CN105648033A - Method for determining trypsin in pancreatic tissue sample of exercise mouse - Google Patents
Method for determining trypsin in pancreatic tissue sample of exercise mouse Download PDFInfo
- Publication number
- CN105648033A CN105648033A CN201610099005.3A CN201610099005A CN105648033A CN 105648033 A CN105648033 A CN 105648033A CN 201610099005 A CN201610099005 A CN 201610099005A CN 105648033 A CN105648033 A CN 105648033A
- Authority
- CN
- China
- Prior art keywords
- peptide
- dna
- trypsin
- solution
- electrode
- 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
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000012588 trypsin Substances 0.000 title claims abstract description 34
- 108090000631 Trypsin Proteins 0.000 title claims abstract description 33
- 102000004142 Trypsin Human genes 0.000 title claims abstract description 33
- 210000004923 pancreatic tissue Anatomy 0.000 title claims abstract description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 44
- 239000010931 gold Substances 0.000 claims abstract description 24
- 229910052737 gold Inorganic materials 0.000 claims abstract description 24
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 230000008836 DNA modification Effects 0.000 claims description 12
- 241000700159 Rattus Species 0.000 claims description 11
- 241000699670 Mus sp. Species 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 6
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 6
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 2
- -1 PBST compound Chemical class 0.000 claims description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000008366 buffered solution Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 2
- 230000012447 hatching Effects 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 108091005601 modified peptides Proteins 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229910052697 platinum Inorganic materials 0.000 claims description 2
- 238000005498 polishing Methods 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000012488 sample solution Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000010408 sweeping Methods 0.000 claims description 2
- 230000009182 swimming Effects 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 claims description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 abstract description 17
- 229960000907 methylthioninium chloride Drugs 0.000 abstract description 17
- 238000002848 electrochemical method Methods 0.000 abstract description 6
- 125000003277 amino group Chemical group 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 238000009396 hybridization Methods 0.000 abstract description 3
- 230000011664 signaling Effects 0.000 abstract description 3
- 238000011895 specific detection Methods 0.000 abstract description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 108010054147 Hemoglobins Proteins 0.000 description 4
- 102000001554 Hemoglobins Human genes 0.000 description 4
- 102000016943 Muramidase Human genes 0.000 description 4
- 108010014251 Muramidase Proteins 0.000 description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 4
- 102000057297 Pepsin A Human genes 0.000 description 4
- 108090000284 Pepsin A Proteins 0.000 description 4
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 4
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 229960000274 lysozyme Drugs 0.000 description 4
- 239000004325 lysozyme Substances 0.000 description 4
- 235000010335 lysozyme Nutrition 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010038061 Chymotrypsinogen Proteins 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960001322 trypsin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
Abstract
The invention belongs to the field of electrochemical techniques and discloses measurement of trypsin content with a peptide as a recognition unit and methylene blue as a signaling molecule. A glutaraldehyde method is used, the peptide and DNA are cross-linked under the action of a terminal amino group of the peptide and an amino group of DNA, and peptide/DNA is obtained; then the surface of a gold electrode is modified with peptide/DNA under the action between an Au atom and a terminal sulfhydryl group of the peptide; H1 and H2 are added, a hybridization chain reaction is performed, DNA double strands are formed, and then methylene blue is added and cut in the DNA double strands; when the target trypsin exists, the trypsin cuts the peptide, the DNA double strands cut with methylene blue fall off from the electrode accordingly, and electrochemical signals are weakened. Determination of the target trypsin is realized according to intensity of the electrochemical signals, and an electrochemical method for high-sensitivity specific detection of the trypsin in a pancreatic tissue sample of an exercise mouse is established and has the characteristics of simplicity, low cost and high sensitivity.
Description
Technical field
The invention belongs to technical field of electrochemistry, for signaling molecule, trypsin amount is detected for recognition unit and methylene blue with peptide. It is specifically related to a kind of tryptic method in mensuration exercised rats pancreatic tissue sample.
Background technology
Trypsin is a kind of serine protein hydrolase. In vertebrates, it not only plays the effect of digestive enzyme, and can also limit the precursor decomposing other enzymes such as chymotrypsinogen, procarboxypeptidase, phospholipid proenzyme, plays activation. Trypsin is the protease that specificity is the strongest, and in the amino acid range determining protein, it becomes indispensable instrument.
The tryptic method of mensuration grown up at present mainly has ultraviolet spectrophotometry (ZhangL, DuJ.Asensitiveandlabel-freetrypsincolorimetricsensorwith cytochromecasasubstrate [J] .Biosensors&Bioelectronics, 2016, 79:347-352.), liquid crystal method (ChuangCH, LinYC, ChenWL, etal.Detectingtrypsinatliquidcrystal/aqueousinterfacebyu singsurface-immobilizedbovineserumalbumin. [J] .Biosensors&Bioelectronics, 2015, 78.), fluorescence method (Mu Lixuan, Shi Wensheng. the preparation method of trypsin fluorescence chemical sensor:, CN104152141A [P] .2014, Jia Lan, Chen Song, Liu Xiang, Hou Wenjuan, Zhang Yaodong. a kind of fluorescent probe for trypsin detection and preparation method thereof:, CN104946249A [P] .2015), electrochemical method (Ru-PingL, Xiao-CuiT, PingQ, etal.MultiplexedElectrochemicalDetectionofTrypsinandChym otrypsinBasedonDistinguishableSignalNanoprobes [J] .AnalyticalChemistry, 2014,86 (18): 9256-9263, QiangYi, QicaiLiu, FengGao, QingquanChen, GuinaWang.ApplicationofanElectrochemicalImmunosensorwith aMWCNT/PDAAModifiedElectrodeforDetectionofSerumTrypsin [J] .Sensors, 2014, 14 (6): 10203-10212.), QCM method (StoytchevaM, ZlatevR, CosnierS, etal.Highsensitivetrypsinactivityevaluationapplyinganano structuredQCM-sensor [J] .Biosensors&Bioelectronics, 2012, 41 (1): 862 866,BayramogluG, AricaMY.Developmentofasensitivemethodforselectionofaffin ityligandfortrypsinusingquartzcrystalmicrobalancesensor. [J] .Bioprocess&BiosystemsEngineering, 2012,35 (3): 423-431.), optical colorimetry (ZaccheoBA, CrooksRM.Self-PoweredSensorforNaked-EyeDetectionofSerumT rypsin [J] .AnalyticalChemistry, 2011,83 (4): 1185-8.) etc. But, these methods are respectively arranged with its shortcoming.
The present invention utilizes glutaraldehyde method, by peptide terminal amino group and DNA amino effect, peptide and DNA is cross-linked, obtains peptide/DNA; Then utilize and act between Au atom and peptide terminal sulfhydryl group, by peptide/DNA modification in gold electrode surfaces; Adding H1 and H2, occur hybridization chain reaction to form DNA double chain, be subsequently adding methylene blue, methylene blue cuttage is in the DNA double chain formed; When there being object trypsin to exist, peptide cutting is caused that cuttage has the DNA double chain of methylene blue to come off from electrode by trypsin, electrochemical signals reduces, power according to electrochemical signals realizes the tryptic mensuration of object, establish a kind of to the electrochemical method of trypsin high sensitivity specific detection in exercised rats pancreatic tissue sample, method has simply, and cost is low, highly sensitive feature.
Summary of the invention
The present invention provides a kind of method of trypsin amount in specific detection exercised rats pancreatic tissue sample, utilizes glutaraldehyde method, by peptide terminal amino group and DNA amino effect, peptide and DNA is cross-linked, obtains peptide/DNA; Then utilize and act between Au atom and peptide terminal sulfhydryl group, by peptide/DNA modification in gold electrode surfaces; Adding H1 and H2, first occur hybridization chain reaction to form DNA double chain, be subsequently adding methylene blue, methylene blue cuttage is in the DNA double chain formed; When there being object trypsin to exist, peptide cutting is caused that cuttage has the DNA double chain of methylene blue to come off from electrode by trypsin, and electrochemical signals reduces, and realizes the tryptic mensuration of object according to the power of electrochemical signals.
Technical scheme
A kind of measure tryptic electrochemical method in exercised rats pancreatic tissue sample, using methylene blue as signaling molecule, utilize with the trypsin specificity dissection to peptide, measure tryptic content with electrochemical method detection method. Determination step is as follows:
(1) preparation of peptide/DNA1
Take 0.1��20 �� g peptide solid and be dispersed in the PBS buffer solution containing 5% glutaraldehyde, adding the DNA1 solution that 100 �� L concentration are 0.1��1000mM after continuously stirred 2h again in solution, 4 DEG C of vibrations obtain peptide modifying DNA 1 after hatching 12h, i.e. peptide/DNA1, preserves at 4 DEG C.
(2) prepared by peptide/DNA modification gold electrode
First by gold electrode respectively with 1.0 ��m, 0.3 ��m, 0.05 ��m of alumina powder polishing, successively with distilled water, ethanol and distilled water ultrasonic 5min respectively, finally at the H of 0.1M2SO4Solution is circulated voltammetric scan until i-v curve is stable; At 1��60 �� L peptide/DNA solution of clean gold electrode surfaces dropping (1) gained, at 25 DEG C, assemble 120min. The gold electrode surfaces deionized water having modified peptide/DNA is cleaned three times, dries up with high pure nitrogen. Then gold electrode is immersed in the sulfydryl hexanol solution of 1mM, at 25 DEG C, close 60min, obtain peptide/DNA modification gold electrode after fully cleaning, save backup at 4 DEG C.
(3) H1, H2 and MB self-assembling peptides/DNA modification gold electrode
Take 1��60 �� L mixed solution containing H1 and the H2 of 0.001��400 ��M and drip the peptide in (3) gained/DNA modification gold electrode surfaces. After at room temperature reaction a period of time, then with the PBST solution washing three times of 50 �� L. Then take 1��60 �� L MB solution containing 0.001��400mM again and drip in electrode surface, after at room temperature reaction 30 minutes, then with the PBST solution washing three times of 50 �� L. Obtain H1, H2 and MB self-assembling peptides/DNA modification gold electrode.
(4) electrochemical method determining trypsin
1��60 �� L trypsin sample solution is added drop-wise to H1, H2 and MB self-assembling peptides/DNA modification gold electrode surfaces, at room temperature reacts 30min. Then, electrode surface is repeatedly rinsed with phosphate buffer solution three times. With above-mentioned the electrode obtained for working electrode, saturated calomel electrode is reference electrode, platinum electrode is auxiliary electrode, utilizes differential pulse voltammetry (DPV) speed of sweeping with 1��1000mV/s in phosphate buffer solution to be scanned, and electric potential scanning ranges for-0.4 to-0.1V. According to electrochemical signals, trypsin is carried out quantitatively.
(5) exercised rats pancreatic tissue sample measures
Mouse movement mode adopts disposable exhausted swimming, and water temperature controls at 32 �� 1 DEG C, and mice pool specifications is long 100cm �� 50cm �� 50cm, and mice continues to sink ten seconds to surface and exhausts standard for power. Mice broken end is drawn materials, and after adding homogenate medium by 1:10, is made into tissue homogenate, the frozen centrifugation 15min when 3000 revs/min, takes supernatant and measures trypsin by (4) described method.
Experimental water of the present invention is redistilled water.
Phosphate buffer solution compound method used in the present invention is: dissolve 0.2gKH in one liter of water2PO4And 2.9gNa2HPO4Obtain the phosphate buffer of 0.1MpH7.4.
PBS compound method used in the present invention is: by 0.2gKH2PO4, 8.0gNaCl and 0.2gKCl be dissolved in one liter of water, obtain the phosphate buffered solution that 0.15MpH is 7.4, i.e. PBS.
PBST compound method used in the present invention is: adding Tween-20 in phosphate buffer solution, the volume ratio making Tween-20 is 0.05%, obtains PBST solution.
The electrochemical workstation of the present invention selects CHI660D type electrochemical workstation (Shanghai Chen Hua Instrument Ltd.).
The remarkable result of the present invention
The present invention have studied the relation between the trypsin of variable concentrations and electrochemical signals, obtains the tryptic standard curve of detection, the range of linearity and linear equation. When tryptic concentration is at 2.0ngmL-1-700ngmL-1Between time, along with the change of trypsinase concentration, DPV intensity has significant change. Calculating the tryptic linear equation of detection is that (Ipa is DPV peak current intensity to I=0.47085C-1.25723, unit nA; C is tryptic concentration, unit ngmL-1; R2=0.9997), detection is limited to 0.5ngmL-1��
The precision of this assay method is by being 3ngmL to concentration-1Trypsin carry out 11 parallel assays and calculate, relative standard deviation respectively 2.1%, it was shown that the assay method of the present invention has good repeatability.
The method is measured tryptic selectivity by the compositions such as thrombin, pepsin, lysozyme, alkali phosphatase, bovine serum albumin, peroxide oxidation enzyme, glucoseoxidase and the hemoglobin having examined in animal body and often contain in exercised rats pancreatic tissue sample.Test result indicate that, when tryptic concentration is 20ngmL-1Time, the error thrombin of 1000 times, pepsin, lysozyme, alkali phosphatase, bovine serum albumin, peroxide oxidation enzyme, glucoseoxidase and hemoglobin in the scope of 5% all do not disturb the method to tryptic response. Showing that method has high selectivity, this method has the feasibility being applied to authentic sample detection.
Accompanying drawing explanation
Fig. 1. trypsin measuring principle figure.
Fig. 2. trypsin canonical plotting. Abscissa is trypsinase concentration, and unit is ngmL-1, vertical coordinate Ipa is DPV peak current intensity.
Fig. 3. the method testing result to sample. (A) thrombin, (B) pepsin, (C) lysozyme, (D) alkali phosphatase, (E) bovine serum albumin, (F) peroxide oxidation enzyme, (G) glucoseoxidase, (H) hemoglobin and (I) trypsin.
Specific implementation method
The following is the specific embodiment that the present invention relates to, technical scheme is done to be described further, but protection scope of the present invention is not limited to these embodiments. All changes without departing substantially from present inventive concept or equivalent replacement are included within protection scope of the present invention.
Obtain trypsin standard curve according to the step (1) to (4) of technical scheme and see Fig. 2, its Methylene Blue (MB); Sulfydryl hexanol (MCH) is all purchased from Aladdin reagent company limited, and trypsin, thrombin, pepsin, lysozyme, alkali phosphatase, bovine serum albumin, peroxide oxidation enzyme, glucoseoxidase and hemoglobin are purchased from Fluka company.
Peptide used and DNA buy from Beijing SBS Genetech biological engineering company limited.
Preferably, the sequence of described peptide is GGRGGC.
Preferably, described DNA1 partial sequence is 5 '-TGAGGTAGTAGGTTGTATAGTT-3 '.
Preferably, described H1 partial sequence is 5 '-AGGGCGGGTGGGTTGTATAGTAGGCAAAGTAACTATACAACCTACTACCTCATGGG T-3 '.
Preferably, described H2 partial sequence is 5 '-TGGGTACTTTGCCTACTATACAATGAGGTAGTAGGTTGTATAGTAGGGTAGGGCGG G-3 '.
Trypsin amount in action mice pancreatic tissue sample has been measured by the method according to the invention, and adopt standard addition method that method has been evaluated, the sample determination response rate is 94.3 103.2%, measurement result is in Table 1, and the method for the present invention has the advantages that precision is high in trypsin detects.
Tryptic measurement result in table 1 mice pancreatic tissue sample.
| Numbering | Contenta,b | Addition | Measured amount | The response rate (%) |
| 1 | 3.2 | 3.00 | 6.03 | 94.3 |
| 2 | 12.7 | 10.00 | 23.02 | 103.2 |
| 3 | 15.9 | 15.00 | 31.18 | 96.1 |
a7 measurement results
bUnit: ngmL-1
Claims (4)
1. measure a tryptic method in exercised rats pancreatic tissue sample, comprise the following steps:
(1) take 0.1��20 �� g peptide solid and be dispersed in the PBS buffer solution containing 5% glutaraldehyde, adding the DNA1 solution that 100 �� L concentration are 0.1��1000mM after continuously stirred 2h again in solution, 4 DEG C of vibrations obtain peptide modifying DNA 1 after hatching 12h, i.e. peptide/DNA1, preserves at 4 DEG C;
(2) first by gold electrode respectively with 1.0 ��m, 0.3 ��m, the polishing of 0.05 ��m of alumina powder, successively with distilled water, ethanol and distilled water ultrasonic 5min respectively, finally at the H of 0.1M2SO4Solution is circulated voltammetric scan until i-v curve is stable; At 1��60 �� L peptide/DNA solution of clean gold electrode surfaces dropping (1) gained, at 25 DEG C, assemble 120min;The gold electrode surfaces deionized water having modified peptide/DNA is cleaned three times, dries up with high pure nitrogen; Then gold electrode is immersed in the sulfydryl hexanol solution of 1mM, at 25 DEG C, close 60min, obtain peptide/DNA modification gold electrode after fully cleaning, save backup at 4 DEG C;
(3) take 1��60 �� L mixed solution containing H1 and the H2 of 0.001��400 ��M and drip the peptide in (3) gained/DNA modification gold electrode surfaces; After at room temperature reaction a period of time, then with the PBST solution washing three times of 50 �� L; Then take 1��60 �� L MB solution containing 0.001��400mM again and drip in electrode surface, after at room temperature reaction 30 minutes, then with the PBST solution washing three times of 50 �� L; Obtain H1, H2 and MB self-assembling peptides/DNA modification gold electrode;
(4) 1��60 �� L trypsin sample solution is added drop-wise to H1, H2 and MB self-assembling peptides/DNA modification gold electrode surfaces, at room temperature reacts 30min; Then, electrode surface is repeatedly rinsed with phosphate buffer solution three times; With above-mentioned the electrode obtained for working electrode, saturated calomel electrode is reference electrode, and platinum electrode is auxiliary electrode, utilizes differential pulse voltammetry DPV speed of sweeping with 1��1000mV/s in phosphate buffer solution to be scanned, and electric potential scanning ranges for-0.4 to-0.1V; According to electrochemical signals, trypsin is carried out quantitatively;
(5) mouse movement mode adopts disposable exhausted swimming, and water temperature controls at 32 �� 1 DEG C, and mice pool specifications is long 100cm �� 50cm �� 50cm, and mice continues to sink ten seconds to surface and exhausts standard for power; Mice broken end is drawn materials, and after adding homogenate medium by 1:10, is made into tissue homogenate, the frozen centrifugation 15min when 3000 revs/min, takes supernatant and measures trypsin by (4) described method;
The partial sequence of wherein said peptide and the partial sequence of DNA are as follows:
The partial sequence of described peptide is GGRGGC;
Described DNA1 partial sequence is 5 '-TGAGGTAGTAGGTTGTATAGTT-3 ';
Described H1 partial sequence is 5 '-AGGGCGGGTGGGTTGTATAGTAGGCAAAGTAACTATACAACCTACTACCTCATGGG T-3 ';
Described H2 partial sequence is 5 '-TGGGTACTTTGCCTACTATACAATGAGGTAGTAGGTTGTATAGTAGGGTAGGGCGG G-3 '.
2. tryptic method in a kind of mensuration exercised rats pancreatic tissue sample according to claim 1, it is characterised in that described phosphate buffer solution compound method is: dissolve 0.2gKH in one liter of water2PO4And 2.9gNa2HPO4Obtain the phosphate buffer of 0.1MpH7.4.
3. tryptic method in a kind of mensuration exercised rats pancreatic tissue sample according to claim 1, it is characterised in that described PBS compound method is: by 0.2gKH2PO4, 8.0gNaCl and 0.2gKCl be dissolved in one liter of water, obtain the phosphate buffered solution that 0.15MpH is 7.4, i.e. PBS.
4. tryptic method in a kind of mensuration exercised rats pancreatic tissue sample according to claim 1, it is characterized in that described PBST compound method is: phosphate buffer solution will add Tween-20, the volume ratio making Tween-20 is 0.05%, obtains PBST solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610099005.3A CN105648033B (en) | 2016-02-23 | 2016-02-23 | A method of trypsase in measurement exercised rats pancreatic tissue sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610099005.3A CN105648033B (en) | 2016-02-23 | 2016-02-23 | A method of trypsase in measurement exercised rats pancreatic tissue sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105648033A true CN105648033A (en) | 2016-06-08 |
| CN105648033B CN105648033B (en) | 2019-06-04 |
Family
ID=56488715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610099005.3A Expired - Fee Related CN105648033B (en) | 2016-02-23 | 2016-02-23 | A method of trypsase in measurement exercised rats pancreatic tissue sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105648033B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113495091A (en) * | 2021-05-28 | 2021-10-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005108612A2 (en) * | 2003-11-28 | 2005-11-17 | Genorx, Inc. | Nanoscale biosensor device, system and technique |
| CN103439296A (en) * | 2013-07-31 | 2013-12-11 | 南昌大学 | Construction and application of aptamer sensor based on Au NPs and DNA circulation double amplification technique |
| CN104328192A (en) * | 2014-11-05 | 2015-02-04 | 南京大学 | Nuclease amplified high-sensitivity electrochemical immunoassay method |
-
2016
- 2016-02-23 CN CN201610099005.3A patent/CN105648033B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005108612A2 (en) * | 2003-11-28 | 2005-11-17 | Genorx, Inc. | Nanoscale biosensor device, system and technique |
| CN103439296A (en) * | 2013-07-31 | 2013-12-11 | 南昌大学 | Construction and application of aptamer sensor based on Au NPs and DNA circulation double amplification technique |
| CN104328192A (en) * | 2014-11-05 | 2015-02-04 | 南京大学 | Nuclease amplified high-sensitivity electrochemical immunoassay method |
Non-Patent Citations (6)
| Title |
|---|
| ALAN K. H. CHENG等: "Aptamer-Based Biosensors for Label-Free Voltammetric Detection of Lysozyme", 《ANAL. CHEM.》 * |
| HELLAS C.M. YAU等: "Electrochemical properties of DNA-intercalating doxorubicin and methylene blue on n-hexadecyl mercaptan-doped 5"-thiol-labeled DNA-modified gold electrodes", 《BIOSENSORS AND BIOELECTRONICS》 * |
| PINAR KARA等: "Electrochemical genosensor for the detection of interaction between methylene blue and DNA", 《ELECTROCHEMISTRY COMMUNICATIONS》 * |
| YUN DAI等: "Adenosine-triggered elimination of methylene blue noncovalently bound to immobilized functional dsDNA-aptamer constructs", 《THE JOURNAL OF PHYSICAL CHEMISTRY B》 * |
| 李凌等主编: "附表A-10、A-11", 《生物化学与分子生物学实验指导》 * |
| 许世超 等: "以亚甲基蓝为杂交指示剂的DNA电化学传感器", 《精细化工》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113495091A (en) * | 2021-05-28 | 2021-10-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
| CN113495091B (en) * | 2021-05-28 | 2024-01-23 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105648033B (en) | 2019-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Electrochemical assay of active prostate-specific antigen (PSA) using ferrocene-functionalized peptide probes | |
| CN101581691B (en) | Preparation method of modified glassy carbon electrode as glucose sensor and application thereof | |
| CN101458223A (en) | Preparation of quantitative rapid detecting sensor of microcapsule algae toxin and applications | |
| Cao et al. | A simple and general approach to assay protease activity with electrochemical technique | |
| Ostatná et al. | Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes | |
| CN103940890B (en) | Preparation method and application of DNA-AuNPs (gold nanoparticles) nanometer network structure | |
| CN101458215A (en) | Electrochemical luminescent adaptorprotein sensor of poly-pyridine ruthenium complex compound method for making same | |
| CN102262115A (en) | Electrochemical immunosensor for melamine content determination, and preparation method and application thereof | |
| Zhang et al. | An electrochemical sensor for detecting triglyceride based on biomimetic polydopamine and gold nanocomposite | |
| CN101655473B (en) | Preparation method of nanogold immunoelectrode | |
| Liu et al. | A sensitive, non-damaging electrochemiluminescent aptasensor via a low potential approach at DNA-modified gold electrodes | |
| CN110220961A (en) | A kind of L-arginine detection method and sensor based on the complex film modified electrode of polypeptide | |
| CN107543850A (en) | Detect biology sensor of trypsase and its preparation method and application | |
| CN110220960A (en) | A kind of detection method and sensor of L-arginine | |
| Wang et al. | Ultrasensitive electrochemical DNA biosensor based on a tetrahedral structure and proximity-dependent surface hybridization | |
| Deng et al. | Label-free electrochemical sensing platform for the detection of protease | |
| CN110441535A (en) | A kind of preparation method of the electrochemical immunosensor based on Pd NCs functionalization CuInOS detection Procalcitonin | |
| Khaloo et al. | Voltammetric and potentiometric study of cysteine at cobalt (II) phthalocyanine modified carbon-paste electrode | |
| Olgaç et al. | Development and characterisation of cysteine-based gold electrodes for the electrochemical biosensing of the SARS-CoV-2 spike antigen | |
| CN105648033A (en) | Method for determining trypsin in pancreatic tissue sample of exercise mouse | |
| Dorčák et al. | Interaction of biomacromolecules with surfaces viewed by electrochemical methods | |
| CN104865299A (en) | Preparation method and application of nanometer gold palladium/3aminopropyl triethoxy silanization manganic oxide PSA (prostate-specific antigen) sensor | |
| CN104020200A (en) | Electrochemical method for measuring melamine | |
| CN210376224U (en) | Sensor for detecting L-arginine | |
| CN109470757B (en) | Electrochemical detection method for prostate specific antigen detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190604 |