CN107153092A - 温敏响应高分子阵列的制备方法及其在样品预处理中的应用 - Google Patents
温敏响应高分子阵列的制备方法及其在样品预处理中的应用 Download PDFInfo
- Publication number
- CN107153092A CN107153092A CN201710512030.4A CN201710512030A CN107153092A CN 107153092 A CN107153092 A CN 107153092A CN 201710512030 A CN201710512030 A CN 201710512030A CN 107153092 A CN107153092 A CN 107153092A
- Authority
- CN
- China
- Prior art keywords
- sample
- array
- temperature sensitive
- temperature
- responsive polymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229920000642 polymer Polymers 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000010612 desalination reaction Methods 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 238000002493 microarray Methods 0.000 claims abstract description 12
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 10
- 230000010148 water-pollination Effects 0.000 claims abstract description 10
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 9
- 125000000304 alkynyl group Chemical group 0.000 claims abstract description 8
- 150000003839 salts Chemical class 0.000 claims abstract description 7
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims abstract description 6
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 claims abstract description 6
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical class CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims abstract description 6
- 231100000719 pollutant Toxicity 0.000 claims abstract description 6
- MRNHPUHPBOKKQT-UHFFFAOYSA-N indium;tin;hydrate Chemical compound O.[In].[Sn] MRNHPUHPBOKKQT-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 53
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 229920002521 macromolecule Polymers 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
- 239000012498 ultrapure water Substances 0.000 claims description 9
- 102000007079 Peptide Fragments Human genes 0.000 claims description 8
- 108010033276 Peptide Fragments Proteins 0.000 claims description 8
- 238000011065 in-situ storage Methods 0.000 claims description 8
- 238000011010 flushing procedure Methods 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 5
- AFVLVVWMAFSXCK-VMPITWQZSA-N alpha-cyano-4-hydroxycinnamic acid Chemical group OC(=O)C(\C#N)=C\C1=CC=C(O)C=C1 AFVLVVWMAFSXCK-VMPITWQZSA-N 0.000 claims description 5
- NKNDPYCGAZPOFS-UHFFFAOYSA-M copper(i) bromide Chemical compound Br[Cu] NKNDPYCGAZPOFS-UHFFFAOYSA-M 0.000 claims description 5
- 239000003999 initiator Substances 0.000 claims description 5
- 235000015511 Liquidambar orientalis Nutrition 0.000 claims description 4
- 235000000126 Styrax benzoin Nutrition 0.000 claims description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 230000007704 transition Effects 0.000 claims description 4
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 3
- NZNMSOFKMUBTKW-UHFFFAOYSA-N Cyclohexanecarboxylic acid Natural products OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 claims description 3
- 239000004870 Styrax Substances 0.000 claims description 3
- 239000000178 monomer Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 229930005346 hydroxycinnamic acid Natural products 0.000 claims description 2
- 235000010359 hydroxycinnamic acids Nutrition 0.000 claims description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 150000003254 radicals Chemical class 0.000 claims description 2
- 230000002269 spontaneous effect Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 244000028419 Styrax benzoin Species 0.000 claims 1
- 150000001262 acyl bromides Chemical class 0.000 claims 1
- 238000004220 aggregation Methods 0.000 claims 1
- 230000002776 aggregation Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 3
- 101710141544 Allatotropin-related peptide Proteins 0.000 abstract description 2
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 abstract description 2
- 230000000977 initiatory effect Effects 0.000 abstract 1
- 229920002627 poly(phosphazenes) Polymers 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 241000736148 Styrax Species 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical class OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 3
- 238000010183 spectrum analysis Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 206010016825 Flushing Diseases 0.000 description 2
- 229920002518 Polyallylamine hydrochloride Polymers 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- VTXVGVNLYGSIAR-UHFFFAOYSA-N decane-1-thiol Chemical compound CCCCCCCCCCS VTXVGVNLYGSIAR-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 229910052738 indium Inorganic materials 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000010526 radical polymerization reaction Methods 0.000 description 2
- 238000009938 salting Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004115 Sodium Silicate Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000007740 vapor deposition Methods 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical class OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F120/52—Amides or imides
- C08F120/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
Abstract
本发明涉及温敏响应高分子阵列的制备方法及其在样品预处理中的应用，基于光影印技术和炔基与巯基的点击化学技术，在氧化铟锡玻片(ITO玻片)构建羟基亲水性微阵列，再在亲水微阵列内基于表面引发原子转移自由基聚合生成高分子聚(N‑异丙基丙烯酰胺)(PNIPAM)的可控亲水性阵列。当低于临界温度时高分子阵列其亲水性作用使得样品汇聚在玻片表面，高于临界温度时蛋白质/肽段通过疏水作用与疏水性的高分子阵列相吸附，而共存的盐和污染物则被冲走，从而实现样品靶上自除盐和高效富集的预处理，进而可对目标样品进行MALDI‑MS一步快速检测。本发明制备及操作简单、普适性高、分析时间短、成本低廉，具有一定的生物医学应用价值。
Description
技术领域
本发明涉及温敏响应高分子阵列的制备方法及其在样品预处理中的应用，具体包括直接在氧化铟锡玻片(ITO玻片)表面生长高分子聚(N-异丙基丙烯酰胺)(PNIPAM)来制备可控亲水性阵列，并将其用于样品的预处理。
背景技术
生物质谱是蛋白质组学研究的重要技术手段，在蛋白样品的处理过程中非挥发性盐的存在会抑制样品的离子化，从而造成检测的灵敏度的降低甚至无法检测，因此，对蛋白质或多肽样品进行除盐往往是质谱分析前必不可少的步骤。
目前，对蛋白质/肽段样品的除盐方法主要是基于固相萃取技术，即蛋白或多肽保留在吸附材料上，盐类不吸附而被除去，然后将保留的蛋白或肽段洗脱下来进行检测，该方法虽然已推广应用，但是由于处理过程中需要多次的样品吸附、洗脱、转移，所以存在步骤复杂、所需时间长、而且会造成样品的损失和潜在的样品污染等缺点。近年来，利用疏水聚合物的表面化技术实现了样品高效靶上原位除盐与富集后直接用于基质辅助激光解吸电离飞行时间质谱(MALDI-MS)检测，该方法虽然可避免样品额外的损失以及污染物的引入，但刚性的憎水高分子会在表面形成一层致密的单分子层进而会限制样品与吸附剂的充分接触，所以对样品的表面富集及检测的灵敏度会造成影响。
聚异丙基丙烯酰胺(PNIPAM)是一种温敏型聚合物，当环境温度低于其最低临界温度LCST(32℃)时，亲水基团与水分子呈现较强的氢键作用，PNIPAM聚合物可溶于水，聚合物链在水中呈现伸展结构。温度高于LCST时，氢键破坏，异丙基和碳链的疏水作用导致PNIPAM聚合物链呈现收缩结构。近年来，PNIPAM修饰的纳米颗粒已被用于温度可控的样品的富集和释放，但由于缺少表面阵列的汇聚效应，所以在样品的富集及检测灵敏度方面仍然存在限制。因此，设计制备一种特殊的高分子阵列、实现样品的简单快速除盐及高灵敏检测势在必行。
发明内容
针对现有技术中存在的缺点与不足，本发明的目的在于提供一种温敏响应高分子阵列的制备方法及其在样品预处理中的应用。本发明制备及操作简单、普适性高、分析时间短、成本低廉，具有一定的生物医学应用价值。
本发明解决其技术问题所采用的技术方案是：
温敏响应高分子阵列的制备方法，利用光影印技术和炔基与巯基的点击化学技术，在氧化铟锡玻片(ITO玻片)构建羟基(OH)亲水性微阵列；利用羟基与2-溴异丁酰溴(BIBB)间的共价反应形成原位自由基聚合反应的引发剂，再在溴化亚铜(CuBr)和2,2’-联吡啶(Bpy)的催化下，单体N-异丙基丙烯酰胺(NIPAM)在ITO玻片表面聚合生成所述温敏反应高分子阵列，即高分子聚(N-异丙基丙烯酰胺)(PNIPAM)的可控亲水性阵列。
进一步地，1-硫代甘油通过光引发剂安息香二乙醚，使巯基(SH)与ITO玻片上的炔基共价结合，形成羟基亲水性微阵列。
进一步地，利用表面原位生成的所述引发剂，在常温有机相条件下引发表面原子转移自由基聚合反应。
温敏响应高分子阵列的制备方法制得的高分子阵列的应用，所述温敏反应高分子阵列可通过温度转变自发进行表面除盐及样品富集，并用于对样品的预处理。当低于临界温度时，所述温敏响应高分子阵列的亲水性作用使得样品汇聚在ITO玻片表面，而高于临界温度时，样品中蛋白质或肽段通过疏水作用与疏水性的所述温敏响应高分子阵列相吸附，而共存的盐和污染物则被冲走，从而实现样品靶上自除盐和高效富集，即完成对样品的预处理。
对样品的预处理包括如下步骤：
(1)将样品溶液滴在含所述高分子PNIPAM的可控亲水性阵列的ITO玻片上，并在60℃条件下温育30min-1h；
(2)用冲洗液对样品进行2-3次冲洗；将基质溶液滴在样品处，并在4℃下蒸干溶液；
(3)通过基质辅助激光解吸电离飞行时间质谱(MALDI-MS)进行信号采集，从质谱图中得到样品蛋白质或肽段信息。
进一步地，所述样品溶液的体积为1μL；冲洗液为60℃超纯水，体积为2μL；基质溶液的配比如下：α-氰基-4-羟基肉桂酸(CHCA)固体溶解到含有体积百分数分别为50％的乙腈、0.01％的三氟乙酸和49.9％的超纯水的溶剂中，配成最终浓度为10mg/ml的溶液，体积为1μL。
本发明测定目标蛋白质/肽段的原理如下：
本发明设计的温敏响应高分子阵列的制备方法以及将其用于样品预处理的原理如图1和2所示：通过光影印技术和炔基与巯基的点击化学技术，在ITO玻片构建OH亲水性微阵列，进而在亲水微阵列内基于表面引发原子转移自由基聚合生成温敏响应高分子PNIPAM阵列；当低于临界温度时高分子阵列基于亲水性作用使得样品汇聚在玻片表面，而高于临界温度时蛋白质/肽段通过疏水作用与疏水性的高分子相吸附，而共存的盐和污染物则被热水冲走，实现了样品的预处理(样品靶上自除盐和高效富集)，进而通过MALDI-MS测得的特征峰，可以实现蛋白质/肽段的快速检测。
本发明通过光影印和点击化学技术在ITO玻片构建亲水性微阵列，结合原位自由基聚合反应生成高分子PNIPAM的可控亲水性阵列，提出一种简单、快速、高灵敏的靶上自除盐方法；相对现有质谱前的除盐方法，本发明具有以下特点：
(1)利用光影印和点击化学技术同时结合原位自由基聚合反应生成高分子的可控亲水性阵列，制备简单、成本低廉、重复性好。
(2)利用温敏型高分子阵列进行高效靶上自除盐，不需要进行样品的转移，可以避免了样品额外的损失以及污染物的引入。
(3)本发明构建了亲疏水微阵列，可以通过强汇聚作用使样品自动富集在较小的亲水区域，可以实现样品高效靶上原位除盐与富集后直接用于MALDI-MS检测，免去多次样品转移造成的样品损失和污染，从而提高检测灵敏度，使其更适于低丰度蛋白质的检测。
(4)本发明普适性高、分析时间短、方法非常简便，具有一定的生物医学应用价值。
附图说明
图1为高分子PNIPAM的可控亲水性阵列的制备方法示意图；
图2为可控亲水性阵列对蛋白质/肽段样品自除盐的示意图。
具体实施方式
实施例1：结合附图1，详述高分子PNIPAM的可控亲水性阵列的制备过程。
(1)将ITO玻片经过聚丙烯胺盐酸盐(PAH)和硅酸钠溶液交替喷射，用超纯水冲洗，氮气吹干，在电阻炉中450°煅烧4h。100μL硅烷化试剂(O-(propargyl)-N-(triethoxysilylpropyl)carbamate)和0.5g MgSO4·7H2O经过气相沉积反应在ITO玻片表面修饰炔基。
(2)在上述ITO玻片上滴入50vol％ 1-硫代甘油和2wt％安息香二乙醚(DMPAP)的四氢呋喃溶液，铺上光掩膜在365nm 14.4mW·cm-2紫外光下照射20s，依次用四氢呋喃、超纯水冲洗，氮气吹干(即1-硫代甘油通过光引发剂安息香二乙醚，使巯基与ITO玻片上的炔基共价结合，形成-OH亲水性微阵列，DMPAP是光引发剂，相当于催化剂，可加速紫外光下巯基与炔基的反应)；
再滴入5vol％全氟癸硫醇和2wt％DMPAP的乙醇溶液，铺上反向光掩膜在365nm150mW·cm-2紫外光下照射60s，依次用乙醇、超纯水冲洗，氮气吹干(全氟癸硫醇使得亲水的羟基阵列周边憎水性更强，这样形成的阵列由于阵列里外的亲憎水性差距更大，使形成的阵列效果更明显)。
(3)将上述ITO玻片置于0.08M BIBB和0.1M三乙醇胺(TEA)的N,N-二甲基甲酰胺(DMF)溶液中，室温下反应30min，DMF冲洗、超纯水冲洗，氮气吹干，可生成原位自由基聚合反应的引发剂。
(4)将步骤(3)中ITO玻片置于含2M NIPAM单体和9mM CuBr、18mM2,2’-bipyridine(Bpy)的除氧超纯水中，通入氮气，室温下反应30min，超纯水冲洗，氮气吹干，生成高分子PNIPAM的可控亲水性阵列。
实施例2：结合附图2，以细胞色素C(cyt-c)的酶解液为例，说明该PNIPAM的可控亲水性阵列在蛋白质/肽段自除盐中的应用。
(1)配制样品液：把1mL含50mM NH4HCO3的0.5mg/mL cyt-c溶液与100μL含50mMNH4HCO3的0.2mg/ml胰蛋白酶溶液混合，在37°恒温箱中温育24h。用50mM NH4HCO3溶液稀释使它们的最终浓度分别为4pmol/μL、400fmol/μL、40fmol/μL。(本实施例的样品是蛋白质的盐溶液，要分析酶解之后的各个肽段，必须先用酶将蛋白酶解成肽段，再进行除盐和富集，质谱分析；如果样品是纯蛋白的盐溶液，那就直接不用利用酶处理，除盐之后质谱分析的是蛋白的质荷比)。
(2)分别滴加1μL上述样品液于含PNIPAM可控亲水性阵列的ITO玻片上，并在60℃条件下温育30min。
(3)用2μL 60℃超纯水对样品进行3次冲洗，再将1μL基质溶液(基质溶液的配比：α-氰基-4-羟基肉桂酸(CHCA)固体溶解到含有体积百分数分别为50％的乙腈、0.01％的三氟乙酸和49.9％的超纯水的溶剂中，配成最终浓度为10mg/ml的溶液；基质溶液作用如下：将被分析物的溶液和某种基质溶液混合，蒸发溶剂，使被分析物与基质成为晶体或半晶体，再用一定波长的脉冲式激光进行照射,基质吸收激光能量后,均匀地传递给待分析物，使待分析物瞬间气化并离子化，离子质量的不同导致其飞行速度不同而分离)滴在样品处，并在4℃下蒸干溶液，通过质谱仪进行质谱信号采集。
(4)从质谱图得到样品蛋白质信息。
现有文献报道过利用磁性纳米材料除盐富集肽段的覆盖率是62％，通过本发明实施例除盐之后测定的cyt-c的酶解液肽段峰的覆盖率是67.5％，不但检测的灵敏度有所提高，而且通过本实施例预处理样品(除盐)的方法非常简便，实用性更强。
Claims (7)
1.温敏响应高分子阵列的制备方法，其特征在于，利用光影印技术和炔基与巯基的点击化学技术，在氧化铟锡玻片(ITO玻片)构建羟基(OH)亲水性微阵列；利用羟基与2-溴异丁酰溴(BIBB)间的共价反应形成原位自由基聚合反应的引发剂，再在溴化亚铜(CuBr)和2,2’-联吡啶(Bpy)的催化下，单体N-异丙基丙烯酰胺(NIPAM)在ITO玻片表面聚合生成所述温敏反应高分子阵列，即高分子聚(N-异丙基丙烯酰胺)(PNIPAM)的可控亲水性阵列。
2.如权利要求1所述的温敏响应高分子阵列的制备方法，其特征在于，所述羟基亲水性微阵列的制备：1-硫代甘油通过光引发剂安息香二乙醚，使巯基(SH)与ITO玻片上的炔基共价结合，形成羟基亲水性微阵列。
3.如权利要求1所述的温敏响应高分子阵列的制备方法，其特征在于，利用表面原位生成的所述引发剂，在常温有机相条件下引发表面原子转移自由基聚合反应。
4.权利要求1所述的温敏响应高分子阵列的制备方法制得的高分子阵列的应用，其特征在于，所述温敏反应高分子阵列可通过温度转变自发进行表面除盐及样品富集，并用于对样品的预处理。
5.如权利要求4所述的应用，其特征在于，当低于临界温度时，所述温敏响应高分子阵列的亲水性作用使得样品汇聚在ITO玻片表面，而高于临界温度时，样品中蛋白质或肽段通过疏水作用与疏水性的所述温敏响应高分子阵列相吸附，而共存的盐和污染物则被冲走，从而实现样品靶上自除盐和高效富集，即完成对样品的预处理。
6.如权利要求4或5所述的应用，其特征在于，对样品的预处理包括如下步骤：
(1)将样品溶液滴在含所述高分子PNIPAM的可控亲水性阵列的ITO玻片上，并在60℃条件下温育30min-1h；
(2)用冲洗液对样品进行2-3次冲洗；将基质溶液滴在样品处，并在4℃下蒸干溶液；
(3)通过基质辅助激光解吸电离飞行时间质谱(MALDI-MS)进行信号采集，从质谱图中得到样品蛋白质或肽段信息。
7.如权利要求6所述的应用，其特征在于，所述样品溶液的体积为1μL；冲洗液为60℃超纯水，体积为2μL；基质溶液的配比如下：α-氰基-4-羟基肉桂酸(CHCA)固体溶解到含有体积百分数分别为50％的乙腈、0.01％的三氟乙酸和49.9％的超纯水的溶剂中，配成最终浓度为10mg/ml的溶液，体积为1μL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710512030.4A CN107153092B (zh) | 2017-06-28 | 2017-06-28 | 温敏响应高分子阵列的制备方法及其在样品预处理中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710512030.4A CN107153092B (zh) | 2017-06-28 | 2017-06-28 | 温敏响应高分子阵列的制备方法及其在样品预处理中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107153092A true CN107153092A (zh) | 2017-09-12 |
| CN107153092B CN107153092B (zh) | 2019-07-23 |
Family
ID=59795062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710512030.4A Active CN107153092B (zh) | 2017-06-28 | 2017-06-28 | 温敏响应高分子阵列的制备方法及其在样品预处理中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107153092B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362890A (zh) * | 2018-02-08 | 2018-08-03 | 南京大学 | 基于含硼梳形聚合物图案化靶板富集、除盐和鉴定糖肽的方法 |
| CN109354637A (zh) * | 2018-10-13 | 2019-02-19 | 菏泽学院 | 聚n-异丙基丙烯酰胺油凝胶合成方法 |
-
2017
- 2017-06-28 CN CN201710512030.4A patent/CN107153092B/zh active Active
Non-Patent Citations (4)
| Title |
|---|
| ERICK YU等: ""Poly(N-isopropylacrylamide)-gated Fe3O4/SiO2 core shell nanoparticles for the temperature triggered release of lysozyme"", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
| KENICHI NAGASE等: ""Preparation of Thermoresponsive Cationic Copolymer Brush Surfaces and Application of the Surface to Separation of Biomolecules"", 《BIOMACROMOLECULES》 * |
| OSCAR NORBERG等: ""Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry"", 《BIOSENSORS AND BIOELECTRONICS》 * |
| XIAOYUN QIU等: ""Fabrication of dual-responsive cellulose-based membrane via simplified surface-initiated ATRP"", 《CARBOHYDRATE POLYMERS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108362890A (zh) * | 2018-02-08 | 2018-08-03 | 南京大学 | 基于含硼梳形聚合物图案化靶板富集、除盐和鉴定糖肽的方法 |
| CN109354637A (zh) * | 2018-10-13 | 2019-02-19 | 菏泽学院 | 聚n-异丙基丙烯酰胺油凝胶合成方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107153092B (zh) | 2019-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Djozan et al. | Evaluation of a new method for chemical coating of aluminum wire with molecularly imprinted polymer layer. Application for the fabrication of triazines selective solid-phase microextraction fiber | |
| AU774336B2 (en) | Probes for a gas phase ion spectrometer | |
| US20060000772A1 (en) | Separation apparatus and separation method | |
| Worrall et al. | Purification of contaminated peptides and proteins on synthetic membrane surfaces for matrix-assisted laser desorption/ionization mass spectrometry | |
| Huang et al. | Simultaneous determination of iodide and iodate in povidone iodine solution by ion chromatography with homemade and exchange capacity controllable columns and column-switching technique | |
| Asiabi et al. | On-line electrochemically controlled in-tube solid phase microextraction of inorganic selenium followed by hydride generation atomic absorption spectrometry | |
| Sangsuwan et al. | Patterned poly (acrylic acid) brushes containing gold nanoparticles for peptide detection by surface-assisted laser desorption/ionization mass spectrometry | |
| CN107153092A (zh) | 温敏响应高分子阵列的制备方法及其在样品预处理中的应用 | |
| CN104725559B (zh) | 嗜硫色谱材料及其制备方法和应用 | |
| KR101592517B1 (ko) | 레이저 탈착 이온화 질량 분석용 기판 및 그 제조방법 | |
| CN104258826A (zh) | 一种活性/可控介孔材料表面离子印迹聚合物及其制备方法与应用 | |
| CN111474283A (zh) | 对目标化合物进行定性/定量检测的方法和试剂盒 | |
| WO2004031759A1 (ja) | 質量分析用プレート、その調製方法およびその用途 | |
| Miah et al. | Rapid CE-UV evaluation of polypyrrole-coated magnetic nanoparticles for selective binding of endocrine disrupting compounds and pharmaceuticals by aromatic interactions | |
| US9607816B2 (en) | Two-dimensional separation and imaging technique for the rapid analysis of biological samples | |
| Wojciechowski et al. | Ion emission from water ice due to energetic particle bombardment | |
| Rahimi et al. | Application of copolymer coated frits for solid-phase extraction of poly cyclic aromatic hydrocarbons in water samples | |
| CN110895271A (zh) | 一种快速检测生物基质样品中百草枯的方法 | |
| Deng et al. | Sensitivity and robustness enhancements by using a V-shape ion funnel in FTICR-MS | |
| WO2004031757A1 (ja) | 物質の分離方法 | |
| CN114749221A (zh) | 一种蛋白质组学样品前处理装置及其制备方法与应用 | |
| CN107014891B (zh) | 一种用于激光解吸离子化质谱的螺旋二十四面体结构基底 | |
| Liu et al. | Dual-use ionization source for direct and sensitive detection of trace carbamates residue | |
| CN113686832B (zh) | 一种磺胺类抗生素的超灵敏类识别点阵阵列检测方法 | |
| JP6685587B2 (ja) | 質量分析用イオン化支援剤及びこれを使用した質量分析方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |