步骤 S05: 采用热氧化法， 在硅纳米线阵列 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; Step S05: using a thermal oxidation method, a passivation layer of a certain thickness (for example, 3 nm to 15 nm) is grown on the silicon nanowire array 4, and a TaN/Ta 2 0 5 layer is formed by an atomic layer deposition process, wherein, passivation is performed. The layer structure includes a SiON layer 5 and a TaN/Ta 2 0 5 layer 6 in order from top to bottom;
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。 Step S06: removing the TaN/Ta 2 0 5 layer 6 between the silicon nanowires in the silicon nanowire array 4 by a plasma dry etching process.
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。 实施例 2 同时检测急性心梗 miRNAs与蛋白标记物  Then, the spotting machine points the probe and the antibody in the silicon nanowire array, respectively, and the arrangement is shown in Fig. 1. Example 2 Simultaneous detection of acute myocardial infarction miRNAs and protein markers
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。  Simultaneous detection of acute myocardial infarction-associated miRNAs and protein markers in plasma samples by modifying different probes or antibodies on silicon nanowires using silicon nanowire chips that simultaneously detect acute myocardial infarction miRNAs and protein markers Research purposes.
其检测方法如下：  The detection method is as follows:
1 ) 每一条硅纳米线在上样前检测 0-5V (梯度 0.1V) 电压下的电流值； 1) Each silicon nanowire detects the current value at 0-5V (gradient 0.1V) voltage before loading;
2) 每一条硅纳米线在上样后检测 0-5V (梯度 0.1V) 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； 2) Each silicon nanowire detects the current value at a voltage of 0-5V (gradient 0.1V) after loading; 3) calculates the resistance value of each silicon nanowire before and after loading according to the voltage-current ratio;
4) 每一条硅纳米线在上样前后的电阻值比作为有效参数；  4) The ratio of resistance values of each silicon nanowire before and after loading is taken as an effective parameter;
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 0- 1 M浓度范围内多个点与电阻值比作图；  5) Pre-acquisition of different concentration standard curves of various miRNAs and protein markers on the same batch of silicon nanowire chips, that is, taking a ratio of multiple points in the concentration range of 0-1 M to the resistance value;
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。  6) The resistance value of each measurement is compared with the standard curve of the specific miRNA and protein label, and the concentration of the specific miRNA and protein label of the sample to be tested is estimated.
图 5 为其中一条硅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 ΙρΜ)样后电阻增大。 Figure 5 is a graph of voltage and current before and after sample on a silicon nanowire, showing the upper miRNA (this For example, U6 is perfectly matched to ΙρΜ) and the resistance increases.
实施例 3 本发明硅纳米线芯片的检测应用  Example 3 Detection Application of Silicon Nanowire Chip of the Invention
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。 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。 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。 步骤如下：  Since the miRNAs and protein markers differ in the phase of changes in the plasma samples of patients with myocardial infarction, the present invention lengthens the time window for diagnosing myocardial infarction, for example, 1-72 hours, especially increased early (within 2 hours) diagnosis. possibility. A 200 uL plasma sample was bound to a single-stranded DNA probe or antibody modified on the surface of different silicon nanowires, resulting in a change in the resistance of each silicon nanowire before and after loading. Since each of the silicon nanowires modifies probes against different miRNAs or antibodies against different protein markers, the absolute value of each specific miRNA or protein marker can be obtained by analyzing the change in resistance and comparing the standard curve. Proceed as follows:
1) 获取待测血浆样品。  1) Obtain the plasma sample to be tested.
收集急性心肌梗塞患者出现胸痛症状后 1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 1所制备的硅纳米线芯片； Plasma samples extracted at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after the symptoms of chest pain occurred in patients with acute myocardial infarction were collected, and the silicon nanowire chips prepared in Example 1 were loaded;
2)在 37度摇床中杂交半小时； 2) Hybriding in a 37 degree shaker for half an hour;
3) 1XSSC清洗四遍；  3) 1XSSC cleaning four times;
4) 芯片吹干；  4) The chip is blown dry;
5) 每一条硅纳米线在上样后检测 0-5V (梯度 0.1V) 电压下的电流值； 5) Each silicon nanowire is tested for current value at 0-5V (gradient 0.1V) after loading;
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； 6) Calculating the resistance value of each silicon nanowire after loading according to the voltage-current ratio;
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；  7) The ratio of the resistance value of each silicon nanowire after loading to the previously determined resistance value before loading is taken as an effective parameter;
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。  8) Calculate the concentration of specific miRNA and protein markers of the sample to be tested against the standard curve of miRNA and protein markers.
实验结果显示： miR- miR-133a、 miR-208a、 miR-29a、 miR-499 在胸痛症状后 1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。 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。 The experimental results show: miR- miR-133a, miR-208a, miR-29a, miR-499 The plasma samples obtained 1 hour after the symptoms of chest pain showed a significant increase from normal plasma, peaked in the samples taken 2-8 hours after the chest pain symptoms, and basically fell in the samples taken 24 hours after the chest pain symptoms. However, c TnT and cTnl showed a significant increase in plasma samples obtained 4 hours after the symptoms of chest pain compared with normal plasma, and continued to increase in samples taken 8-24 hours after chest pain symptoms.
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。 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。  The above is only the preferred embodiment of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the invention should be determined by the scope of the invention as defined by the appended claims.

Claims

权利要求 Rights request
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 silicon nanowire chip for simultaneous detection of miRNAs and protein markers, characterized in that the chip includes: an integrated arrangement of at least one single-stranded DNA probe in a silicon nanowire array, and a protein marker Integrated arrangement of antibodies in silicon nanowire arrays.
2、 如权利要求 1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。 2. The silicon nanowire chip for simultaneous detection of miRNAs and protein markers as claimed in claim 1, wherein the single-stranded DNA probe includes a complete matching probe for miRNAs and a single base mismatch probe. needle, negative control probe, C. elegans 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-l、 miR-133a、 miR-145、 miR-146a、 miR-206、 miR-208a、 miR-21、 miR-29a、 miR-499探 针。 3. The silicon nanowire chip for simultaneous detection of miRNAs and protein markers as claimed in claim 2, wherein the probes for miRNAs include miR-1, miR-133a, miR-145, and miR-146a. ,miR-206,miR-208a,miR-21,miR-29a,miR-499 probes.
4、 如权利要求 1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、 cTnK CK-MM、 CK-MB的特异性抗体、 阴性对照抗体、 牛血清白蛋白抗体。 4. The silicon nanowire chip for simultaneous detection of miRNAs and protein markers as claimed in claim 1, wherein the protein marker antibodies include specific antibodies against cTnT, cTnK CK-MM, and CK-MB. , negative control antibody, bovine serum albumin antibody.
5、 如权利要求 1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： 5. The silicon nanowire chip for simultaneous detection of miRNAs and protein markers as claimed in claim 1, characterized in that the chip structure includes:
半导体衬底； semiconductor substrate;
二氧化硅隔离层， 其生长在所述半导体衬底上； A silicon dioxide isolation layer grown on the semiconductor substrate;
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； A polysilicon layer grown on the silicon dioxide isolation layer; the polysilicon layer includes a patterned silicon nanowire array;
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/Ta205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。 Passivation layer, which grows on the polysilicon layer; The passivation layer structure includes a SiON layer and a TaN/Ta 2 0 5 layer from bottom to top; wherein, the TaN/Ta 2 0 5 layer only covers the The surface and sidewall of each silicon nanowire in the silicon 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 1000 A〜5000 A。 6. The silicon nanowire chip according to claim 5, wherein the thickness of the silicon dioxide isolation layer is 1000 Å to 5000 Å.
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 5θ Α〜100θ Α。 7. The silicon nanowire chip according to claim 5, wherein the polysilicon layer is thick The degree is 5θΑ~100θΑ.
8、 如权利要求 5所述的硅纳米线芯片， 其特征在于， 所述硅纳米线的 线宽范围为 5nm〜130nm; 其厚度为 5nm〜100nm。 8. The silicon nanowire chip according to claim 5, wherein the silicon nanowire has a line width ranging from 5 nm to 130 nm; and a thickness ranging from 5 nm to 100 nm.
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 Ta205层的厚度为 10 A〜50 A。 9. The silicon nanowire chip according to claim 5, wherein the thickness of the SiON layer is 10 A~50 A; the thickness of the TaN layer is 10 A~50 A; the Ta 2 0 5 The thickness of the layer is 10 A~50 A.
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： 10. A method for preparing a silicon nanowire chip for simultaneous detection of miRNAs and protein markers, characterized by comprising the following steps:
步骤 S01 : 提供半导体衬底； Step S01: Provide a semiconductor substrate;
步骤 S02: 在所述半导体衬底上生长二氧化硅隔离层； Step S02: grow a silicon dioxide isolation layer on the semiconductor substrate;
步骤 S03 : 在所述二氧化硅隔离层上生长多晶硅层； Step S03: Grow a polysilicon layer on the silicon dioxide isolation layer;
步骤 S04: 图形化所述多晶硅层以形成硅纳米线阵列； Step S04: Pattern the polysilicon layer to form a silicon nanowire array;
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； Step S05: Grow a passivation layer on the silicon nanowire array, wherein the passivation layer structure includes a SiON layer and a TaN/Ta 2 0 5 layer from bottom to top;
步骤 S06: 去除硅纳米线阵列中各硅纳米线之间的 TaN层和 Ta205层。 Step S06: Remove the TaN layer and Ta 2 0 5 layer between the silicon nanowires in the silicon nanowire array.
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 preparation method according to claim 10, wherein the silicon dioxide isolation layer growth process in step S02 is a wet oxygen 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 preparation method according to claim 10, wherein the forming of the silicon nanowire array in step S04 is completed by a plasma dry etching process.
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 preparation method of claim 10, wherein the SiON layer in step S05 is grown on the surface of the silicon nanowire array by thermal oxidation, and the TaN/Ta 2 0 5 layer is formed by atomic It is grown by layer deposition process.
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 去除工艺是采用等离子干法刻蚀工艺。 14. The preparation method according to claim 10, characterized in that the removal process in step S06 adopts a plasma dry etching process.
15、一种利用如权利要求 1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： 15. A detection method for simultaneously detecting miRNAs and protein markers using the silicon nanowire chip as claimed in claim 1, characterized by:
1 ) 每一条硅纳米线在上样前检测 0-5V (梯度 0.1V) 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 0.1V) 电压下的电流值； 1) The current value of each silicon nanowire at a voltage of 0-5V (gradient 0.1V) is detected before loading; 2) After loading the sample, detect the current value of each silicon nanowire at a voltage of 0-5V (gradient 0.1V);
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； 3) Calculate the resistance value of each silicon nanowire before and after loading the sample based on the voltage-to-current ratio;
4) 每一条硅纳米线在上样前后的电阻值比作为有效参数； 4) The resistance value ratio of each silicon nanowire before and after loading is used as an effective parameter;
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 0- 1M浓度范围内多个点与电阻值比作图； 5) Obtain in advance different concentration standard curves of multiple miRNAs and protein markers on the same batch of silicon nanowire chips, and plot multiple points in the 0-1M concentration range with the resistance value ratio;
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。 6) Compare the resistance value ratio of each measurement with the standard curve of specific miRNA and protein markers to obtain the concentration of specific miRNA and protein markers in the sample to be tested.
16、 如权利要求 1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 1小 时 -72小时内检测到急性心肌梗塞 miRNAs的变化。 16. Application of the silicon nanowire chip in detecting acute myocardial infarction miRNAs according to claim 1, characterized in that the silicon nanowire chip detects acute myocardial infarction within 1 hour to 72 hours after the symptoms of acute myocardial infarction appear. Changes in infarcted miRNAs.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI696703B (en) * 2015-09-30 2020-06-21 中央研究院 Method and kit for making diagnosis of myocardial infarction

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9362108B2 (en) 2012-11-01 2016-06-07 Shanghai Ic R&D Center Co., Ltd Silicon nanowire bio-chip structure
CN103018429B (en) * 2012-11-01 2017-03-15 上海集成电路研发中心有限公司 A kind of structure and its manufacture method for silicon nano-wire biological detection chip
FR3004463A1 (en) * 2013-04-11 2014-10-17 Genethon EXPRESSION SYSTEM FOR SELECTIVE GENE THERAPY
CN104077471A (en) * 2014-06-13 2014-10-01 华东师范大学 Data analysis method used for silicon nanowire chip to detect biomolecule
WO2017107025A1 (en) * 2015-12-21 2017-06-29 黄荣堂 Detection device
CN109402129B (en) * 2018-09-30 2021-09-10 中山大学 Angiostrongylus cantonensis Acsod3 gene and application thereof in treating or relieving angiostrongylus cantonensis disease

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006321829A (en) * 2005-05-17 2006-11-30 Kazuhiko Ishihara Copolymer and method for producing the same
WO2009145359A1 (en) * 2008-05-30 2009-12-03 Canon Kabushiki Kaisha Graft polymer-containing substrate, method for producing the same, target substance-detecting element, and target substance detection kit
CN102354669A (en) * 2011-10-25 2012-02-15 上海华力微电子有限公司 Production method of silicon nano-wire device
CN102356162A (en) * 2009-02-04 2012-02-15 得克萨斯系统大学董事会 Dual targeting of mir-208 and mir-499 in the treatment of cardiac disorders
CN102416184A (en) * 2011-12-08 2012-04-18 哈尔滨医科大学 Application of antisense locked nucleotide sequence of microRNA-1 in preparation of medicines used for preventing or treating heart failure after myocardial infarction
CN102437189A (en) * 2011-11-30 2012-05-02 上海华力微电子有限公司 Silicon nanowire device and manufacturing method thereof
CN102435747A (en) * 2011-10-26 2012-05-02 中国科学院苏州纳米技术与纳米仿生研究所 Acute myocardial infarction diagnosis-oriented biosensor and preparation method thereof
CN102481310A (en) * 2009-05-20 2012-05-30 得克萨斯系统大学董事会 Identification Of Micro-rnas Involved In Post-myocardial Infarction Remodeling And Heart Failure

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070052037A1 (en) * 2005-09-02 2007-03-08 Hongfa Luan Semiconductor devices and methods of manufacture thereof
WO2008070058A2 (en) * 2006-12-04 2008-06-12 Purdue Research Foundation Method and apparatus for detection of molecules using sensor array
CN101592627B (en) * 2009-03-19 2012-12-05 中国科学院苏州纳米技术与纳米仿生研究所 Method for manufacturing and integrating multichannel high-sensitive biosensor
US9029132B2 (en) * 2009-08-06 2015-05-12 International Business Machines Corporation Sensor for biomolecules
CN101993820A (en) * 2009-08-21 2011-03-30 殷勤伟 Method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs
CA2783536A1 (en) * 2009-12-09 2011-06-16 Aviir, Inc. Biomarker assay for diagnosis and classification of cardiovascular disease
CN102110648B (en) * 2009-12-24 2013-05-01 中国科学院微电子研究所 Method for preparing bulk silicon gate-all-around metal oxide semiconductor field effect transistors
KR101259355B1 (en) * 2010-05-12 2013-04-30 광운대학교 산학협력단 Silicon nanowire bio-sensor
CN102719556A (en) * 2011-03-29 2012-10-10 北京五加和分子医学研究所有限公司 AAV vector-based high-throughput miRNA activity detection method, and applications thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006321829A (en) * 2005-05-17 2006-11-30 Kazuhiko Ishihara Copolymer and method for producing the same
WO2009145359A1 (en) * 2008-05-30 2009-12-03 Canon Kabushiki Kaisha Graft polymer-containing substrate, method for producing the same, target substance-detecting element, and target substance detection kit
CN102356162A (en) * 2009-02-04 2012-02-15 得克萨斯系统大学董事会 Dual targeting of mir-208 and mir-499 in the treatment of cardiac disorders
CN102481310A (en) * 2009-05-20 2012-05-30 得克萨斯系统大学董事会 Identification Of Micro-rnas Involved In Post-myocardial Infarction Remodeling And Heart Failure
CN102354669A (en) * 2011-10-25 2012-02-15 上海华力微电子有限公司 Production method of silicon nano-wire device
CN102435747A (en) * 2011-10-26 2012-05-02 中国科学院苏州纳米技术与纳米仿生研究所 Acute myocardial infarction diagnosis-oriented biosensor and preparation method thereof
CN102437189A (en) * 2011-11-30 2012-05-02 上海华力微电子有限公司 Silicon nanowire device and manufacturing method thereof
CN102416184A (en) * 2011-12-08 2012-04-18 哈尔滨医科大学 Application of antisense locked nucleotide sequence of microRNA-1 in preparation of medicines used for preventing or treating heart failure after myocardial infarction

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG GUO-JUN ET AL.,: "SILICON NANOWIRE BIOSENSOR AND ITS APPLICATIONS IN DISEASE DIAGNOSTICS: A REVIEW", ANALYTICA CHIMICA ACTA, vol. 749, 24 October 2012 (2012-10-24), pages 1 - 15, XP028943830, DOI: doi:10.1016/j.aca.2012.08.035 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI696703B (en) * 2015-09-30 2020-06-21 中央研究院 Method and kit for making diagnosis of myocardial infarction

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