WO2014048117A1 - Chip, preparation method and use thereof and method for screening drug - Google Patents

Chip, preparation method and use thereof and method for screening drug Download PDF

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Publication number
WO2014048117A1
WO2014048117A1 PCT/CN2013/075362 CN2013075362W WO2014048117A1 WO 2014048117 A1 WO2014048117 A1 WO 2014048117A1 CN 2013075362 W CN2013075362 W CN 2013075362W WO 2014048117 A1 WO2014048117 A1 WO 2014048117A1
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WO
WIPO (PCT)
Prior art keywords
chip
group
polyethylene glycol
solid support
glycol modified
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PCT/CN2013/075362
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French (fr)
Chinese (zh)
Inventor
朱劲松
何建安
陈新颖
彭开美
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广州高通生物技术有限公司
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Publication of WO2014048117A1 publication Critical patent/WO2014048117A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/34—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
    • C03C17/42—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions at least one coating of an organic material and at least one non-metal coating

Definitions

  • Chip preparation method, use and method for screening medicine
  • the invention relates to the field of drug screening, in particular to a chip, a preparation method, a use and a method for screening a drug. Background technique
  • the present invention provides a chip, a preparation method, a use, and a method for screening a drug.
  • the chip provided by the invention has good anti-protein non-specific adsorption ability, can fix a plurality of small molecular substances in parallel, and is used for screening drugs with complex components, and can effectively analyze small molecular compounds only by using a trace amount of compounds. Bioactivity with specific disease targets, sensitivity! 3 ⁇ 4.
  • the present invention provides the following technical solutions:
  • the invention provides a chip comprising a solid support, a modified polyethylene glycol at both ends, and a photocrosslinking agent; the polyethylene glycol modified at both ends is modified at one end by a riveting group and the other end is modified by a binding group.
  • the polyethylene glycol; the solid support is linked to the riveting group; the photocrosslinking agent is bonded to the binding group; the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
  • Binding group selected from -COOH, -OH, -NH 2, -OCH 3.
  • the chip provided by the invention is introduced into the photocrosslinker on the surface of the solid support by the polyethylene glycol modified at both ends, and is converted into a highly reactive intermediate carbene by ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ).
  • Carbene groups can bind small molecules in a variety of ways without the need for small molecules with specific functional groups.
  • the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
  • the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
  • the photocrosslinking agent is coupled to the binding group via an acid amine condensation reaction.
  • the polyethylene glycol modified at both ends of the chip provided by the present invention has a molecular weight of 100 to 5000. In other embodiments of the present invention, the polyethylene glycol modified at both ends of the chip provided by the present invention has a molecular weight of 1000 to 4000.
  • the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is 1500 to 3400.
  • the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 2000 to 2800.
  • the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 100 to 500.
  • the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 500 to 1,000.
  • the molecular weight of the modified polyethylene glycol at both ends of the chip provided by the present invention is from 1000 to 2000.
  • the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 2000 to 5000.
  • the solid support is a glass substrate or a gold-plated glass substrate.
  • the invention also provides a method for preparing a chip, comprising the following steps:
  • Step 1 The polyethylene glycol is respectively connected to the riveting group and the binding group to obtain polyethylene glycol modified at both ends;
  • Step 2 The solid support is pretreated and connected to the riveting group
  • Step 3 After activating the binding group, the photocrosslinking agent is coupled with an acid amine condensation reaction, and the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
  • the binding group is selected from the group consisting of -COOH, -OH, -Li 2 .
  • the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
  • the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
  • the polyethylene glycol modified at both ends has a molecular weight of 100 to 5000.
  • the present invention provides a modified polyethylene chip at both ends of the chip.
  • the molecular weight of the alcohol is from 1000 to 4000.
  • the method for preparing a chip provided by the present invention has a molecular weight of 1500 to 3400 modified at both ends.
  • the method for preparing a chip provided by the present invention has a molecular weight of from 2,000 to 2,800 for the polyethylene glycol modified at both ends.
  • the polyethylene glycol modified at both ends in the method for preparing a chip provided by the present invention has a molecular weight of 100 to 500.
  • the polyethylene glycol modified at both ends of the method for preparing a chip provided by the present invention has a molecular weight of 500 to 1000.
  • the method for preparing a chip according to the present invention has a molecular weight of from 1000 to 2000 for the polyethylene glycol modified at both ends.
  • the method for preparing a chip provided by the present invention has a molecular weight of from 2,000 to 5,000 for the polyethylene glycol modified at both ends.
  • the solid support in the method of fabricating the chip provided by the present invention, is a glass substrate or a gold-plated glass substrate.
  • the present invention also provides a chip produced by the above preparation method.
  • the invention also provides the use of the above chip for screening drugs.
  • the invention also provides a method for screening drugs based on a chip, comprising the following steps: Step 1: mixing a sample to be tested with a chip, drying it, placing it under ultraviolet light, washing away the unbound analyte, and obtaining a first chip. , inactivating the unbound site, mixing with the target, and obtaining a first response value by surface plasmon resonance detection;
  • Step 2 The ligand not bound to the target is mixed with the chip as a negative control, dried and exposed to ultraviolet light, and the unconjugated negative control is washed away to obtain a second chip, and the unbound site is inactivated.
  • the second response value is obtained by surface plasmon resonance detection;
  • Step 3 obtaining a ratio of the first response value to the second response value, and determining whether the analyte and the target are combined according to the ratio; when the ratio is not less than 3, The analyte is a drug that binds to the target; when the ratio is less than 3, the analyte is a ligand that does not bind to the target;
  • the chip comprises a solid support, a polyethylene glycol modified at both ends, and a photocrosslinker; the polyethylene glycol modified at both ends is a polyethylene glycol modified at one end by a riveting group and the other end is modified by a binding group; The solid support is attached to the riveting group, and the photocrosslinking agent is linked to the binding group;
  • the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
  • Binding group selected from -COOH, -OH, -NH 2, -OCH 3.
  • the method for screening a drug based on a chip includes the following steps:
  • Step 1 The sample to be tested is spotted on a specific area of the chip by using a high-precision spotter. After drying, it is placed in ultraviolet light for photocrosslinking, and the unbound analyte is washed away, and the unbound site is inactivated and the target is inactivated. Mixing, obtaining a small molecule array, and obtaining a first response value by surface plasmon resonance detection;
  • Step 2 using a high-precision spotting instrument to spot the negative control on a specific area of the chip, drying and then placing ultraviolet light for photocrosslinking Washing off the unbound negative control, inactivating the unbound site, and mixing with the target to obtain a small molecule array, and obtaining a second response value by surface plasmon resonance detection;
  • Step 3 Obtain a ratio of the first response value to the second response value, and determine whether the analyte and the target are combined according to the ratio; when the ratio is not less than 3, the analyte is a drug that binds to the target; when the ratio is less than 3, The analyte is a ligand that does not bind to the target;
  • the chip comprises a solid support, a modified polyethylene glycol at both ends, and a photocrosslinker; the polyethylene glycol modified at both ends is a polyethylene glycol modified at one end by a riveting group and the other end is modified by a binding group; The support is attached to the riveting group, and the photocrosslinking agent is linked to the binding group;
  • the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
  • Binding group selected from -COOH, -OH, -NH 2.
  • the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
  • the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
  • the polyethylene glycol at both ends of the chip has a molecular weight of 100 to 5000.
  • the molecular weight of the modified polyethylene glycol in the chip has a molecular weight of 1000 to 4000.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 1500-3400.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 2000 to 2800.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 100-500.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 500 to 1000.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 1000 to 2000.
  • the molecular weight of the polyethylene glycol at both ends of the chip is 2000 to 5000.
  • the solid support of the chip is a glass substrate or a gold-plated glass substrate.
  • the drug is a traditional Chinese medicine compound or a natural product.
  • the invention provides a chip, a preparation method and a drug screening method.
  • the chip comprises a solid support, a self-assembling compound attached to a solid support, and a photocrosslinker coupled to a binding group in the self-assembling compound; the self-assembling compound comprises a riveting group attached to the solid support, and a rugged group-linked polyethylene glycol and a binding group attached to the polyethylene glycol.
  • the invention also provides a method of screening for a drug. The method first dissolves the fraction of the traditional Chinese medicine compound and the natural product in a suitable organic solvent, and then samples the sample in a specific region of the above-mentioned photo-crosslinking group-modified SPR chip by a high-precision spotting device, and after drying, is exposed. Small molecules were immobilized in ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ). Finally, SPR bioassay technology is used to rapidly screen the drug components that interact with the target protein.
  • the chip provided by the invention only needs an extremely small amount (ng grade) of compound to effectively analyze the biological activity of the small molecule compound and the specific disease target, and the sample amount used is small; however, the present invention uses the self-assembling compound to polymerize
  • the diol and the binding group are covalently immobilized on the surface of the chip, reduce background noise, improve signal-to-noise ratio, and have good anti-protein non-specific adsorption ability;
  • the present invention provides a photo-crosslinking agent on the surface of the chip, which is irradiated by ultraviolet light. Down conversion to a more reactive intermediate carbene, the carbene group can bind small molecules in a variety of ways without the need for small molecules with specific functional groups.
  • the screening method can realize parallel fixation of natural drug molecules of various structures;
  • the chip provided by the invention can directly fix up to 800 kinds of compounds directly to specific regions of the SPR chip, and then utilizes the advantages of high-throughput, rapid, dynamic analysis of the SPR technology to rapidly screen the effective components of the traditional Chinese medicine compound and the natural product. Fast detection and low cost.
  • Example 1 shows the surface protein adsorption of the chip provided in Example 1 and the conventional chip of the control group; wherein, line 1 indicates that the surface of the control chip adsorbs protein to cause SPR signal, and line 2 indicates that the surface of the test chip adsorbs protein to cause SPR signal;
  • Example 10 shows the results of SPR detection of the chip provided by the present invention and the negative control chip (ligand not bound to the target) in Example 10; wherein line 1 indicates the analyte R18, and line 2 indicates the analyte FOBISIN 101, line 3 A negative control is shown.
  • the invention discloses a chip, a preparation method, a use and a method for screening a medicine, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described in the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the medicine, the natural small molecule, the raw material and the reagent used in the method, the preparation method, the use and the method for screening the medicine provided by the present invention are all commercially available.
  • the surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
  • the solid support was immersed in the ethanol solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 450 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-COOH is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained.
  • the photocrosslinking reagent is a phenyl azide compound.
  • the surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
  • a solution of self-assembled compound of HS-PEG-OCH 3 (molecular weight 350) and 13 ⁇ 4-? 0-011 (molecular weight 100) was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM.
  • the solid support was immersed in the ethanol solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 100 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen.
  • the surface of -0H is carboxylated by succinic anhydride, and after activation by EDC/NHSS, the photocrosslinking reagent is fixed to obtain a chip.
  • the photocrosslinking reagent is an aziridine compound.
  • the surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
  • the surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
  • the photocrosslinking reagent is fixed, that is, the chip is obtained.
  • the photocrosslinking reagent is a benzophenone compound.
  • test group 243 natural ligand small molecules were taken as the analytes, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chip prepared in Example 1 using a Genetix Q-Array Mini, at room temperature. After standing, after drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the test object was fixed by photocrosslinking to obtain a test group chip.
  • Control group The substrate was heated to 70-80 ° C with distilled water H 2 O 30 mL; 6 mL of ammonia water was added; 6 mL of hydrogen peroxide was added; the chip to be washed was placed, and treated at 70-80 ° C for 10 min; Distilled water, washed with ethanol, and blown dry with nitrogen. A certain amount of 11-mercapto-1-undecanoic acid (hereinafter abbreviated as MUA) was weighed and dissolved in absolute ethanol to prepare a 1 mM solution. The above cleaned chip was immersed in its newly prepared solution at room temperature overnight (15 hours).
  • MUA 11-mercapto-1-undecanoic acid
  • the overnight chip was taken out, soaked in ethanol and placed on a shaker at room temperature for 15 minutes, then rinsed three times with deionized water and ethanol, dried with nitrogen, stored in a refrigerator, and set aside.
  • the above chip was immersed in 10 mL of EDC/NHS solution at room temperature for 30 minutes to fix the photocrosslinking reagent trifluoromethylaryldiazirines (TADs).
  • TADs photocrosslinking reagent trifluoromethylaryldiazirines
  • 243 natural small molecules were dissolved in DMSO as test substances, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a high-precision spotter Genetix Q-Array Mini.
  • the sample amount was InL, and it was left at room temperature. After drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the test substance was fixed by photocross
  • line 1 showed that the surface of the control chip adsorbed protein caused SPR
  • the signal corresponds to 85 RU
  • line 2 shows that the SPR signal is 55 RU corresponding to the adsorption protein on the surface of the test chip.
  • the results show that the test group chip can significantly reduce the non-specific adsorption of protein, which can improve the sensitivity and specificity of detection.
  • the chip prepared by using the inventive examples 2 to 8 adopts the above method, and as a result, the test group chip can significantly reduce the non-specific adsorption of the protein, thereby improving the sensitivity and specificity of the detection.
  • Example 10 The chip provided by the invention is used for drug screening
  • Test group 243 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
  • ultraviolet light wavelength 365, energy 4 J/cm 2
  • Control group The ligand small molecule biotin not bound to the 14-3-3 target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted in the example using a high-precision spotter Genetix Q-Array Mini.
  • the surface of the photocrosslinking reagent in the chip prepared from 1 to 8 is spotted in InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and fixed by photocrosslinking. Measure the object and get the second chip.
  • test group obtained the first detection value
  • control group obtained the second detection value.
  • the test results are shown in Figure 2. Among them, line 1 indicates the analyte R18, line 2 indicates the analyte FOBISIN101, and line 3 indicates the negative control.
  • the 14-3-3 target proteins all had significant effects, while the negative control did not show significant signal changes with the 14-3-3 target protein.
  • the first detected value has a significant difference compared to the second detected value.
  • the ratio of the first detected value to the second detected value is greater than three. It can be seen that the analyte R18 and the test substance FOBISIN101 can specifically bind to the 14-3-3 target protein and can be used as a treatment for related diseases. Treatment drugs.
  • Example 11 The chip provided by the invention is used for drug screening
  • Test group 300 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
  • ultraviolet light wavelength 365, energy 4 J/cm 2
  • Control group The ligand small molecule biotin not bound to the Plasmepsins II target protein was used as a negative control, dissolved in DMSO, and the test object was spotted in Examples 1 to 8 using a high-precision spotter Genetix Q-Array Mini.
  • the surface of the photocrosslinking reagent in the obtained chip was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the photo-crosslinking fixed the analyte. Obtain a second chip.
  • the first detected value has a significant difference compared to the second detected value.
  • the ratio of the first detected value to the second detected value is greater than three. It can be seen that the test substance P01 and the test substance P02 can specifically bind to the Plasmepsins II target protein, and can be used as a drug for treating related diseases.
  • Example 12 The chip provided by the invention is used for drug screening
  • Test group 341 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
  • ultraviolet light wavelength 365, energy 4 J/cm 2
  • Control group The ligand small molecule biotin that does not bind to the histone deacetylase 5 (HDAC5) target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted using a high-precision spotter Genetix Q-Array Mini.
  • the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and photocrosslinked. The object to be tested obtains the second chip.
  • the first detected value has a significant difference compared to the second detected value.
  • the ratio of the first detected value to the second detected value is greater than three. From this, it can be seen that the analyte HA1 and the analyte HA2 can specifically bind to the histone deacetylase 5 (HDAC5) target protein, and can be used as a medicine for treating a related disease.
  • Example 13 The chip provided by the present invention is used for drug screening
  • Test group 301 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
  • Control group The ligand small molecule biotin not bound to the beta-site APP Cleaving Enzyme 1 (BACE1) target protein was used as a negative control, dissolved in DMSO, and the analyte was measured using a high-precision spotter Genetix Q-Array Mini.
  • the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 was spotted, and the spotting amount was InL, which was left at room temperature, dried, and exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes. The photocrosslinking fixes the analyte to obtain a second chip.
  • the first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three.
  • test substance KA2 and the test substance FA2 can specifically bind to the beta-site APP Cleaving Enzyme 1 (BACE1) target protein, and can be used as a drug for treating a related disease.
  • BACE1 beta-site APP Cleaving Enzyme 1
  • Test group 260 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
  • Control group The ligand small molecule biotin not bound to the COX-2 target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted in Example 1 using a high-precision spotter Genetix Q-Array Mini. 8 The surface of the photocrosslinking reagent in the obtained chip was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and photocrosslinked to fix the analyte. , get the second chip.
  • test substance C08 and COX-2 target protein had significant effects, while the negative pair There was no significant signal change between the vehicle and the COX-2 target protein.
  • the first detected value has a significant difference compared to the second detected value.
  • the ratio of the first detected value to the second detected value is greater than three. From this, it can be seen that the test substance C08 can specifically bind to the COX-2 target protein, and can be used as a drug for treating a related disease.

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Abstract

The present invention relates to the field of drug screening, and in particular to a chip, preparation method and use thereof and method for screening drug. The chip provided in the present invention has a good capacity for resisting nonspecific protein adsorption, and can immobilize a variety of small molecular substances in parallel, and it is further used for screening drugs having complicated ingredients, and has high sensitivity with only traces of compounds being required for effectively analyzing the bioactivities of small molecular substances and specific disease targets.

Description

一种芯片、 制备方法、 用途及筛选药物的方法  Chip, preparation method, use and method for screening medicine
本申请要求于 2012 年 09 月 27 日提交中国专利局、 申请号为 201210371255.X, 发明名称为"一种芯片、 制备方法、 用途及筛选药物的 方法"的中国专利申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域  This application claims priority to Chinese Patent Application No. 201210371255.X, filed on September 27, 2012, entitled "A Chip, Preparation Method, Use, and Method for Screening Drugs", all of which are The content is incorporated herein by reference. Technical field
本发明涉及药物筛选领域， 特别涉及一种芯片、 制备方法、 用途及筛 选药物的方法。 背景技术  The invention relates to the field of drug screening, in particular to a chip, a preparation method, a use and a method for screening a drug. Background technique
中药是我国几千年来的文化沉淀，具有悠久的历史和文化渊源。然而， 中药的开发仍然依赖于多年来的传统方法，并以原料药为基石出，缺乏系统 的现代科学的基础研究。 大多数的中药的作用机制不清楚、 中药药方的关 键组分不明确， 而且可能存在无法意料的副作用。这些因素阻碍了中药进 军国际天然药物市场的主流。 中药要走向世界， 进入国际市场， 必须实现 中药现代化，与世界现代医学接轨。研究人员希望寻找出治疗特定疾病的 中药配方中的某种关键组分，解开传统中医的潜在功效之谜，为传统中药 的发展开辟新的道路。例如采用超临界萃取等技术对中药成分的提取，然 后通过生物实验（如： 细胞实验）确认某一种馏分中是否含有有效活性成 分， 作为下一步细分、 提纯的决策依据。 这种方法操作繁瑣、 耗时费力、 效率低。  Traditional Chinese medicine is a cultural deposit in China for thousands of years and has a long history and cultural origin. However, the development of traditional Chinese medicine still relies on traditional methods for many years, and is based on APIs, lacking systematic basic research in modern science. The mechanism of action of most Chinese medicines is unclear, the key components of Chinese medicine prescriptions are unclear, and there may be unpredictable side effects. These factors have hindered the mainstreaming of Chinese medicine into the international natural medicine market. To go to the world and enter the international market, traditional Chinese medicine must realize the modernization of traditional Chinese medicine and integrate it with the world's modern medicine. Researchers hope to find a key component of traditional Chinese medicine formula for treating specific diseases, unlock the mystery of the potential efficacy of traditional Chinese medicine, and open up a new path for the development of traditional Chinese medicine. For example, the extraction of traditional Chinese medicine ingredients by supercritical extraction techniques, and then through biological experiments (such as: cell experiments) to confirm whether a certain fraction contains effective active ingredients, as a decision basis for further subdivision and purification. This method is cumbersome, time consuming, and inefficient.
经济快速的高通量陣选已成为当今药物陣选的主流。 在过去的几年 中，世界上著名的制药公司纷纷与以高通量药物筛选技术为核心的中小型 生物科技公司结盟或合作，采用高通量或超高通量药物筛选技术进行先导 物分子的筛选。 其中以小分子阵列芯片技术的应用最为广泛。 哈佛大学 Schreiber课题组首次报道了小分子化合物微阵列芯片， 主要用于筛选能 与特定蛋白质特异性结合的化合物。他们将玻片表面进行化学处理，使其 衍生化产生活性基团，然后采用高精度点样仪吸取约 1 nL的溶于合适有机 溶剂 （例如 DMSO )样品点样于玻片特定的区域， 利用表面活性基团与 小分子特定基团的相互作用将小分子共价固定于芯片表面。 通过这种方 式， 可以实现小分子阵列， 而且满足高密度地固定于玻片表面。 虽然这种 特异性固定策略非常适合于固定合成化合物，但是天然化合物的结构通常 是多样性，如果采用单一的固定方式难以将多种小分子固定于同一张芯片 中， 尤其是部分环状小分子并没有连接臂用于固定。 因此， 天然化合物的 固定是小分子阵列的技术瓶颈。 发明内容 The rapid economic high-throughput array has become the mainstream of today's drug array. In the past few years, the world's leading pharmaceutical companies have partnered or collaborated with small and medium-sized biotechnology companies with high-throughput drug screening technologies to use high-throughput or ultra-high-throughput drug screening technologies for lead molecules. Screening. Among them, small molecule array chip technology is the most widely used. The Schreiber team at Harvard University first reported small molecule compound microarray chips, which are mainly used to screen compounds that specifically bind to specific proteins. They chemically treated the surface of the slide to derivatize it to produce reactive groups, and then used a high-precision spotter to draw about 1 nL of the sample dissolved in a suitable organic solvent (such as DMSO) to spot the specific area of the slide. Surface active groups and The interaction of small molecule-specific groups covalently immobilizes small molecules on the surface of the chip. In this way, a small molecule array can be realized, and it is required to be fixed to the surface of the slide at a high density. Although this specific immobilization strategy is very suitable for immobilizing synthetic compounds, the structure of natural compounds is usually versatile. It is difficult to immobilize multiple small molecules in the same chip, especially partial small molecules, if a single immobilization method is used. There is no connecting arm for fixing. Therefore, the immobilization of natural compounds is a technical bottleneck of small molecule arrays. Summary of the invention
有鉴于此，本发明提供一种芯片、制备方法、用途及筛选药物的方法。 本发明提供的芯片，具有艮好的抗蛋白质非特异性吸附能力， 能够并行固 定多种小分子物质，进而用于筛选具有复杂组分的药物，仅仅需要微量的 化合物就能够有效地分析小分子化合物与具体疾病靶标的生物活性，灵敏 度! ¾。  In view of this, the present invention provides a chip, a preparation method, a use, and a method for screening a drug. The chip provided by the invention has good anti-protein non-specific adsorption ability, can fix a plurality of small molecular substances in parallel, and is used for screening drugs with complex components, and can effectively analyze small molecular compounds only by using a trace amount of compounds. Bioactivity with specific disease targets, sensitivity! 3⁄4.
为了实现上述发明目的， 本发明提供以下技术方案：  In order to achieve the above object, the present invention provides the following technical solutions:
本发明提供了一种芯片， 包括固体支持物、两端修饰的聚乙二醇和光 交联剂； 两端修饰的聚乙二醇为一端由铆定基团修饰、另一端由结合基团 修饰的聚乙二醇；固体支持物与铆定基团连接、光交联剂与结合基团连接； 铆定基团选自 -SH、 -S-S -、 -SiCl3; The invention provides a chip comprising a solid support, a modified polyethylene glycol at both ends, and a photocrosslinking agent; the polyethylene glycol modified at both ends is modified at one end by a riveting group and the other end is modified by a binding group. The polyethylene glycol; the solid support is linked to the riveting group; the photocrosslinking agent is bonded to the binding group; the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
结合基团选自 -COOH、 -OH、 -NH2、 -OCH3。 Binding group selected from -COOH, -OH, -NH 2, -OCH 3.
本发明提供的芯片在固体支持物表面通过两端修饰的聚乙二醇引入 光交联剂， 在紫外光（波长 365nm, 能量 4J/cm2 ) 照射下转化为反应活 性较高的中间体卡宾，卡宾基团能以多种方式结合小分子而无需小分子带 有特定功能基团。 The chip provided by the invention is introduced into the photocrosslinker on the surface of the solid support by the polyethylene glycol modified at both ends, and is converted into a highly reactive intermediate carbene by ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ). Carbene groups can bind small molecules in a variety of ways without the need for small molecules with specific functional groups.
作为优选， 光交联剂选自苯基叠氮类化合物、 吖丙啶类化合物、二苯 曱酮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
优选地， 光交联剂选自苯基叠氮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
作为优选， 光交联剂与结合基团经酸胺缩合反应偶联。  Preferably, the photocrosslinking agent is coupled to the binding group via an acid amine condensation reaction.
在本发明的一些实施例中，本发明提供的芯片中两端修饰的聚乙二醇 的分子量为 100 ~ 5000。 在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 1000 ~ 4000。 In some embodiments of the present invention, the polyethylene glycol modified at both ends of the chip provided by the present invention has a molecular weight of 100 to 5000. In other embodiments of the present invention, the polyethylene glycol modified at both ends of the chip provided by the present invention has a molecular weight of 1000 to 4000.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 1500 ~ 3400。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is 1500 to 3400.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 2000 ~ 2800。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 2000 to 2800.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 100 ~ 500。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 100 to 500.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 500 ~ 1000。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 500 to 1,000.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 1000 ~ 2000。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol at both ends of the chip provided by the present invention is from 1000 to 2000.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 2000 ~ 5000。  In still other embodiments of the present invention, the molecular weight of the modified polyethylene glycol in the chip provided by the present invention is from 2000 to 5000.
在本发明的一些实施例中，本发明提供的芯片中， 固体支持物为玻璃 基片或镀金玻璃基片。  In some embodiments of the invention, in the chip provided by the present invention, the solid support is a glass substrate or a gold-plated glass substrate.
本发明还提供了一种芯片的制备方法， 包括如下步骤：  The invention also provides a method for preparing a chip, comprising the following steps:
步骤 1 : 取聚乙二醇分别与铆定基团、 结合基团连接， 获得两端修饰 的聚乙二醇；  Step 1: The polyethylene glycol is respectively connected to the riveting group and the binding group to obtain polyethylene glycol modified at both ends;
步骤 2: 取固体支持物经预处理后与铆定基团连接；  Step 2: The solid support is pretreated and connected to the riveting group;
步骤 3: 活化结合基团后， 与光交联剂经酸胺缩合反应偶联， 即得； 铆定基团选自 -SH、 -S-S -、 -SiCl3; Step 3: After activating the binding group, the photocrosslinking agent is coupled with an acid amine condensation reaction, and the riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
结合基团选自 -COOH、 -OH、 -丽2。 The binding group is selected from the group consisting of -COOH, -OH, -Li 2 .
作为优选， 光交联剂选自苯基叠氮类化合物、 吖丙啶类化合物、二苯 曱酮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
优选地， 光交联剂选自苯基叠氮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
在本发明的一些实施例中，本发明提供的芯片的制备方法中两端修饰 的聚乙二醇的分子量为 100 ~ 5000。  In some embodiments of the present invention, in the method for preparing a chip provided by the present invention, the polyethylene glycol modified at both ends has a molecular weight of 100 to 5000.
在本发明的另一些实施例中，本发明提供的芯片中两端修饰的聚乙二 醇的分子量为 1000 ~ 4000。 In other embodiments of the present invention, the present invention provides a modified polyethylene chip at both ends of the chip. The molecular weight of the alcohol is from 1000 to 4000.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 1500 ~ 3400。  In still other embodiments of the present invention, the method for preparing a chip provided by the present invention has a molecular weight of 1500 to 3400 modified at both ends.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 2000 ~ 2800。  In still other embodiments of the present invention, the method for preparing a chip provided by the present invention has a molecular weight of from 2,000 to 2,800 for the polyethylene glycol modified at both ends.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 100 ~ 500。  In still other embodiments of the present invention, the polyethylene glycol modified at both ends in the method for preparing a chip provided by the present invention has a molecular weight of 100 to 500.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 500 ~ 1000。  In still other embodiments of the present invention, the polyethylene glycol modified at both ends of the method for preparing a chip provided by the present invention has a molecular weight of 500 to 1000.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 1000 ~ 2000。  In still other embodiments of the present invention, the method for preparing a chip according to the present invention has a molecular weight of from 1000 to 2000 for the polyethylene glycol modified at both ends.
在本发明的另一些实施例中，本发明提供的芯片的制备方法中两端修 饰的聚乙二醇的分子量为 2000 ~ 5000。  In still other embodiments of the present invention, the method for preparing a chip provided by the present invention has a molecular weight of from 2,000 to 5,000 for the polyethylene glycol modified at both ends.
在本发明的一些实施例中，本发明提供的芯片的制备方法中， 固体支 持物为玻璃基片或镀金玻璃基片。  In some embodiments of the present invention, in the method of fabricating the chip provided by the present invention, the solid support is a glass substrate or a gold-plated glass substrate.
本发明还提供了上述制备方法制得的芯片。  The present invention also provides a chip produced by the above preparation method.
本发明还提供了上述芯片用于筛选药物的应用。  The invention also provides the use of the above chip for screening drugs.
本发明还提供了一种基于芯片筛选药物的方法， 包括如下步骤： 步骤 1 : 取待测物与芯片混合， 干燥后置于紫外光下， 洗去未结合的 待测物， 获得第一芯片， 灭活未结合位点后与靶标混合， 经表面等离子 体共振检测获得第一响应值；  The invention also provides a method for screening drugs based on a chip, comprising the following steps: Step 1: mixing a sample to be tested with a chip, drying it, placing it under ultraviolet light, washing away the unbound analyte, and obtaining a first chip. , inactivating the unbound site, mixing with the target, and obtaining a first response value by surface plasmon resonance detection;
步骤 2: 取不与靶标结合的配体作为阴性对照物与芯片混合， 干燥后 置于紫外光下曝光， 洗去未结合的所述阴性对照物， 获得第二芯片， 灭活 未结合位点后与靶标混合， 经表面等离子体共振检测获得第二响应值； 步骤 3: 获得第一响应值与第二响应值的比值， 根据比值判断待 测物与靶标是否结合；比值不小于 3时，待测物为与靶标结合的药物； 比值小于 3时， 待测物为不与靶标结合的配体；  Step 2: The ligand not bound to the target is mixed with the chip as a negative control, dried and exposed to ultraviolet light, and the unconjugated negative control is washed away to obtain a second chip, and the unbound site is inactivated. After mixing with the target, the second response value is obtained by surface plasmon resonance detection; Step 3: obtaining a ratio of the first response value to the second response value, and determining whether the analyte and the target are combined according to the ratio; when the ratio is not less than 3, The analyte is a drug that binds to the target; when the ratio is less than 3, the analyte is a ligand that does not bind to the target;
芯片包括固体支持物、 两端修饰的聚乙二醇和光交联剂； 两端修饰 的聚乙二醇为一端由铆定基团修饰、 另一端由结合基团修饰的聚乙二醇； 固体支持物与铆定基团连接， 光交联剂与结合基团连接； The chip comprises a solid support, a polyethylene glycol modified at both ends, and a photocrosslinker; the polyethylene glycol modified at both ends is a polyethylene glycol modified at one end by a riveting group and the other end is modified by a binding group; The solid support is attached to the riveting group, and the photocrosslinking agent is linked to the binding group;
铆定基团选自 -SH、 -S-S -、 -SiCl3; The riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
结合基团选自 -COOH、 -OH、 -NH2、 -OCH3。 Binding group selected from -COOH, -OH, -NH 2, -OCH 3.
具体可以为，本发明提供的一种基于芯片筛选药物的方法， 包括如下 步骤：  Specifically, the method for screening a drug based on a chip provided by the present invention includes the following steps:
步骤 1 : 采用高精度点样仪将待测物点样于芯片的特定区域， 干燥后 置于紫外光进行光交联，洗去未结合的待测物，灭活未结合位点后与靶标 混合， 获得小分子阵列， 经表面等离子体共振检测获得第一响应值； 步骤 2: 采用高精度点样仪将阴性对照物点样于芯片的特定区域， 干 燥后置于紫外光进行光交联，洗去未结合的阴性对照物，灭活未结合位点 后与靶标混合， 获得小分子阵列， 经表面等离子体共振检测获得第二响 应值；  Step 1: The sample to be tested is spotted on a specific area of the chip by using a high-precision spotter. After drying, it is placed in ultraviolet light for photocrosslinking, and the unbound analyte is washed away, and the unbound site is inactivated and the target is inactivated. Mixing, obtaining a small molecule array, and obtaining a first response value by surface plasmon resonance detection; Step 2: using a high-precision spotting instrument to spot the negative control on a specific area of the chip, drying and then placing ultraviolet light for photocrosslinking Washing off the unbound negative control, inactivating the unbound site, and mixing with the target to obtain a small molecule array, and obtaining a second response value by surface plasmon resonance detection;
步骤 3: 获得第一响应值与第二响应值的比值， 根据所述比值判 断待测物与靶标是否结合； 比值不小于 3时， 待测物为与靶标结合的 药物； 比值小于 3时， 待测物为不与靶标结合的配体；  Step 3: Obtain a ratio of the first response value to the second response value, and determine whether the analyte and the target are combined according to the ratio; when the ratio is not less than 3, the analyte is a drug that binds to the target; when the ratio is less than 3, The analyte is a ligand that does not bind to the target;
芯片包括固体支持物、 两端修饰的聚乙二醇和光交联剂； 两端修饰 的聚乙二醇为一端由铆定基团修饰、 另一端由结合基团修饰的聚乙二醇； 固体支持物与铆定基团连接， 光交联剂与结合基团连接；  The chip comprises a solid support, a modified polyethylene glycol at both ends, and a photocrosslinker; the polyethylene glycol modified at both ends is a polyethylene glycol modified at one end by a riveting group and the other end is modified by a binding group; The support is attached to the riveting group, and the photocrosslinking agent is linked to the binding group;
铆定基团选自 -SH、 -S-S -、 -SiCl3; The riveting group is selected from the group consisting of -SH, -SS-, -SiCl 3 ;
结合基团选自 -COOH、 -OH、 -NH2。 Binding group selected from -COOH, -OH, -NH 2.
作为优选， 光交联剂选自苯基叠氮类化合物、 吖丙啶类化合物、二苯 曱酮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of a phenyl azide compound, an aziridine compound, and a benzophenone compound.
优选地， 光交联剂选自苯基叠氮类化合物。  Preferably, the photocrosslinking agent is selected from the group consisting of phenyl azide compounds.
在本发明的一些实施例中，本发明提供的基于芯片的筛选药物的方法 中， 芯片中两端爹饰的聚乙二醇的分子量为 100 ~ 5000。  In some embodiments of the present invention, in the chip-based method for screening drugs provided by the present invention, the polyethylene glycol at both ends of the chip has a molecular weight of 100 to 5000.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端修饰的聚乙二醇的分子量为 1000 ~ 4000。  In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the modified polyethylene glycol in the chip has a molecular weight of 1000 to 4000.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 1500 ~ 3400。 在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 2000 ~ 2800。 In other embodiments of the present invention, in the chip-based method for screening drugs, the molecular weight of the polyethylene glycol at both ends of the chip is 1500-3400. In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the polyethylene glycol at both ends of the chip is 2000 to 2800.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 100 ~ 500。  In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the polyethylene glycol at both ends of the chip is 100-500.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 500 ~ 1000。  In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the polyethylene glycol at both ends of the chip is 500 to 1000.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 1000 ~ 2000。  In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the polyethylene glycol at both ends of the chip is 1000 to 2000.
在本发明的另一些实施例中，本发明提供的基于芯片的筛选药物的方 法中， 芯片中两端爹饰的聚乙二醇的分子量为 2000 ~ 5000。  In still another embodiment of the present invention, in the method for screening a drug based on a chip, the molecular weight of the polyethylene glycol at both ends of the chip is 2000 to 5000.
在本发明的一些实施例中，本发明提供的基于芯片的筛选药物的方法 中， 芯片的固体支持物为玻璃基片或镀金玻璃基片。  In some embodiments of the invention, in the method of chip-based screening of drugs provided by the present invention, the solid support of the chip is a glass substrate or a gold-plated glass substrate.
作为优选， 药物为中药复方或天然产物。  Preferably, the drug is a traditional Chinese medicine compound or a natural product.
本发明提供了一种芯片、制备方法及药物筛选方法。该芯片包括固体 支持物、与固体支持物连接的自组装化合物以及与自组装化合物中的结合 基团偶联的光交联剂； 自组装化合物包括与固体支持物连接的铆定基团、 与铆定基团连接的聚乙二醇以及与聚乙二醇连接的结合基团。本发明还提 供了一种筛选药物的方法。该方法首先将中药复方、天然产物的馏分溶于 合适的有机溶剂中，然后利用高精度的点样仪将样品点样于上述光交联基 团修饰的 SPR芯片特定的区域， 干燥后， 暴露于紫外光（波长 365nm, 能量 4J/cm2 ) 中固定小分子。 最后利用 SPR生物检测技术快速筛选与靶 向蛋白有相互作用的药物成分。 The invention provides a chip, a preparation method and a drug screening method. The chip comprises a solid support, a self-assembling compound attached to a solid support, and a photocrosslinker coupled to a binding group in the self-assembling compound; the self-assembling compound comprises a riveting group attached to the solid support, and a rugged group-linked polyethylene glycol and a binding group attached to the polyethylene glycol. The invention also provides a method of screening for a drug. The method first dissolves the fraction of the traditional Chinese medicine compound and the natural product in a suitable organic solvent, and then samples the sample in a specific region of the above-mentioned photo-crosslinking group-modified SPR chip by a high-precision spotting device, and after drying, is exposed. Small molecules were immobilized in ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ). Finally, SPR bioassay technology is used to rapidly screen the drug components that interact with the target protein.
本发明提供的芯片， 仅仅需要极其微量（ng级别） 的化合物就能够 有效地分析小分子化合物与具体疾病靶标的生物活性，所用样品量少；此 夕卜，本发明采用自组装化合物将聚乙二醇、结合基团共价固定于芯片的表 面，降低背景噪音，提高信噪比，具有艮好的抗蛋白质非特异性吸附能力； 本发明提供的芯片表面引入光交联剂，在紫外光照射下转化为反应活性较 高的中间体卡宾，卡宾基团能以多种方式结合小分子而无需小分子带有特 定功能基团。 该筛选方法可以实现各种结构的天然药物分子的并行固定； 本发明提供的芯片可以将高达 800多种化合物直接固定于 SPR芯片的特 定区域， 然后利用 SPR技术的高通量、 快速、 动力学分析等优点， 快速 筛选中药复方、 天然产物的有效组分， 检测迅速， 成本低廉。 附图说明 The chip provided by the invention only needs an extremely small amount (ng grade) of compound to effectively analyze the biological activity of the small molecule compound and the specific disease target, and the sample amount used is small; however, the present invention uses the self-assembling compound to polymerize The diol and the binding group are covalently immobilized on the surface of the chip, reduce background noise, improve signal-to-noise ratio, and have good anti-protein non-specific adsorption ability; the present invention provides a photo-crosslinking agent on the surface of the chip, which is irradiated by ultraviolet light. Down conversion to a more reactive intermediate carbene, the carbene group can bind small molecules in a variety of ways without the need for small molecules with specific functional groups. The screening method can realize parallel fixation of natural drug molecules of various structures; The chip provided by the invention can directly fix up to 800 kinds of compounds directly to specific regions of the SPR chip, and then utilizes the advantages of high-throughput, rapid, dynamic analysis of the SPR technology to rapidly screen the effective components of the traditional Chinese medicine compound and the natural product. Fast detection and low cost. DRAWINGS
图 1示实施例 1提供的芯片与对照组普通芯片的表面蛋白质吸附情 况； 其中， 线 1示对照组芯片表面吸附蛋白引起 SPR信号， 线 2示试验 组芯片表面吸附蛋白引起 SPR信号；  1 shows the surface protein adsorption of the chip provided in Example 1 and the conventional chip of the control group; wherein, line 1 indicates that the surface of the control chip adsorbs protein to cause SPR signal, and line 2 indicates that the surface of the test chip adsorbs protein to cause SPR signal;
图 2示实施例 10中本发明提供的芯片与阴性对照芯片 （不与靶标结 合的配体） 的 SPR检测结果； 其中线 1 示待测物 R18, 线 2示待测物 FOBISIN 101 , 线 3示阴性对照物。 具体实施方式  2 shows the results of SPR detection of the chip provided by the present invention and the negative control chip (ligand not bound to the target) in Example 10; wherein line 1 indicates the analyte R18, and line 2 indicates the analyte FOBISIN 101, line 3 A negative control is shown. detailed description
本发明公开了一种芯片、 制备方法、 用途及筛选药物的方法， 本领域 技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是， 所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视 为包括在本发明。 本发明的方法及应用已经通过较佳实施例进行了描述， 相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和 应用进行改动或适当变更与组合， 来实现和应用本发明技术。  The invention discloses a chip, a preparation method, a use and a method for screening a medicine, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described in the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
本发明提供的一种芯片、制备方法、用途及筛选药物的方法中所用药 物、 天然小分子、 原料及试剂均可由市场购得。  The medicine, the natural small molecule, the raw material and the reagent used in the method, the preparation method, the use and the method for screening the medicine provided by the present invention are all commercially available.
下面结合实施例， 进一步阐述本发明： 本发明提供的芯片的制备  The invention will be further illustrated below in conjunction with the embodiments: Preparation of the chip provided by the present invention
在洁净的玻璃基片表面蒸镀或磁控溅射 1-2 nm铬膜， 再在铬膜的表 面蒸镀或者磁控溅射约 50 nm的金膜。  The surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入上述制得的镀金玻璃基片，于 70 ~ 80°C下 处理 10 min,冷却 10 min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。 配置 HS-PEG-OCH3(分子量为 350)和 HS-PEG-COOH (分子量为 450) 的自组装化合物的乙醇溶液，其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的乙醇溶液中， 室温下放置 15小 时， 取出连接有分子量为 450的自组装化合物的固体支持物， 用蒸馏水、 乙醇充分清洗、 氮气吹干。 表面的 -COOH经由 EDC/NHSS活化后， 固定 光交联试剂， 即得芯片。 光交联试剂为苯基叠氮类化合物。 实施例 2本发明提供的芯片的制备 Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into the gold-plated glass substrate prepared above, treat it at 70 ~ 80 °C for 10 min, cool for 10 min, add distilled water, The mixture was washed with ethanol and dried with nitrogen to obtain a solid support. A solution of HS-PEG-OCH 3 (molecular weight 350) and HS-PEG-COOH (molecular weight 450) of the self-assembling compound in ethanol was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM. The solid support was immersed in the ethanol solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 450 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-COOH is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a phenyl azide compound. Example 2 Preparation of Chip Provided by the Invention
在洁净的玻璃基片表面蒸镀或磁控溅射 1-2 nm铬膜， 再在铬膜的表 面蒸镀或者磁控溅射约 50 nm的金膜。  The surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入上述制得的镀金玻璃基片， 于 70〜80°C下 处理 lO min, 冷却 lO min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into the gold-plated glass substrate prepared above, treat it at 70~80 °C for 10 min, cool for 10 min, add distilled water, The mixture was washed with ethanol and dried with nitrogen to obtain a solid support.
配置 HS-PEG-OCH3(分子量为 350)和1¾-? 0-011(分子量为 100)的自 组装化合物的乙醇溶液， 其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将 固体支持物浸泡入上述自组装化合物的乙醇溶液中，室温下放置 15小时， 取出连接有分子量为 100的自组装化合物的固体支持物，用蒸馏水、 乙醇 充分清洗、 氮气吹干。 表面的 -0H经丁二酸酐羧基化， 后经 EDC/NHSS 活化后， 固定光交联试剂， 即得芯片。 光交联试剂为吖丙啶类化合物。 实施例 3本发明提供的芯片的制备 A solution of self-assembled compound of HS-PEG-OCH 3 (molecular weight 350) and 13⁄4-? 0-011 (molecular weight 100) was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM. The solid support was immersed in the ethanol solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 100 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. The surface of -0H is carboxylated by succinic anhydride, and after activation by EDC/NHSS, the photocrosslinking reagent is fixed to obtain a chip. The photocrosslinking reagent is an aziridine compound. Example 3 Preparation of Chip Provided by the Invention
在洁净的玻璃基片表面蒸镀或磁控溅射 1-2 nm铬膜， 再在铬膜的表 面蒸镀或者磁控溅射约 50 nm的金膜。  The surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入上述制得的镀金玻璃基片， 于 70〜80°C下 处理 lO min, 冷却 lO min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into the gold-plated glass substrate prepared above, treat it at 70~80 °C for 10 min, cool for 10 min, add distilled water, The mixture was washed with ethanol and dried with nitrogen to obtain a solid support.
配置 HS-PEG-OCH3(分子量为 350)和 HS-PEG-COOH-NH2(分子量为 339)的自组装化合物的乙醇溶液， 其中二者的摩尔比为 10:1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的乙醇溶液中， 室温下放 置 15小时， 取出连接有上述分子的自组装化合物的固体支持物， 用蒸馏 水、 乙醇充分清洗、 氮气吹干。 表面的 -NH2经由催化剂 EDC、 DMAP活 化后， 固定光交联试剂， 即得芯片。 光交联试剂为二苯曱酮类化合物。 实施例 4本发明提供的芯片的制备 An ethanol solution of self-assembled compound of HS-PEG-OCH 3 (molecular weight 350) and HS-PEG-COOH-NH 2 (molecular weight 339) was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM. Soak the solid support in the ethanol solution of the above self-assembled compound and let it stand at room temperature. After 15 hours, the solid support of the self-assembled compound to which the above molecule was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface of -NH 2 is activated by the catalysts EDC and DMAP, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a benzophenone compound. Example 4 Preparation of Chip Provided by the Invention
在洁净的玻璃基片表面蒸镀或磁控溅射 1-2 nm铬膜， 再在铬膜的表 面蒸镀或者磁控溅射约 50 nm的金膜。  The surface of the clean glass substrate is vapor-deposited or magnetron-sputtered with a 1-2 nm chromium film, and then deposited on the surface of the chromium film or magnetron-sputtered with a gold film of about 50 nm.
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入上述制得的镀金玻璃基片， 于 70〜80°C下 处理 lO min, 冷却 lO min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into the gold-plated glass substrate prepared above, treat it at 70~80 °C for 10 min, cool for 10 min, add distilled water, The mixture was washed with ethanol and dried with nitrogen to obtain a solid support.
配置 -S-S-PEG-OCH3(分子量为 1500)和 -S-S-PEG-COOH(分子量为 1000)的自组装化合物的乙醇溶液， 其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的乙醇溶液中， 室温下放 置 15小时，取出连接有分子量为 1000的自组装化合物的固体支持物，用 蒸馏水、 乙醇充分清洗、 氮气吹干。 表面的 -COOH经由 EDC/NHSS活化 后， 固定光交联试剂， 即得芯片。 光交联试剂为苯基叠氮类化合物。 实施例 5本发明提供的芯片的制备 An ethanol solution of self-assembled compound of -SS-PEG-OCH 3 (molecular weight 1500) and -SS-PEG-COOH (molecular weight 1000) was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM. The solid support was immersed in the ethanol solution of the above self-assembled compound, allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 1000 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-COOH is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a phenyl azide compound. Example 5 Preparation of Chip Provided by the Invention
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入玻璃基片， 于 70〜80°C下 处理 lO min, 冷却 10 min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put it into a glass substrate, treat it at 70~80 °C for 10 min, cool for 10 min, add distilled water, wash with ethanol, and blow with nitrogen. Dry to make a solid support.
配置 Cl3-Si-PEG-OCH3(分子量为 100)和 Cl3-Si-PEG-COOH (分子量为 500)的自组装化合物的曱苯溶液， 其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的曱苯溶液中， 室温下放 置 15小时， 取出连接有分子量为 10000的自组装化合物的固体支持物， 用蒸馏水、 乙醇充分清洗、 氮气吹干。 表面的 -COOH经由 EDC/NHSS活 化后， 固定光交联试剂， 即得芯片。 光交联试剂为苯基叠氮类化合物。 实施例 6本发明提供的芯片的制备 a solution of a self-assembled compound of Cl 3 -Si-PEG-OCH 3 (molecular weight of 100) and Cl 3 -Si-PEG-COOH (molecular weight of 500), wherein the molar ratio of the two is 10:1, The concentration is lmM. The solid support was immersed in the toluene solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 10,000 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-COOH is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a phenyl azide compound. Example 6 Preparation of Chip Provided by the Invention
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入玻璃基片， 于 70〜80°C下 处理 lO min, 冷却 10 min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put it into a glass substrate, treat it at 70~80 °C for 10 min, cool for 10 min, add distilled water, wash with ethanol, and blow with nitrogen. Dry to make a solid support.
配置 Cl3-Si-PEG-OCH3(分子量为 450)和 Cl3-Si-PEG-OH (分子量为 436) 的自组装化合物的曱苯溶液，其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的曱苯溶液中， 室温下放置 15小 时，取出连接有分子量为 8000的自组装化合物的固体支持物，用蒸馏水、 乙醇充分清洗、 氮气吹干。 表面的 -OH 经由丁二酸酐羧基化后， 再经由 EDC/NHSS 活化后， 固定光交联试剂， 即得芯片。 光交联试剂为吖丙啶 类化合物。 实施例 7本发明提供的芯片的制备 a solution of a self-assembled compound of Cl 3 -Si-PEG-OCH 3 (molecular weight 450) and Cl 3 -Si-PEG-OH (molecular weight 436) in a molar ratio of 10:1, total The concentration is lmM. The solid support was immersed in the toluene solution of the above self-assembled compound, allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 8,000 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface -OH is carboxylated by succinic anhydride and then activated by EDC/NHSS, the photocrosslinking reagent is immobilized to obtain a chip. The photocrosslinking reagent is an aziridine compound. Example 7 Preparation of Chip Provided by the Invention
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入玻璃基片， 于 70〜80°C下 处理 10 min, 冷却 10 min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into a glass substrate, treat at 70~80 °C for 10 min, cool for 10 min, add distilled water, wash with ethanol, and blow with nitrogen. Dry to make a solid support.
配置 Cl3-Si-PEG-OCH3(分子量为 2000)和 Cl3-Si-PEG-NH2(分子量为 3400)的自组装化合物的水溶液， 其中二者的摩尔比为 10:1 , 总浓度为 lmM。 将固体支持物浸泡入上述自组装化合物的水溶液中， 室温下放置 15小时， 取出连接有分子量为 3400的自组装化合物的固体支持物， 用蒸 馏水、 乙醇充分清洗、 氮气吹干。 表面的 -NH2经由 EDC/NHSS活化后， 固定光交联试剂， 即得芯片。 光交联试剂为二苯曱酮类化合物。 实施例 8本发明提供的芯片的制备 An aqueous solution of a self-assembling compound of Cl 3 -Si-PEG-OCH 3 (molecular weight 2000) and Cl 3 -Si-PEG-NH 2 (molecular weight: 3400), wherein the molar ratio of the two is 10:1, the total concentration For lmM. The solid support was immersed in an aqueous solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 3,400 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-NH 2 is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a benzophenone compound. Example 8 Preparation of Chip Provided by the Invention
取蒸馏水 30 mL加热至 70 ~ 80 °C , 加入氨水 6 mL、 双氧水 6 mL, 放入玻璃基片， 于 70〜80°C下 处理 10 min, 冷却 10 min, 加入蒸馏水、 乙醇清洗， 氮气吹干， 制得固体支持物。  Take 30 mL of distilled water to 70 ~ 80 °C, add 6 mL of ammonia water and 6 mL of hydrogen peroxide, put into a glass substrate, treat at 70~80 °C for 10 min, cool for 10 min, add distilled water, wash with ethanol, and blow with nitrogen. Dry to make a solid support.
配置 Cl3-Si-PEG-OCH3(分子量为 450)和 Cl3-Si-PEG-COOH的自组装 化合物的水溶液， 其中二者的摩尔比为 10: 1 , 总浓度为 lmM。 将固体支 持物浸泡入上述自组装化合物的水溶液中， 室温下放置 15小时， 取出连 接有分子量为 5000的自组装化合物的固体支持物， 用蒸馏水、 乙醇充分 清洗、 氮气吹干。 表面的 -COOH经由 EDC/NHSS活化后， 固定光交联试 剂， 即得芯片。 光交联试剂为苯基叠氮类化合物。 实施例 9蛋白非特异性吸附的检测 An aqueous solution of a self-assembling compound of Cl 3 -Si-PEG-OCH 3 (molecular weight 450) and Cl 3 -Si-PEG-COOH was prepared, wherein the molar ratio of the two was 10:1 and the total concentration was lmM. Solid branch The substrate was immersed in an aqueous solution of the above self-assembled compound, and allowed to stand at room temperature for 15 hours, and a solid support to which a self-assembled compound having a molecular weight of 5000 was attached was taken out, washed thoroughly with distilled water and ethanol, and dried with nitrogen. After the surface-COOH is activated by EDC/NHSS, the photocrosslinking reagent is fixed, that is, the chip is obtained. The photocrosslinking reagent is a phenyl azide compound. Example 9 Detection of non-specific adsorption of proteins
试验组： 取 243个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1制得的芯片中光交联试 剂表面， 室温下放置， 干燥后， 在紫外光（波长 365nm, 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得试验组芯片。 The test group: 243 natural ligand small molecules were taken as the analytes, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chip prepared in Example 1 using a Genetix Q-Array Mini, at room temperature. After standing, after drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the test object was fixed by photocrosslinking to obtain a test group chip.
对照组： 将基片用蒸馏水 H2O 30 mL加热至 70〜80°C ; 加入氨水 6 mL; 加入双氧水 6 mL; 放入待洗芯片， 70〜80°C下 处理 10 min; 冷却 lO min; 蒸馏水、 乙醇清洗， 氮气吹干。 称取的一定量 11-巯基 -1-十一酸 (以下简称为 MUA), 溶于无水乙醇配成 1 mM的溶液。 将上述清洗好的 芯片浸泡其新配制的溶液中， 室温过夜 ( 15小时）。 取出过夜的芯片， 用 乙醇浸泡并置于摇床上， 室温 15分钟， 然后分别用去离子水及乙醇冲洗 3 次， 用氮气吹干， 储存于冰箱， 备用。 取上述芯片浸泡在 10 mL 的 EDC/NHS 溶 液 中 ， 室 温 30 分钟 ， 固 定 光 交联 试 剂 trifluoromethylaryldiazirines (TADs)。将 243个天然小分子作为待测物溶于 DMSO中， 利用高精度点样仪 Genetix Q-Array Mini将待测物点样于实施 例 1至 8制得的芯片中光交联试剂表面， 点样量为 InL, 室温下放置， 干 燥后， 在紫外光（波长 365nm, 能量 4J/cm2 )下暴露 45分钟， 光交联固 定待测物， 获得对照组芯片。 Control group: The substrate was heated to 70-80 ° C with distilled water H 2 O 30 mL; 6 mL of ammonia water was added; 6 mL of hydrogen peroxide was added; the chip to be washed was placed, and treated at 70-80 ° C for 10 min; Distilled water, washed with ethanol, and blown dry with nitrogen. A certain amount of 11-mercapto-1-undecanoic acid (hereinafter abbreviated as MUA) was weighed and dissolved in absolute ethanol to prepare a 1 mM solution. The above cleaned chip was immersed in its newly prepared solution at room temperature overnight (15 hours). The overnight chip was taken out, soaked in ethanol and placed on a shaker at room temperature for 15 minutes, then rinsed three times with deionized water and ethanol, dried with nitrogen, stored in a refrigerator, and set aside. The above chip was immersed in 10 mL of EDC/NHS solution at room temperature for 30 minutes to fix the photocrosslinking reagent trifluoromethylaryldiazirines (TADs). 243 natural small molecules were dissolved in DMSO as test substances, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a high-precision spotter Genetix Q-Array Mini. The sample amount was InL, and it was left at room temperature. After drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the test substance was fixed by photocrosslinking to obtain a control chip.
取试验组芯片及对照组芯片， 分别用 DMF、 乙醇充分清洗， 洗掉未 固定的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结 合的活化位点失活； 然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = 7.4)作为流动相， 待基线稳定后， 通入 14-3-3 靶标蛋白 （10 μΜ) 250s。本发明实施例 1提供的芯片与对照组普通芯片的表面蛋白质吸附情 况比较结果如图 1所示。其中，线 1示对照组芯片表面吸附蛋白引起 SPR 信号相应为 85 RU, 线 2示试验组芯片表面吸附蛋白引起 SPR信号相应 为 55 RU。 结果表明试验组芯片能显著降低蛋白质的非特异性吸附，从而 能提高检测的灵敏度和特异性。 Take the test chip and the control chip, wash them thoroughly with DMF and ethanol, wash away the unfixed analyte, and dry it with nitrogen and soak it in ethanolamine (1M, pH = 8.5) for 30 min to make the unbound activation site. Inactivation; then follow the SPR operating manual to install the chip with PBS buffer solution (pH = 7.4) as the mobile phase. After the baseline is stabilized, introduce 14-3-3 target protein (10 μΜ) for 250 s. The results of comparing the surface protein adsorption of the chip provided in Example 1 of the present invention with the common chip of the control group are shown in FIG. Among them, line 1 showed that the surface of the control chip adsorbed protein caused SPR The signal corresponds to 85 RU, and line 2 shows that the SPR signal is 55 RU corresponding to the adsorption protein on the surface of the test chip. The results show that the test group chip can significantly reduce the non-specific adsorption of protein, which can improve the sensitivity and specificity of detection.
用本发明实施例 2至 8制得的芯片采用上述方法，结果同上，试验组 芯片能显著降低蛋白质的非特异性吸附，从而能提高检测的灵敏度和特异 性。 实施例 10本发明提供的芯片用于药物的筛选  The chip prepared by using the inventive examples 2 to 8 adopts the above method, and as a result, the test group chip can significantly reduce the non-specific adsorption of the protein, thereby improving the sensitivity and specificity of the detection. Example 10 The chip provided by the invention is used for drug screening
试验组： 取 243个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交 联试剂表面， 室温下放置， 干燥后， 在紫外光（波长 365, 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第一芯片。 Test group: 243 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
对照组： 不与 14-3-3靶标蛋白结合的配体小分子生物素作为阴性对 照物， 溶于 DMSO中， 利用高精度点样仪 Genetix Q-Array Mini将待测物 点样于实施例 1至 8制得的芯片中光交联试剂表面， 点样量为 InL, 室温 下放置， 干燥后， 在紫外光（波长 365nm, 能量 4J/cm2 )下暴露 45分钟， 光交联固定待测物， 获得第二芯片。 Control group: The ligand small molecule biotin not bound to the 14-3-3 target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted in the example using a high-precision spotter Genetix Q-Array Mini. The surface of the photocrosslinking reagent in the chip prepared from 1 to 8 is spotted in InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and fixed by photocrosslinking. Measure the object and get the second chip.
分别取第一芯片及第二芯片， 用 DMF、 乙醇充分清洗， 洗掉未固定 的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结合的 活化位点失活；然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = Take the first chip and the second chip separately, wash thoroughly with DMF and ethanol, wash away the unfixed analyte, and dry it with nitrogen and soak it in ethanolamine (1M, pH = 8.5) for 30 min to make the unbound activation site. Inactivated; then follow the SPR operating manual to install the chip in PBS buffer solution (pH =
7.4)作为流动相，待基线稳定后，分别通入 14-3-3靶标蛋白 （10 μΜ) 250s, 试验组获得第一检测值， 对照组获得第二检测值。 检测结果如图 2所示。 其中， 线 1示待测物 R18, 线 2示待测物 FOBISIN101 , 线 3示阴性对照 物。 7.4) As the mobile phase, after the baseline was stabilized, 14-3-3 target protein (10 μΜ) was introduced for 250 s, the test group obtained the first detection value, and the control group obtained the second detection value. The test results are shown in Figure 2. Among them, line 1 indicates the analyte R18, line 2 indicates the analyte FOBISIN101, and line 3 indicates the negative control.
结果表明， 待测物 R18与 14-3-3靶标蛋白、 待测物 FOBISIN101与 The results showed that the analytes R18 and 14-3-3 target proteins, the analytes FOBISIN101 and
14-3-3靶标蛋白均具有显著作用， 而阴性对照物与 14-3-3靶标蛋白没有 明显的信号变化。 第一检测值与第二检测值相比， 具有显著差异。 第一检 测值与第二检测值的比值大于 3。 由此可知， 待测物 R18、 待测物 FOBISIN101能够与 14-3-3靶标蛋白特异性结合，可以用作相关疾病的治 疗药物。 实施例 11 本发明提供的芯片用于药物的筛选 The 14-3-3 target proteins all had significant effects, while the negative control did not show significant signal changes with the 14-3-3 target protein. The first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three. It can be seen that the analyte R18 and the test substance FOBISIN101 can specifically bind to the 14-3-3 target protein and can be used as a treatment for related diseases. Treatment drugs. Example 11 The chip provided by the invention is used for drug screening
试验组： 取 300个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交 联试剂表面， 室温下放置， 干燥后， 在紫外光（波长 365 , 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第一芯片。 Test group: 300 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
对照组： 不与 Plasmepsins II靶标蛋白结合的配体小分子生物素作为 阴性对照物， 溶于 DMSO中， 利用高精度点样仪 Genetix Q-Array Mini 将待测物点样于实施例 1至 8制得的芯片中光交联试剂表面， 点样量为 InL, 室温下放置， 干燥后， 在紫外光（波长 365nm, 能量 4J/cm2 )下暴 露 45分钟， 光交联固定待测物， 获得第二芯片。 Control group: The ligand small molecule biotin not bound to the Plasmepsins II target protein was used as a negative control, dissolved in DMSO, and the test object was spotted in Examples 1 to 8 using a high-precision spotter Genetix Q-Array Mini. The surface of the photocrosslinking reagent in the obtained chip was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the photo-crosslinking fixed the analyte. Obtain a second chip.
分别取第一芯片及第二芯片， 用 DMF、 乙醇充分清洗， 洗掉未固定 的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结合的 活化位点失活；然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = 7.4)作为流动相，待基线稳定后，分别通入 Plasmepsins II靶标蛋白 （ 10 μΜ) 250s, 试验组获得第一检测值， 对照组获得第二检测值。  Take the first chip and the second chip separately, wash thoroughly with DMF and ethanol, wash away the unfixed analyte, and dry it with nitrogen and soak it in ethanolamine (1M, pH = 8.5) for 30 min to make the unbound activation site. Inactivation; then, according to the SPR operation manual, the chip was loaded with PBS buffer solution (pH = 7.4) as the mobile phase. After the baseline was stabilized, the Plasmepsins II target protein (10 μΜ) was introduced for 250 s, and the test group obtained the first detection value. The control group obtained the second detection value.
结果表明， 待测物 P01与 Plasmepsins II靶标蛋白、 待测物 P02与 Plasmepsins II靶标蛋白均具有显著作用， 而阴性对照物与 Plasmepsins II 靶标蛋白没有明显的信号变化。第一检测值与第二检测值相比，具有显著 差异。 第一检测值与第二检测值的比值大于 3。 由此可知， 待测物 P01、 待测物 P02能够与 Plasmepsins II靶标蛋白特异性结合， 可以用作治疗相 关疾病的药物。 实施例 12本发明提供的芯片用于药物的筛选  The results showed that the P01 and Plasmepsins II target proteins, the P02 and Plasmepsins II target proteins had significant effects, while the negative control had no obvious signal changes with the Plasmepsins II target protein. The first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three. It can be seen that the test substance P01 and the test substance P02 can specifically bind to the Plasmepsins II target protein, and can be used as a drug for treating related diseases. Example 12 The chip provided by the invention is used for drug screening
试验组： 取 341个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交 联试剂表面， 室温下放置， 干燥后， 在紫外光（波长 365 , 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第一芯片。 对照组：不与 histone deacetylase 5 (HDAC5)靶标蛋白结合的配体小分 子生物素作为阴性对照物， 溶于 DMSO 中， 利用高精度点样仪 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交联试剂表 面，点样量为 InL,室温下放置，干燥后，在紫外光（波长 365 ,能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第二芯片。 Test group: 341 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip. Control group: The ligand small molecule biotin that does not bind to the histone deacetylase 5 (HDAC5) target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted using a high-precision spotter Genetix Q-Array Mini. The surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and photocrosslinked. The object to be tested obtains the second chip.
分别取第一芯片及第二芯片， 用 DMF、 乙醇充分清洗， 洗掉未固定 的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结合的 活化位点失活；然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = 7.4)作为流动相， 待基线稳定后， 分别通入 histone deacetylase 5 (HDAC5) 靶标蛋白 （10 μΜ) 250s,试验组获得第一检测值，对照组获得第二检测值。  Take the first chip and the second chip separately, wash thoroughly with DMF and ethanol, wash away the unfixed analyte, and dry it with nitrogen and soak it in ethanolamine (1M, pH = 8.5) for 30 min to make the unbound activation site. Inactivation; then, according to the SPR operation manual, the chip was loaded with PBS buffer solution (pH = 7.4) as the mobile phase. After the baseline was stabilized, the histone deacetylase 5 (HDAC5) target protein (10 μΜ) was passed for 250 s, and the test group was obtained. The first detected value and the control group obtained the second detected value.
结果表明， 待测物 HA1与 histone deacetylase 5 (HDAC5)靶标蛋白、 待测物 HA2与 histone deacetylase 5 (HDAC5)靶标蛋白均具有显著作用， 而阴性对照物与 histone deacetylase 5 (HDAC5)靶标蛋白没有明显的信号 变化。 第一检测值与第二检测值相比， 具有显著差异。 第一检测值与第二 检测值的比值大于 3。由此可知，待测物 HA1、待测物 HA2能够与 histone deacetylase 5 (HDAC5)靶标蛋白特异性结合， 可以用作治疗相关疾病的药 物。 实施例 13本发明提供的芯片用于药物的筛选  The results showed that the HA1 and the histone deacetylase 5 (HDAC5) target protein, the analyte HA2 and the hisine deacetylase 5 (HDAC5) target protein had significant effects, while the negative control and the histone deacetylase 5 (HDAC5) target protein were not obvious. The signal changes. The first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three. From this, it can be seen that the analyte HA1 and the analyte HA2 can specifically bind to the histone deacetylase 5 (HDAC5) target protein, and can be used as a medicine for treating a related disease. Example 13 The chip provided by the present invention is used for drug screening
试验组： 取 301个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交 联试剂表面， 室温下放置，干燥后，在紫外光（波长 365nm, 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第一芯片。 Test group: 301 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
对照组：不与 beta-site APP Cleaving Enzyme 1 (BACE1)靶标蛋白结合的配 体小分子生物素作为阴性对照物， 溶于 DMSO 中， 利用高精度点样仪 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交联 试剂表面， 点样量为 InL, 室温下放置， 干燥后， 在紫外光（波长 365nm, 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第二芯片。 Control group: The ligand small molecule biotin not bound to the beta-site APP Cleaving Enzyme 1 (BACE1) target protein was used as a negative control, dissolved in DMSO, and the analyte was measured using a high-precision spotter Genetix Q-Array Mini. The surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 was spotted, and the spotting amount was InL, which was left at room temperature, dried, and exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes. The photocrosslinking fixes the analyte to obtain a second chip.
分别取第一芯片及第二芯片， 用 DMF、 乙醇充分清洗， 洗掉未固定 的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结合的 活化位点失活；然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = 7.4)作为流动相， 待基线稳定后， 分别通入 beta-site APP Cleaving Enzyme l (BACEl)靶标蛋白 （10 μΜ) 250s, 试验组获得第一检测值， 对照组获得 第二检测值。 Take the first chip and the second chip separately, wash them thoroughly with DMF and ethanol, and wash them off. The test substance, after drying with nitrogen, was soaked in ethanolamine (1M, pH = 8.5) for 30 min to inactivate the unbound activation site; then, according to the SPR operating manual, the chip was mounted in PBS buffer solution (pH = 7.4). As the mobile phase, after the baseline was stabilized, the beta-site APP Cleaving Enzyme l (BACE1) target protein (10 μΜ) was passed for 250 s, the first test value was obtained in the test group, and the second test value was obtained in the control group.
结果表明， 待测物 KA2与 beta-site APP Cleaving Enzyme 1 (BACE1) 靶标蛋白、 待测物 FA2与 beta-site APP Cleaving Enzyme 1 (BACE1)靶标 蛋白均具有显著作用， 而阴性对照物与 beta-site APP Cleaving Enzyme 1 (BACE 1 )靶标蛋白没有明显的信号变化。 第一检测值与第二检测值相比， 具有显著差异。 第一检测值与第二检测值的比值大于 3。 由此可知， 待测 物 KA2、 待测物 FA2能够与 beta-site APP Cleaving Enzyme 1 (BACE1)靶 标蛋白特异性结合， 可以用作治疗相关疾病的药物。 实施例 14本发明提供的芯片用于药物的筛选  The results showed that the analyte KA2 and the beta-site APP Cleaving Enzyme 1 (BACE1) target protein, the analyte FA2 and the beta-site APP Cleaving Enzyme 1 (BACE1) target protein all had significant effects, while the negative control and beta- There was no significant signal change in the site APP Cleaving Enzyme 1 (BACE 1 ) target protein. The first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three. It can be seen that the test substance KA2 and the test substance FA2 can specifically bind to the beta-site APP Cleaving Enzyme 1 (BACE1) target protein, and can be used as a drug for treating a related disease. Example 14 The chip provided by the invention is used for drug screening
试验组： 取 260个天然配体小分子作为待测物， 溶于 DMSO中， 利 用 Genetix Q-Array Mini将待测物点样于实施例 1至 8制得的芯片中光交 联试剂表面， 室温下放置， 干燥后， 在紫外光（波长 365, 能量 4J/cm2 ) 下暴露 45分钟， 光交联固定待测物， 获得第一芯片。 Test group: 260 natural ligand small molecules were taken as the analyte, dissolved in DMSO, and the test object was spotted on the surface of the photocrosslinking reagent in the chips prepared in Examples 1 to 8 using a Genetix Q-Array Mini. After standing at room temperature, after drying, it was exposed to ultraviolet light (wavelength 365, energy 4 J/cm 2 ) for 45 minutes, and the analyte was fixed by photocrosslinking to obtain a first chip.
对照组： 不与 COX-2靶标蛋白结合的配体小分子生物素作为阴性对 照物， 溶于 DMSO中， 利用高精度点样仪 Genetix Q-Array Mini将待测物 点样于实施例 1至 8制得的芯片中光交联试剂表面， 点样量为 InL, 室温 下放置， 干燥后， 在紫外光（波长 365nm, 能量 4J/cm2 )下暴露 45分钟， 光交联固定待测物， 获得第二芯片。 Control group: The ligand small molecule biotin not bound to the COX-2 target protein was used as a negative control, dissolved in DMSO, and the sample to be tested was spotted in Example 1 using a high-precision spotter Genetix Q-Array Mini. 8 The surface of the photocrosslinking reagent in the obtained chip was spotted with InL, placed at room temperature, dried, exposed to ultraviolet light (wavelength 365 nm, energy 4 J/cm 2 ) for 45 minutes, and photocrosslinked to fix the analyte. , get the second chip.
分别取第一芯片及第二芯片， 用 DMF、 乙醇充分清洗， 洗掉未固定 的待测物， 氮气吹干后浸泡入乙醇胺 (1M, pH = 8.5) 30 min, 使未结合的 活化位点失活；然后按照 SPR的操作手册安装芯片以 PBS緩冲溶液 (pH = 7.4)作为流动相，待基线稳定后，分别通入 14-3-3靶标蛋白 （10 μΜ) 250s, 试验组获得第一检测值， 对照组获得第二检测值。  Take the first chip and the second chip separately, wash thoroughly with DMF and ethanol, wash away the unfixed analyte, and dry it with nitrogen and soak it in ethanolamine (1M, pH = 8.5) for 30 min to make the unbound activation site. Inactivation; then, according to the SPR operation manual, the chip was installed with PBS buffer solution (pH = 7.4) as the mobile phase. After the baseline was stabilized, 14-3-3 target protein (10 μΜ) was introduced for 250 s, respectively. One test value, the control group obtained the second test value.
结果表明，待测物 C08与 COX-2靶标蛋白具有显著作用， 而阴性对 照物与 COX-2靶标蛋白没有明显的信号变化。 第一检测值与第二检测值 相比，具有显著差异。第一检测值与第二检测值的比值大于 3。 由此可知， 待测物 C08能够与 COX-2靶标蛋白特异性结合，可以用作治疗相关疾病 的药物。 以上对本发明所提供的一种芯片、制备方法、用途及筛选药物的方法 阐述， 以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。 应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下， 还可以对本发明进行若干改进和修饰 ,这些改进和修饰也落入本发明权利 要求的保护范围内。 The results showed that the test substance C08 and COX-2 target protein had significant effects, while the negative pair There was no significant signal change between the vehicle and the COX-2 target protein. The first detected value has a significant difference compared to the second detected value. The ratio of the first detected value to the second detected value is greater than three. From this, it can be seen that the test substance C08 can specifically bind to the COX-2 target protein, and can be used as a drug for treating a related disease. The above description of a chip, a preparation method, a use, and a method for screening a drug provided by the present invention are merely for assisting in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims

权 利 要 求 Rights request
1、 一种芯片， 其特征在于， 包括固体支持物、 两端修饰的聚乙二醇 和光交联剂； 所述两端修饰的聚乙二醇为一端由铆定基团修饰、另一端由 结合基团修饰的聚乙二醇； 所述固体支持物与所述铆定基团连接，所述光 交联剂与所述结合基团连接； 1. A chip, characterized in that it includes a solid support, polyethylene glycol modified at both ends, and a photocrosslinking agent; the polyethylene glycol modified at both ends is modified with a riveting group at one end and a riveting group at the other end. Polyethylene glycol modified with a binding group; the solid support is connected to the riveting group, and the photo-crosslinking agent is connected to the binding group;
所述铆定基团选自 -SH、 -S-S -、 -SiCl3; The anchoring group is selected from -SH, -SS-, -SiCl 3 ;
所述结合基团选自 -COOH、 -OH、 -NH2、 -OCH3。 The binding group is selected from -COOH, -OH, -NH 2 , -OCH 3 .
2、 根据权利要求 1所述的芯片， 其特征在于， 所述两端修饰的聚乙 二醇的分子量为 100 ~ 5000。 2. The chip according to claim 1, wherein the molecular weight of the polyethylene glycol modified at both ends is 100 to 5000.
3、 根据权利要求 1所述的芯片， 其特征在于， 所述光交联剂选自苯 基叠氮类化合物、 吖丙啶类化合物、 二苯曱酮类化合物。 3. The chip according to claim 1, characterized in that the photo-crosslinking agent is selected from phenyl azide compounds, aziridine compounds, and benzophenone compounds.
4、 一种芯片的制备方法， 其特征在于， 包括如下步骤： 4. A method for preparing a chip, characterized in that it includes the following steps:
步骤 1 : 取聚乙二醇分别与铆定基团、 结合基团连接， 获得两端修饰 的聚乙二醇； Step 1: Take polyethylene glycol and connect it to the riveting group and the binding group respectively to obtain polyethylene glycol modified at both ends;
步骤 2: 取固体支持物经预处理后与所述铆定基团连接； Step 2: Take the solid support and connect it to the riveting group after pretreatment;
步骤 3: 活化所述结合基团后， 与光交联剂经酸胺缩合反应偶联， 即 付； Step 3: After activating the binding group, it is coupled with the photo-crosslinking agent through an acid-amine condensation reaction, and the payment is completed;
所述铆定基团选自 -SH、 -S-S -、 -SiCl3; The anchoring group is selected from -SH, -SS-, -SiCl 3 ;
所述结合基团选自 -COOH、 -OH、 -NH2。 The binding group is selected from -COOH, -OH, -NH2 .
5、 根据权利要求 4所述的制备方法， 其特征在于， 所述两端修饰的 聚乙二醇的分子量为 100 ~ 5000。 5. The preparation method according to claim 4, wherein the molecular weight of the polyethylene glycol modified at both ends is 100 to 5000.
6、 根据权利要求 4所述的制备方法， 其特征在于， 所述光交联剂选 自苯基叠氮类化合物、 吖丙啶类化合物、 二苯曱酮类化合物。 6. The preparation method according to claim 4, characterized in that the photo-crosslinking agent is selected from the group consisting of phenyl azide compounds, aziridine compounds, and benzophenone compounds.
7、 根据权利要求 4至 6任一项所述的制备方法制得的芯片。 7. A chip prepared according to the preparation method according to any one of claims 4 to 6.
8、 根据权利要求 1至 3任一项或权利要求 7所述的芯片用于筛选药 物的应用。 8. Application of the chip according to any one of claims 1 to 3 or claim 7 for screening drugs.
9、 一种基于芯片筛选药物的方法， 其特征在于， 包括如下步骤： 步骤 1 : 取待测物与芯片混合， 干燥后置于紫外光下经光交联后， 洗 去未结合的待测物， 灭活未结合位点后与靶标混合， 经表面等离子体共 振检测获得第一响应值； 9. A method for screening drugs based on a chip, which is characterized by including the following steps: Step 1: Mix the test substance with the chip, dry it, place it under ultraviolet light for photo-cross-linking, and wash it. Remove the unbound test substance, inactivate the unbound site and mix it with the target, and obtain the first response value through surface plasmon resonance detection;
步骤 2: 取不与靶标结合的配体作为阴性对照物与芯片混合， 干燥后 置于紫外光下经光交联后，洗去未结合的阴性对照物，灭活未结合位点后 与靶标混合， 经表面等离子体共振检测获得第二响应值； Step 2: Take the ligand that does not bind to the target and mix it with the chip as a negative control. After drying, place it under UV light for photo-cross-linking. Wash away the unbound negative control, inactivate the unbound site, and then mix it with the target. Mix, and obtain the second response value through surface plasmon resonance detection;
步骤 3: 获得所述第一响应值与所述第二响应值的比值， 根据所 述比值判断所述待测物与所述靶标是否结合； 所述比值不小于 3时， 所述待测物为与所述靶标结合的药物； 所述比值小于 3时， 所述待测 物为不与靶标结合的配体； Step 3: Obtain the ratio of the first response value to the second response value, and determine whether the test object binds to the target based on the ratio; when the ratio is not less than 3, the test object is a drug that binds to the target; when the ratio is less than 3, the test substance is a ligand that does not bind to the target;
所述芯片包括固体支持物、 两端修饰的聚乙二醇和光交联剂； 所述 两端修饰的聚乙二醇为一端由铆定基团修饰、另一端由结合基团修饰的聚 乙二醇； 所述固体支持物与所述铆定基团连接，所述光交联剂与所述结合 基团连接； The chip includes a solid support, polyethylene glycol modified at both ends, and a photocrosslinking agent; the polyethylene glycol modified at both ends is a polyethylene glycol modified with a riveting group at one end and a binding group at the other end. Diol; The solid support is connected to the riveting group, and the photo-crosslinking agent is connected to the binding group;
所述铆定基团选自 -SH、 -S-S -、 -SiCl3; The anchoring group is selected from -SH, -SS-, -SiCl 3 ;
所述结合基团选自 -COOH、 -OH、 -NH2、 -OCH3。 The binding group is selected from -COOH, -OH, -NH 2 , -OCH 3 .
10、根据权利要求 9所述的方法， 其特征在于， 所述两端修饰的聚乙 二醇的分子量为 100 ~ 5000。 10. The method according to claim 9, characterized in that the molecular weight of the polyethylene glycol modified at both ends is 100 ~ 5000.
11、根据权利要求 9所述的方法， 其特征在于， 所述光交联剂选自苯 基叠氮类化合物、 吖丙啶类化合物、 二苯曱酮类化合物。 11. The method according to claim 9, characterized in that the photo-crosslinking agent is selected from the group consisting of phenyl azide compounds, aziridine compounds, and benzophenone compounds.
PCT/CN2013/075362 2012-09-27 2013-05-09 Chip, preparation method and use thereof and method for screening drug WO2014048117A1 (en)

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