CN107741416A - The SERS probes of Multiple Antibodies mark and substrate and its preparation method and application - Google Patents
The SERS probes of Multiple Antibodies mark and substrate and its preparation method and application Download PDFInfo
- Publication number
- CN107741416A CN107741416A CN201710827106.2A CN201710827106A CN107741416A CN 107741416 A CN107741416 A CN 107741416A CN 201710827106 A CN201710827106 A CN 201710827106A CN 107741416 A CN107741416 A CN 107741416A
- Authority
- CN
- China
- Prior art keywords
- sers
- excretion body
- cancer
- multiple antibodies
- excretion
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 90
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 71
- 239000000758 substrate Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 230000029142 excretion Effects 0.000 claims abstract description 85
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 63
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 63
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 33
- 210000002966 serum Anatomy 0.000 claims abstract description 33
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 29
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 20
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000012986 modification Methods 0.000 claims abstract description 14
- 230000004048 modification Effects 0.000 claims abstract description 14
- 229920001690 polydopamine Polymers 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 229960003638 dopamine Drugs 0.000 claims abstract description 7
- 208000035269 cancer or benign tumor Diseases 0.000 claims abstract description 6
- 239000005357 flat glass Substances 0.000 claims description 28
- 239000008367 deionised water Substances 0.000 claims description 19
- 229910021641 deionized water Inorganic materials 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 11
- 230000003595 spectral effect Effects 0.000 claims description 10
- 230000035945 sensitivity Effects 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 238000007689 inspection Methods 0.000 claims description 8
- 239000006193 liquid solution Substances 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 230000005284 excitation Effects 0.000 claims description 7
- 238000000703 high-speed centrifugation Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 6
- 239000002105 nanoparticle Substances 0.000 claims description 5
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 239000011258 core-shell material Substances 0.000 claims description 4
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- PQTCMBYFWMFIGM-UHFFFAOYSA-N gold silver Chemical compound [Ag].[Au] PQTCMBYFWMFIGM-UHFFFAOYSA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 2
- 239000012930 cell culture fluid Substances 0.000 claims description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 abstract description 14
- 238000012546 transfer Methods 0.000 abstract description 9
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 238000007405 data analysis Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000006116 polymerization reaction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 15
- 210000004369 blood Anatomy 0.000 description 11
- 239000008280 blood Substances 0.000 description 11
- 238000011160 research Methods 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 7
- 238000009826 distribution Methods 0.000 description 7
- 238000011528 liquid biopsy Methods 0.000 description 7
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 5
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 229960004502 levodopa Drugs 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 102100034190 Glypican-1 Human genes 0.000 description 4
- 101001070736 Homo sapiens Glypican-1 Proteins 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 210000004907 gland Anatomy 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000011896 sensitive detection Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 description 3
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 2
- 206010016825 Flushing Diseases 0.000 description 2
- 206010027457 Metastases to liver Diseases 0.000 description 2
- 208000016222 Pancreatic disease Diseases 0.000 description 2
- 238000001237 Raman spectrum Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 241000208340 Araliaceae Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010073364 Ductal adenocarcinoma of pancreas Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000010956 Glypican Human genes 0.000 description 1
- 108050001154 Glypican Proteins 0.000 description 1
- 108050007238 Glypican-1 Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 240000002825 Solanum vestissimum Species 0.000 description 1
- 235000018259 Solanum vestissimum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000024691 pancreas disease Diseases 0.000 description 1
- 201000008129 pancreatic ductal adenocarcinoma Diseases 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hospice & Palliative Care (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to technical field of molecular biology, specially the SERS probes of Multiple Antibodies mark and substrate and its preparation method and application.The present invention, which uses to embed under the conditions of auto polymerization using dopamine, combines Multiple Antibodies, obtain poly-dopamine and the SERS substrates and SERS label probes of antibody modification, by the way that combination power is immunized, high-sensitivity detection and the diagnosis of the excretion body of pancreatic cancer cell extraction and the serum sample of clinical Patients with Pancreatic Cancer are realized using Raman spectroscopy, statistical data analysis finds that the SERS platforms based on MIF antibody can realize that the neoplasm staging of cancer of pancreas judges.SERS probes label methods in the present invention are simple and convenient, and high for the detection of cancer of pancreas excretion body, rapid sensitive, specificity, required sample size is few, without ultracentrifugal separation process, can be suitably used for clinical diagnosis and neoplasm staging transfer judges.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to Multiple Antibodies mark SERS probes and substrate and its
Preparation method and application.
Background technology
Cancer of pancreas is one of worst malignant tumor of digestive tract of prognosis, and national tumor center registered to national tumour in 2015
The malignant tumour registration materials in 2012 that national each registration office of central collection reports carry out the hair of analysis estimation, wherein cancer of pancreas
Case load is 8.6 ten thousand, the incidence of disease 6.35/105, the 10th is arranged in all generation rate of malignant tumour, its dead number of cases is up to 7.7
Ten thousand, the death rate 5.72/105, occupy the 6th.Cancer of pancreas lacks special clinical manifestation and sensitive early diagnosis index, its
The blood serum designated object that diagnosis and follow-up are commonly used is CA19-9, but specificity is poor.Diagnosis before pancreatic cancer surgery at present and by stages
Depending on iconography includes Spiral CT Enhanced Scanning and magnetic resonance imaging(MRI)Performance, carry out primary tumo(u)r size
(T), N- regional lymph nodes(N), M- DISTANT METASTASES INs (M) by stages(TNM)Judge.The CT scan time is short, and image taking speed is fast, price
Cheaply, but amount of radiation is big, it is impossible to the inspection method as routine.MRI soft tissue resolutions are high, can show details, no spoke
Penetrate, but the review time is grown, the limitation of scanning range is difficult to assess DISTANT METASTASES IN, but final make a definite diagnosis still needs to pathology inspection
Look into.So current urgent need develops some quick, sensitive, specific height, hurtless measure, the cancer of pancreas of affordable and early diagnoses and divide
The new method and new technology that phase judges.
With being deepened continuously to tumor research, researcher has found a small amount of circulating tumor cell be present in tumor patient blood
(CTC)And a small amount of Circulating tumor DNA (ctDNA) discharged by downright bad cancer cell.By detecting CTC and ctDNA in blood
The method for being diagnosed and being monitored to patient tumors is referred to as liquid biopsy(liquid biopsy).Relative to traditional operation
Biopsy and needle biopsy method, liquid biopsy can solve the problem that the difficult point of clinical sampling, meet the needs of to patient's high-frequency detection, and
With compared to aspiration biopsy Small side effects, it is simple to operate, can repeated sampling, cost is low the advantages of.Therefore liquid biopsy is expected to
It will be played in terms of the dynamic monitoring of the early screening, tumor patient of tumour and personalized medicine etc. precisely medical treatment more and more important
Effect.
Excretion body (exosomes) also begins to receive more and more attention as a kind of important vesica in human body.Due to
Excretion body contains Tumor-specific protein matter and miRNA derived from tumour, the important angle of performer in the growth, transfer in tumour
Color, potential diagnosing tumor mark can be used as., can such as the excretion body extracted in blood plasma, serum and urine by from body fluid
Include the related information of tumour, it is easier to realize the early detection to tumour.Researcher separates from Patients with Pancreatic Cancer serum
To excretion body in be found that a kind of glypican -1(GPC1)Glycoprotein, but in other benign pancreatic diseases and just
In the excretion body separated in the blood of ordinary person, GPC1 content is very low.This detection detects sugar antigens than traditional ELISA
CA19-9 is more reliable.The diagnosis of pancreatic cancer method of this Noninvasive established based on excretion body mark, can be very smart
Really early carcinomatous change is diagnosed to be from benign pancreatic disease.In addition, in cancer of pancreas excretion body micro RNA-10b expression
Rise.Also studies have found that excretion body caused by ductal adenocarcinoma of pancreas cell has facilitation to hepatic metastases, and inhibition of metastasis because
Son(MIF)Expression significantly rise.Circulating the MIF in excretion body can be as the biology mark that cancer of pancreas Index for diagnosis and hepatic metastases are predicted
Will thing.Therefore, excretion body is an emerging field of tumor research, and after circulating tumor cell CTC and ctDNA it is another standby
The tumor markers and the target spot for the treatment of and prognosis of concerned liquid biopsy.
Mainly there is nano-particle trace analysis to the quantitatively characterizing method of excretion body at present(NTA), electron microscopic observation pattern, stream
Formula cell counting, ELISA method or Western Blot methods based on excretion body surface protein, sample size needed for these methods
Greatly, sensitivity and specificity are not high, and sample process complexity is time-consuming, and processing flux is small, can not clinically implement, and instrument is held high
It is expensive, be not suitable in clinical expansion.Surface increases raman scattering spectrum(SERS)It is new characterizing method developed in recent years
With signal indicia meanses, strong with signal, signal stabilization does not have quenching phenomenon, is easy to mark, and spectral signal has " fingerprint " special
The advantages that property.The labelling technique of SERS probes just plays in biomedical sectors such as bio-imaging, medical diagnosis on disease, medicament transports
More and more important effect.Migration inhibition factor (MIF) detection is closed using SERS probe labelled immune methods and excretion body phase at present
Cancer of pancreas excretion body simultaneously realizes that the research judged by stages yet there are no document report.
Our research finds, difunctional DOPA is amine-modified and the sheet glass of protection and SERS probes being capable of highly sensitive Gao Te
Single excretion body is detected different in naturely.Therefore, the present invention devises Multiple Antibodies(anti-MIF, anti-GPC1,anti-
EGFR, anti-EpCAM)The SERS probes of mark, with reference to the amine-modified sheet glass of DOPA, realize outer caused by cell line
Secrete the highly sensitive detection of body.SERS immunization methods based on anti-MIF have highest sensitivity, and the system is used for clinical pancreas
The diagnosis of gland cancer serum sample, statistics result being capable of the significantly blood samples of differentiating pancreatic cancer patient and Healthy People, and can be right
The different TNM of cancer of pancreas tumor stage is classified(P1,2 and P3, whether shift), the result goodness of fit with pathological section
It is high.This method only needs 2uL serum samples, and without carrying out high speed centrifugation, the detection time of single sample only spends 15 minutes.This
Outside, the invention is imaged while realizing high flux sample herein in connection with SERS imaging techniques, is easy to clinician quick, accurate
Really carry out the clinical diagnosis of cancer of pancreas.
The content of the invention
It is an object of the invention to propose a kind of Multiple Antibodies of the highly sensitive detection for cancer of pancreas excretion body(anti-
MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63)The SERS probes and substrate of mark
And preparation method thereof, and the application of probe and substrate in the highly sensitive detection of cancer of pancreas excretion body.
The present invention is used for anti-MIF SERS immunization methods the diagnosis of clinical pancreatopathy cancer serum sample, has highest
Sensitivity and specificity, statistics result being capable of the significantly blood samples of differentiating pancreatic cancer patient and Healthy People, and can be to pancreas
The different TNM of gland cancer tumor stage is classified(P1,2 and P3, whether shift), it is high with the result goodness of fit of pathological section.
The SERS probes of Multiple Antibodies mark proposed by the present invention and the preparation method of substrate, are comprised the following steps that:
(1)In gold-silver nanoparticle solution of 2 ~ 20mM core shell structures, anti-MIF, anti-GPC1, anti-are added
Six kinds of antibody of EGFR, anti-EpCAM, anti-CD9, anti-CD63, the concentration of every kind of antibody is 5 ~ 40 μ g/mL, Mei Zhongkang
The addition of body is 30 ~ 150 μ L;And add the μ L of dopamine solution 40 ~ 150 that concentration is 1 ~ 20mg/mL;0.5 is reacted at room temperature
~ 4 hours;Reacted solution is by centrifugation, then is disperseed with deionized water;Obtain the SERS probes of Multiple Antibodies mark;
(2)Take common glass slide to steep 4 ~ 7h in 8 ~ 14M NaOH, then toward concentration is added in solution be 10 ~ 100mg/
Ml 6 ~ 10ml of dopamine solution, above-mentioned culture dish is placed on special shaking table and shakes 0.5 ~ 2h, is done afterwards with deionized water rinsing
Only;
Slide after modification and cleaning is placed in containing six kinds of antibody anti-MIF, anti-GPC1, anti-EGFR, anti-
EpCAM, anti-CD9, anti-CD63 volumetric concentration are 0.2 ~ 2% BSA(Bovine serum albumin(BSA))In, wherein, every kind of antibody
Concentration be 1 ~ 10mg/mL, react 0.5 ~ 3h;After deionized water rinsing 1 ~ 3 time, naturally dry, substrate is obtained.
The SERS probes and substrate of Multiple Antibodies mark prepared by the present invention, the highly sensitive inspection available for cancer of pancreas excretion body
Survey, comprise the following steps that.
(1)Cell culture and the extraction of excretion body
Pancreatic carcinoma PANC-01 is cultivated in the culture mediums of RPMI 1640, culture medium adds 2 ~ 20% tires before the use
Cow's serum, 50 ~ 150 μ g/mL parasiticins and 50 ~ 150 μ g/mL streptomysins, cell are placed in containing 2 ~ 10% CO2In incubator 25 ~
Cultivated under the conditions of 45 DEG C to sufficient amount.
Excretion body is extracted using high speed centrifugation method.First, cell culture fluid is placed in centrifuge in 500 ~ 2000 g(This
In g refer to centrifugal force, represented sometimes with rotating speed, but be related to high speed centrifugation and all represented with centrifugal force g, similarly hereinafter)Centrifugation 2
~ 10min, then 1000 ~ 3000g centrifuge 2 ~ 10min, remove cell fragment, supernatant liquor are taken through 0.22 μm of membrane filtration, by filtrate
It is placed in ultracentrifuge and centrifuges 1 ~ 5 h in 4 DEG C, 120000 ~ 200000 g, takes lower sediment to add PBS(Phosphate-buffered is molten
Liquid)Dissolving, then be placed in ultracentrifuge and centrifuge 1 ~ 5 h in 4 DEG C, 120000 ~ 200000 g, supernatant is abandoned, precipitation is to be enriched with
Excretion body, add PBS dissolving, be placed in -20 DEG C of refrigerators and preserve.
The sign of excretion body is divided the particle diameter distribution of excretion body using the Nanosight NS300 of Malvern company
Analysis, takes 2 ~ 20 μ L excretion bodies, dilutes 10 ~ 200 times with PBS.Nanosight NS300 camera lenses select sCMOS, laser selection
Blue488, at 22.4 ~ 22.6 DEG C, each sample measures 3 ~ 6 times experimental temperature.Using Flied emission transmission electron microscope(TEM)
Observe excretion bodily form looks.5 ~ 20 μ L excretions bodies are taken on 400 mesh carbon film figure layer grids, with filter paper by surplus liquid from Grid Edge
Edge is wiped, and is dried at room temperature for.The Salkowski's solutions of 2 ~ 20 μ L 3% are added, the min of room temperature negative staining 1.5, dye liquor are blotted with filter paper
2 ~ 5min of heating, drying under 65 DEG C of incandescent lamps afterwards.
(2)The SERS probe in detecting cancer of pancreas excretion bodies marked with Multiple Antibodies
The sheet glass that first poly-dopamine is modified(That is substrate)20 ~ 80 points are soaked at 25 ~ 40 DEG C with 0.01 ~ 0.1% BSA solution
Clock, then rinsed 3 ~ 5 times with PBS;Excretion liquid solution is diluted to various concentrations using PBS:5.44Χ102 --2.72Χ1010
Individual excretion body/ml;2 ~ 10ul excretion liquid solutions are taken to drip on the sheet glass for the poly-dopamine modification for being coated with specific antibodies,
React at 25 ~ 40 DEG C 20 ~ 80 minutes, then rinsed 3 ~ 5 times with PBS;1 ~ 10ul is taken to be marked with the SERS probes of specific antibodies again
Corresponding to dripping on sheet glass, reacted 30 ~ 90 minutes at 25 ~ 40 DEG C, then use deionized water rinsing;The glass prepared
Piece is in Raman spectrometer(Such as HoribaJobinYvon companies, Germany)Upper carry out spectral scan, a length of 785nm of excitation light wave.
(3)Interpretation of result.The SERS probes of Multiple Antibodies mark obtained above, there is the Raman signal of surface enhanced,
In 1073.51cm-1There is most strong and narrow Raman signal molecule at placepATP spectral signal peak.
In order to which data show that conveniently, the present invention uses 1073.51cm in SERS spectra figure-1The logarithm value of the raman scattering intensity at place
Analyzed.To various concentrations (5.44 Χ 102 to 2.72Χ1010 Individual excretion body/ml) excretion body use Multiple Antibodies mark
The SERS platforms of note draw standard working curve.As a result show that anti-MIF has maximum sensitivity, anti-GPC1's is sensitive
Spend minimum, anti-MIF, anti-GPC1, anti-EGFR, the test limit of these four antibody platforms of anti-EpCAM(LOD)
9X10 is reached-19mol/L。2uL 5.44Χ1021 excretion body is comprised only in the sample of individual/ml concentration, shows the present invention's
SERS systems can realize the detection of single excretion body.And the MIF ELISA kits of business minimum are only able to detect 2.72
Χ108The excretion body sample of individual/mL concentration, and sample aequum reaches 100uL.Therefore, marked with the Multiple Antibodies of the present invention
SERS probe immune detection cancer of pancreas excretion bodies, than the detection cancer of pancreas excretion body of commercial ELISA Kit, sensitivity improves
6 orders of magnitude.
The SERS probes and substrate of Multiple Antibodies mark provided by the invention can be used for the serum sample one's duty of Patients with Pancreatic Cancer
Analysis, and the neoplasm staging of cancer of pancreas and transfer judge.
(1)Serum sample exempts from comprising the following steps that for centrifugation multispecific antibody detection:
Serum sample dilutes 2 ~ 5 times before use using PBS, then with 0.22um membrane filtration, finally takes 1 ~ 10uL to dilute
Sample afterwards drip to poly-dopamine, antibody modification sheet glass on.Sheet glass reacts 20 ~ 80 minutes at 25 ~ 40 DEG C, then
Rinsed 3 ~ 5 times with PBS;1 ~ 10ul is taken to be marked with the SERS probes of anti-MIF, anti-GPC1, anti-EGFR antibody respectively again
Corresponding to dripping on sheet glass, reacted 30 ~ 90 minutes at 25 ~ 40 DEG C, then use deionized water rinsing;The glass prepared
Piece carries out spectral scan, a length of 785nm of excitation light wave on Raman spectrometer.
(2)From Shanghai, hospital have collected from December, 2012 to 22 Patients with Pancreatic Cancer and 20 during in August, 2016
The serum sample of example Healthy People.The average age of Patients with Pancreatic Cancer is 60.45 ± 9.81 years old, 9 women and 13 males, health
The average age of people's group is 57.75 ± 8.16 years old, wherein 7 women and 13 males.The research is in hospital's medical research
Carried out under the approval of Ethics Committee, all patients have signed informed consent form before sample is collected.Its serum sample is entered
Row exempts to centrifuge multispecific antibody detection.Analysis of statistical data is as follows:1073.51cm is used in resulting SERS spectra figure-1Locate signal
Logarithm value analyzed.Data use Graph Prism 6.0, statistics analysis software SPSS 21.0 and mapping software
Origin7.5 is mapped and analysis of statistical data.
Statistical analysis is used for the testing result of Patients with Pancreatic Cancer and the serum sample of Healthy People.Shapiro-Wilk is examined
Test display Patients with Pancreatic Cancer groupW experiment =0.901, P=0.032<0.10, and Healthy People control groupW control =0.913, P
=0.072<0.10, this illustrates that normal distribution is not presented in this two groups of data.So we use non-ginseng to this two groups of independent samples
Number Mann-Whitney is examined,Z=-5.541, P<0.0001, it is notable on statistical significance to illustrate that this two groups of samples have
Sex differernce.The average value of Patients with Pancreatic Cancer group and Healthy People control group is respectively 3.79 ± 0.11 and 2.08 ± 0.218.More than
As a result Patients with Pancreatic Cancer and health can be distinguished using the method for the SERS probes mark based on antibody MIF in the explanation present invention
People, and term of reference is provided for clinical diagnosis, it is expected to use the clinical liquid biopsy diagnosis field of reality.
Further, the present invention carries out TNM stage according to the logarithm value of SERS signal intensity to 17 Patients with Pancreatic Cancer samples
(Cancer of pancreas sample not comprising 5 without TNM stage), it is divided into two groups of P1-2 and P3.Reported according to histopathology, these diseases
People's sample has been separated into normal, infiltration and not shifted(Infiltrate by pancreas tissue as adipose tissue, nerve, Duodenal Mucosa,
Bile duct), transfer(It is transferred to gland on liver,spleen,kidney, lymph node).SERS data based on anti-MIF pass through Mann-Whitney
Inspection statistics result finds there is statistical significant difference between normal group, P1-2, transfer and the non-transfer group of infiltration(P<
0.05), and coincide with histopathology report.We also surprisingly it has been found that, anti-MIF SERS data also simultaneously can general
P3 neoplasm stagings differentiate with the P1-2 stages, it means that the present invention can be tumour further by stages and realize accurately swell
Knurl diagnosis provides more reference values.In addition, the present invention also uses serum of the business employment MIF ELISA kits to patient
Sample is analyzed.Test result finds have 9 patients to show positive findings in Patients with Pancreatic Cancer blood sample, and normal
Also 8 Healthy Peoples show positive findings in people.It these results suggest that, anit-MIF SERS platforms of the invention can provide
More more accurately diagnosis and neoplasm staging information, and required sample size is few, it is only necessary to 2uL, and ELISA needs 100uL samples.
In addition, the present invention also uses the SERS systems of anti-GPC1 and anti-EGFR modifications to Patients with Pancreatic Cancer and is good for
Health human serum sample carries out test and statistics interpretation of result.As a result the SERS system Ye Neng areas based on both antibody are found
The phase of other Healthy People and cancer of pancreas, Healthy People and PT1 ~ 2, Healthy People and cancer of pancreas infiltration are not shifted, but can not distinguish cancer of pancreas
PT1 ~ 2 and PT3 phases, transfer can not be distinguished and infiltrate non-diverting blood sample.
Compared with existing report, excretion body phase of the invention closes the SERS probes mark of migration inhibition factor (MIF) mark
Method is simple and convenient, and for the detection of cancer of pancreas excretion body, rapid sensitive, specificity is high, and required sample size is few, without high speed from
The separation process of the heart, realizes the detection of clinical Patients with Pancreatic Cancer and Healthy Human Serum sample, and can be used in cancer of pancreas
Shift and judge by stages.The invention is also applied for the development and application of other Multiple Antibodies SERS platforms.
(1)SERS probe synthesis is easy:Sheet glass is modified and the method for SERS labelled antibodies uses gentle embedding
Method is carried out, it is not necessary to complicated instrument, it is not necessary to which the harsh conditions such as high-temperature strong acid highly basic, reaction condition as mild as a dove, and need not
Complicated special chemical reaction reagent.Sheet glass modification is uniform, and SERS mark stability is high, can place the several months and not gather
Collection.
(2)Rapidly and efficiently:Reaction 1 hour is only needed after adding cell line or clinical serum sample, it is effective with regard to that can reach
Capture.Serum sample only needs the membrane filtration with 0.22um, the high speed centrifugation process without complicated and time consumption.SERS signal is collected
Quickly.
(3)High sensitivity:The minimal detectable concentration of excretion body is 9X10-19Only contain 1 excretion in mol/L, 2uL sample
Body, it is possible to realize the detection of single excretion body.
(4)It is specific high:The system is used for the detection of clinical complex samples serum, merely through the mistake of 0.22um filter membranes
Filter, other compositions do not interfere with to detection.Detection for clinical blood sample can significantly differentiating pancreatic cancer patient and Healthy People
Blood sample, it is possible to the early diagnosis for clinical cancer of pancreas.
(5)Available for auxiliary diagnosis:The SERS sonde methods of MIF antibody can be used for the different phase of differentiating pancreatic cancer patient,
Doctor can be aided in be judged by stages.Virus monitory does not have wound to patient, is a kind of noninvasive liquid biopsy method, institute
With the monitoring after operation and curative effect evaluation of the tumour especially suitable for high frequency time.
(6)Expansibility is strong:Except anti-MIF, the SERS methods of other Multiple Antibodies mark can be used for cancer of pancreas
Diagnosis and by stages, but the adhesion due to antibody and specific difference, detection performance can be varied from.But we can select
The stronger antibody of sensitivity more high specific is selected, establishes the SERS labeling methods suitable for other diseases, can also Multiple Antibodies
It is used in combination.
Brief description of the drawings
Fig. 1 is SERS marks, the diagram of excretion body.Wherein, the excretion of (A) pancreatic cancer cell PANC-01 cell lines extraction
The TEM photos of body, (B) are the NTA grain size distributions of the excretion body of pancreatic cancer cell PANC-01 extractions.
Fig. 2 is that the SERS of anti-CD9/CD63/MIF/GPC1 structures marks system to be used to detect PANC-01 cell lines
The Raman spectrogram of the excretion body of extraction.
Fig. 3 is the Raman spectrogram of SERS marks.Wherein, (A) is that anti-MIF is built under different excretion bulk concentrations
The Raman spectrogram of SERS marks,(B)For in 1073.51 cm-1The Raman signal intensity at place is bent with the change of excretion bulk concentration
Line,(C)The linear criterion working curve of raman scattering intensity and excretion bulk concentration.
Fig. 4 is Raman signal intensity distribution histogram and the inspection statistics data analysis of the serum sample group of SERS marks
Figure.Wherein,(A)Raman signal intensity for the Patients with Pancreatic Cancer serum sample group of the SERS marks based on anti-MIF is distributed post
Shape figure,(B)For based on anti-MIF SERS mark Healthy Human Serum sample group Raman signal intensity distribution histogram,
(C)For the Shapiro-Wilk inspection statistics data analysis figures of serum sample group.
The Shapiro-Wilk statistical analysis figures judged by stages with transfer of Fig. 5 Patients with Pancreatic Cancer serum samples.Wherein,
(A)The judgement by stages of cancer of pancreas, PT2-1 and PT3 is distinguished,(B)The infiltration or transfer of cancer of pancreas judge.
Embodiment
Below by specific embodiment, the present invention is further described.
Embodiment 1:
(1)The SERS probes of Multiple Antibodies mark and the preparation of substrate
The μ g/mL of 50 μ L 10 anti-MIF, anti-are separately added into gold-silver nanoparticle solution of 5mM core shell structure
Six kinds of antibody of GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63, the 10mg/mL DOPA with 60 μ L
Amine aqueous solution, react 1 hour at room temperature.Reacted solution is by centrifugation, then is disperseed with deionized water.In addition, take common
Glass slide steep the 5h in 12M NaOH, then toward the dopamine solution 8ml that 20mg/ml is added in solution, by above-mentioned culture
Ware is placed on special shaking table and shakes 1h, is rinsed well afterwards with deionized water.By the slide after modification and cleaning be placed in containing
5mg/mLanti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 1% concentration
2h is reacted in BSA, after deionized water rinsing 3 times, naturally dry.
(2)Cell culture and the extraction of excretion body
Pancreatic carcinoma PANC-01 is cultivated in the culture mediums of RPMI 1640, and culture medium adds 10% tire ox blood before the use
Clearly, 60 μ g/mL parasiticins and 80 μ g/mL streptomysins, cell are placed in containing 6% CO2Cultivated in incubator under the conditions of 35 DEG C to foot
Enough amounts.
Excretion body is extracted using high speed centrifugation method.First, nutrient solution is placed in into centrifuge to centrifuge in 1200 g
5min, then 2000g centrifugations 6min remove cell fragment, take supernatant liquor through 0.22 μm of membrane filtration, by filtrate be placed in hypervelocity from
In scheming 4 DEG C, 140000 g centrifuge 2 h, take lower sediment add PBS dissolving, then be placed in ultracentrifuge 4 DEG C,
140000 g centrifuge 2 h, abandon supernatant, and precipitation is the excretion body of enrichment, adds PBS dissolvings, is placed in -20 DEG C of refrigerators and protects
Deposit.
(3)The sign of excretion body
The particle diameter distribution of excretion body is analyzed using the Nanosight NS300 of Malvern company, takes 5 μ L excretion bodies, is used
PBS dilutes 20 times.Nanosight NS300 camera lenses select sCMOS, and laser selection Blue488, experimental temperature is 22.4 ~ 22.6
DEG C, each sample measures 4 times.Using Flied emission transmission electron microscope(TEM)Observe excretion bodily form looks.Take 10 μ L excretions bodies in
On 400 mesh carbon film figure layer grids, surplus liquid is wiped from grid edge with filter paper, is dried at room temperature for.Add 10 μ L 3%
Salkowski's solution, the min of room temperature negative staining 2, after blotting dye liquor with filter paper under 65 DEG C of incandescent lamps heating, drying 4min.
(4)SERS immune detection cancer of pancreas excretion bodies based on Multiple Antibodies mark
First by the sheet glass of poly-dopamine modification with being soaked at 30 DEG C of 0.05% BSA solution 50 minutes, then with PBS flushings 4 times.
Excretion liquid solution is diluted to different concentration using PBS, takes 3ul excretion liquid solutions to drip to the poly- DOPA for being coated with specific antibodies
On amine-modified sheet glass, react at 36 DEG C 30 minutes, then rinsed 3 times with PBS.3ul is taken to be marked with specific antibodies again
SERS probes are dripped on corresponding sheet glass, are reacted 60 minutes at 35 DEG C, are then used deionized water rinsing afterwards.The glass prepared
Glass piece is in Raman spectrum(HoribaJobinYvon companies, Germany)Upper carry out spectral scan, a length of 785nm of excitation light wave.
(5)Serum sample exempts to centrifuge multispecific antibody detection
It is have collected from Shanghai Changhai Hospital from December, 2012 to 10 Patients with Pancreatic Cancer and 11 health during in August, 2016
The serum sample of people.The average age of Patients with Pancreatic Cancer is 60.45 ± 9.81 years old, 4 women and 6 males, Healthy People group
Average age is 57.75 ± 8.16 years old, wherein 4 women and 7 males.The research is in medical research ethics committee of Changhai hospital
Carried out under the approval of member's meeting, all patients have signed informed consent form before sample is collected.Serum sample uses before use
PBS dilutes 3 times, then with 0.22um membrane filtration, finally takes the sample after 2uL dilutions to drip to poly-dopamine, antibody modification
Sheet glass on.Sheet glass reacts 50 minutes at 30 DEG C, is then rinsed 3 times with PBS;2ul is taken to be marked with anti-respectively again
The SERS probes of MIF, anti-GPC1, anti-EGFR antibody are dripped on corresponding sheet glass, are reacted 50 minutes at 30 DEG C,
Then deionized water rinsing is used;The sheet glass prepared carries out spectral scan on Raman spectrometer, and excitation light wave is a length of
785nm。
(6)1073.51cm is used in SERS spectra figure obtained by analysis of statistical data-1The logarithm value for locating signal is carried out
Analysis.Data use Graph Prism 6.0, statistics analysis software SPSS 21.0 and mapping software Origin7.5 to carry out
Mapping and analysis of statistical data.
Embodiment 2:
(1)The SERS probes of Multiple Antibodies mark and the preparation of substrate
The μ g/mL of 80 μ L 20 anti-MIF, anti-is added in gold-silver nanoparticle solution of 8mM core shell structure
The 10mg/mL dopamines of the antibody such as GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 and 60 μ L are molten
Liquid, react 1 hour at room temperature.Reacted solution is by centrifugation, then is disperseed with deionized water.In addition, take common glass
Glass slide steeps the 5h in 12M NaOH, then the dopamine solution 8ml toward addition 20mg/ml in solution, and above-mentioned culture dish is put
1h is shaken on special shaking table, is rinsed well afterwards with deionized water.Slide after modification and cleaning is placed in containing 10mg/
In mLanti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 1% concentration BSA
React 2h, after deionized water rinsing 3 times, naturally dry.
(2)Cell culture and the extraction of excretion body
Pancreatic carcinoma PANC-01 is cultivated in the culture mediums of RPMI 1640, and culture medium adds 10% tire ox blood before the use
Clearly, 60 μ g/mL parasiticins and 80 μ g/mL streptomysins, cell are placed in containing 6% CO2Cultivated in incubator under the conditions of 35 DEG C to foot
Enough amounts.
Excretion body is extracted using high speed centrifugation method.First, nutrient solution is placed in into centrifuge to centrifuge in 1200 g
5min, then 2000g centrifugations 6min remove cell fragment, take supernatant liquor through 0.22 μm of membrane filtration, by filtrate be placed in hypervelocity from
In scheming 4 DEG C, 140000 g centrifuge 2 h, take lower sediment add PBS dissolving, then be placed in ultracentrifuge 4 DEG C,
140000 g centrifuge 2 h, abandon supernatant, and precipitation is the excretion body of enrichment, adds PBS dissolvings, is placed in -20 DEG C of refrigerators and protects
Deposit.
(3)The sign of excretion body is carried out using the Nanosight NS300 of Malvern company to the particle diameter distribution of excretion body
Analysis, takes 5 μ L excretion bodies, dilutes 20 times with PBS.Nanosight NS300 camera lenses select sCMOS, laser selection Blue488,
At 22.4 ~ 22.6 DEG C, each sample measures 4 times experimental temperature.Using Flied emission transmission electron microscope(TEM)Observe excretion body
Pattern.10 μ L excretions bodies are taken to be wiped surplus liquid from grid edge with filter paper, in room on 400 mesh carbon film figure layer grids
The lower drying of temperature.Add the Salkowski's solutions of 10 μ L 3%, the min of room temperature negative staining 2, after blotting dye liquor with filter paper under 65 DEG C of incandescent lamps
Heating, drying 4min.
(4)SERS immune detection cancer of pancreas excretion bodies based on Multiple Antibodies mark
First by the sheet glass of poly-dopamine modification with being soaked at 30 DEG C of 0.05% BSA solution 50 minutes, then with PBS flushings 4 times.
Excretion liquid solution is diluted to different concentration using PBS, takes 3ul excretion liquid solutions to drip to the poly- DOPA for being coated with specific antibodies
On amine-modified sheet glass, react at 36 DEG C 30 minutes, then rinsed 3 times with PBS.3ul is taken to be marked with specific antibodies again
SERS probes are dripped on corresponding sheet glass, are reacted 60 minutes at 35 DEG C, are then used deionized water rinsing afterwards.The glass prepared
Glass piece is in Raman spectrum(HoribaJobinYvon companies, Germany)Upper carry out spectral scan, a length of 785nm of excitation light wave.
(5)Serum sample exempts to centrifuge multispecific antibody detection
It is have collected from Shanghai Changhai Hospital from December, 2012 to 12 Patients with Pancreatic Cancer and 9 health during in August, 2016
The serum sample of people.The average age of Patients with Pancreatic Cancer is 60.45 ± 9.81 years old, 5 women and 7 males, Healthy People group
Average age is 57.75 ± 8.16 years old, wherein 3 women and 6 males.The research is in medical research ethics committee of Changhai hospital
Carried out under the approval of member's meeting, all patients have signed informed consent form before sample is collected.Serum sample uses before use
PBS dilutes 5 times, then with 0.22um membrane filtration, finally takes the sample after 6uL dilutions to drip to poly-dopamine, antibody modification
Sheet glass on.Sheet glass reacts 70 minutes at 37 DEG C, is then rinsed 5 times with PBS;5ul is taken to be marked with anti-respectively again
The SERS probes of MIF, anti-GPC1, anti-EGFR antibody are dripped on corresponding sheet glass, are reacted 70 minutes at 37 DEG C,
Then deionized water rinsing is used;The sheet glass prepared carries out spectral scan on Raman spectrometer, and excitation light wave is a length of
785nm。
(6)Analysis of statistical data, resulting SERS data use Graph Prism 6.0, statistics analysis software SPSS
21.0 and mapping software Origin7.5 is mapped and analysis of statistical data.
。
Claims (5)
1. the SERS probes of one kind of multiple antibody labelings and the preparation method of substrate, it is characterised in that concretely comprise the following steps:
(1)In gold-silver nanoparticle solution of 2 ~ 20mM core shell structures, anti-MIF, anti-GPC1, anti-are added
Six kinds of antibody of EGFR, anti-EpCAM, anti-CD9, anti-CD63, the concentration of every kind of antibody is 5 ~ 40 μ g/mL, Mei Zhongkang
The addition of body is 30 ~ 150 μ L;And add the μ L of dopamine solution 40 ~ 150 that concentration is 1 ~ 20mg/mL;0.5 is reacted at room temperature
~ 4 hours;Reacted solution is by centrifugation, then is disperseed with deionized water;Obtain the SERS probes of Multiple Antibodies mark:
(2)Take common glass slide to steep 4 ~ 7h in 8 ~ 14M NaOH, then toward concentration is added in solution be 10 ~ 100mg/
Ml 6 ~ 10ml of dopamine solution, above-mentioned culture dish is placed on special shaking table and shakes 0.5 ~ 2h, is done afterwards with deionized water rinsing
Only;
Slide after modification and cleaning is placed in containing six kinds of antibody anti-MIF, anti-GPC1, anti-EGFR, anti-
EpCAM, anti-CD9, anti-CD63 volumetric concentration is in 0.2 ~ 2% BSA, wherein, the concentration of every kind of antibody for 1 ~
10mg/mL, react 0.5 ~ 3h;After deionized water rinsing 1 ~ 3 time, naturally dry, substrate is obtained.
2. the SERS probes and substrate of the Multiple Antibodies mark that the preparation method as described in claim 1 is prepared.
3. the SERS probes of Multiple Antibodies as claimed in claim 2 mark and substrate are in the highly sensitive inspection of cancer of pancreas excretion body
Application in survey, is comprised the following steps that:
(1)Cell culture and the extraction of excretion body
Pancreatic carcinoma PANC-01 is cultivated in the culture mediums of RPMI 1640, culture medium adds 2 ~ 20% tires before the use
Cow's serum, 50 ~ 150g/mL parasiticins and 50 ~ 150g/mL streptomysins, cell are placed in containing 2 ~ 10% CO225 ~ 45 in incubator
Cultivated under the conditions of DEG C to sufficient amount;
Excretion body is extracted using high speed centrifugation method:First, by cell culture fluid be placed in centrifuge in 500 ~ 2000 g centrifuge 2 ~
10min, then 1000 ~ 3000g centrifuge 2 ~ 10min, remove cell fragment, take supernatant liquor to put filtrate through 0.22m membrane filtrations
In ultracentrifuge 4 DEG C, 120000 ~ 200000g centrifuge 1 ~ 5h, take lower sediment add PBS dissolving, then be placed in hypervelocity from
Scheming centrifuges 1 ~ 5h in 4 DEG C, 120000 ~ 200000 g, abandons supernatant, and precipitation is the excretion body of enrichment, adds PBS dissolvings,
It is placed in -20 DEG C of refrigerators and preserves;
(2)The SERS probe in detecting cancer of pancreas excretion bodies marked with Multiple Antibodies
The sheet glass that first poly-dopamine is modified is that substrate soaks 20 ~ 80 points with 0.01 ~ 0.1% BSA solution at 25 ~ 40 DEG C
Clock, then rinsed 3 ~ 5 times with PBS;Excretion liquid solution use PBS be diluted to concentration for:5.44Χ102~2.72Χ1010 It is individual
Excretion body/ml;2 ~ 10ul excretion liquid solutions are taken to drip on the sheet glass for the poly-dopamine modification for being coated with specific antibodies, 25 ~
React at 40 DEG C 20 ~ 80 minutes, then rinsed 3 ~ 5 times with PBS;Take 1 ~ 10ul to be marked with the SERS probes of specific antibodies again to drip to
On corresponding sheet glass, reacted 30 ~ 90 minutes at 25 ~ 40 DEG C, then use deionized water rinsing;The sheet glass prepared exists
Spectral scan, a length of 785nm of excitation light wave are carried out on Raman spectrometer;
(3)Interpretation of result:The SERS probes of Multiple Antibodies mark obtained above, there is the Raman signal of surface enhanced,
1073.51cm-1There is most strong and narrow Raman signal molecule at placepATP spectral signal peak.
4. the SERS probes and substrate of Multiple Antibodies mark according to claim 3 are in the highly sensitive of cancer of pancreas excretion body
Application in detection, it is characterised in that standard is drawn using the SERS platforms of Multiple Antibodies mark to the excretion body of various concentrations
Working curve;As a result showing that anti-MIF has maximum sensitivity, anti-GPC1 sensitivity is minimum, anti-MIF,
The test limit of these four antibody platforms of anti-GPC1, anti-EGFR, anti-EpCAM has reached 9X10-19mol/L;2uL
5.44Χ1021 excretion body is comprised only in the sample of individual/ml concentration, shows that SERS systems can realize the inspection of single excretion body
Survey.
5. the SERS probes and substrate of Multiple Antibodies mark as claimed in claim 2 are well-behaved in the serum sample of Patients with Pancreatic Cancer
Application in analysis, and the neoplasm staging judgement of cancer of pancreas.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710827106.2A CN107741416B (en) | 2017-09-14 | 2017-09-14 | SERS (surface enhanced Raman scattering) probe and substrate marked by multiple antibodies as well as preparation method and application of SERS probe and substrate |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710827106.2A CN107741416B (en) | 2017-09-14 | 2017-09-14 | SERS (surface enhanced Raman scattering) probe and substrate marked by multiple antibodies as well as preparation method and application of SERS probe and substrate |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107741416A true CN107741416A (en) | 2018-02-27 |
CN107741416B CN107741416B (en) | 2020-07-28 |
Family
ID=61235819
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710827106.2A Expired - Fee Related CN107741416B (en) | 2017-09-14 | 2017-09-14 | SERS (surface enhanced Raman scattering) probe and substrate marked by multiple antibodies as well as preparation method and application of SERS probe and substrate |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107741416B (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109030456A (en) * | 2018-08-25 | 2018-12-18 | 复旦大学 | A kind of Surface enhanced Raman spectroscopy detection substrate and its preparation method and application |
CN110452955A (en) * | 2019-07-31 | 2019-11-15 | 昆山晟纳生物科技有限公司 | The detection method of Microrna in a kind of change of serum C TC |
CN111458506A (en) * | 2020-03-08 | 2020-07-28 | 复旦大学 | Colorectal cancer exosome detection method and system based on TdT signal amplification |
CN114354935A (en) * | 2022-01-05 | 2022-04-15 | 厦门大学 | High-sensitivity label-free renal cancer serum detection biological reagent |
CN115671318A (en) * | 2022-10-18 | 2023-02-03 | 海南大学 | Surface-enhanced Raman probe, preparation method thereof and application thereof in tumor diagnosis |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104820093A (en) * | 2014-12-31 | 2015-08-05 | 上海师范大学 | Method for using polydopamine bioassay surface to carry out antigen detection, and applications thereof |
CN106053815A (en) * | 2016-07-01 | 2016-10-26 | 复旦大学附属中山医院 | Application of GPC1 as tumor diagnosis marker |
CN106248648A (en) * | 2016-07-10 | 2016-12-21 | 复旦大学 | Gold is " Raman quiet zone " substrate that core silver is shell and preparation method and application |
CN106950374A (en) * | 2017-04-10 | 2017-07-14 | 南通大学附属医院 | Application of the albumen of Glypican 1 in diagnosis of pancreatic cancer, the detection method of positive excretion bulk concentration and application thereof |
-
2017
- 2017-09-14 CN CN201710827106.2A patent/CN107741416B/en not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104820093A (en) * | 2014-12-31 | 2015-08-05 | 上海师范大学 | Method for using polydopamine bioassay surface to carry out antigen detection, and applications thereof |
CN106053815A (en) * | 2016-07-01 | 2016-10-26 | 复旦大学附属中山医院 | Application of GPC1 as tumor diagnosis marker |
CN106248648A (en) * | 2016-07-10 | 2016-12-21 | 复旦大学 | Gold is " Raman quiet zone " substrate that core silver is shell and preparation method and application |
CN106950374A (en) * | 2017-04-10 | 2017-07-14 | 南通大学附属医院 | Application of the albumen of Glypican 1 in diagnosis of pancreatic cancer, the detection method of positive excretion bulk concentration and application thereof |
Non-Patent Citations (3)
Title |
---|
BINDHU MADHAVAN ET AL.: "Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity", 《INTERNATIONAL JOURNAL OF CANCER》 * |
BRUNO COSTA-SILVA ET AL.: "Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver", 《NATURE CELL BIOLOGY》 * |
W TRISTRAM ARSCOTT ET AL.: "EGFR isoforms in exosomes as a novel method for biomarker discovery in pancreatic cancer", 《BIOMARKERS MED.》 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109030456A (en) * | 2018-08-25 | 2018-12-18 | 复旦大学 | A kind of Surface enhanced Raman spectroscopy detection substrate and its preparation method and application |
CN110452955A (en) * | 2019-07-31 | 2019-11-15 | 昆山晟纳生物科技有限公司 | The detection method of Microrna in a kind of change of serum C TC |
CN111458506A (en) * | 2020-03-08 | 2020-07-28 | 复旦大学 | Colorectal cancer exosome detection method and system based on TdT signal amplification |
CN111458506B (en) * | 2020-03-08 | 2023-01-06 | 复旦大学 | Colorectal cancer exosome detection method and system based on TdT signal amplification |
CN114354935A (en) * | 2022-01-05 | 2022-04-15 | 厦门大学 | High-sensitivity label-free renal cancer serum detection biological reagent |
CN115671318A (en) * | 2022-10-18 | 2023-02-03 | 海南大学 | Surface-enhanced Raman probe, preparation method thereof and application thereof in tumor diagnosis |
CN115671318B (en) * | 2022-10-18 | 2024-09-20 | 海南大学 | Surface-enhanced Raman probe, preparation method thereof and application thereof in tumor diagnosis |
Also Published As
Publication number | Publication date |
---|---|
CN107741416B (en) | 2020-07-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107741416A (en) | The SERS probes of Multiple Antibodies mark and substrate and its preparation method and application | |
Liu et al. | An improved strategy to detect the epithelial-mesenchymal transition process in circulating tumor cells in hepatocellular carcinoma patients | |
Hosseini et al. | Detection of recurrent bladder cancer: NMP22 test or urine cytology? | |
Shapiro et al. | Raman molecular imaging: a novel spectroscopic technique for diagnosis of bladder cancer in urine specimens | |
Cheng et al. | Diagnostic value of different phenotype circulating tumor cells in hepatocellular carcinoma | |
CN104007257B (en) | Method for detecting non-humoral rare karyotes, and kit thereof | |
JP2008209419A (en) | Method for increasing clinical specificity when detecting tumor and their precursor stage by simultaneously measuring at least two different molecular markers | |
JP2008116466A (en) | Method of detecting tumor cell, and precursor thereof in uterine cervical alimentary tract smear | |
WO2007035842A2 (en) | Comprehensive diagnostic testing procedures for personalized anticancer chemotherapy (pac) | |
Campi et al. | Novel liquid biomarkers and innovative imaging for kidney cancer diagnosis: what can be implemented in our practice today? A systematic review of the literature | |
Nehmann et al. | Comparison of two techniques for the screening of human tumor cells in mouse blood: quantitative real-time polymerase chain reaction (qRT-PCR) versus laser scanning cytometry (LSC) | |
CN114127562A (en) | Detection method of tumor cell surface marker molecule PD-L1 | |
WO2021213262A1 (en) | Immunofluorescence test kit for measuring pd-l1 expression in circulating tumor cells in peripheral blood in stomach cancer patient, and measurement method | |
CN106834511A (en) | A kind of kit of the breast cancer detection based on liquid biopsy | |
EP2728359B1 (en) | A method of sequential and multiple immunostaining for detection of various antigens in the same specimens | |
US7838215B2 (en) | Advanced cervical cell screening methods | |
EP3214445B1 (en) | Diagnostic method | |
CN106680515B (en) | It is combined for the polymolecular marker of pulmonary cancer diagnosis | |
CN110139656A (en) | The Keratin 17 of biomarker as bladder cancer | |
CN113777311A (en) | ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma | |
CN113049552A (en) | MUC1 protein quantitative detection method based on exosome detection and single-molecule fluorescence bleaching technology | |
CN115656083B (en) | Extracellular vesicle nano infrared spectrum detection device for tumor detection, malignancy and metastatic assessment and application thereof | |
US20120264110A1 (en) | Automated pap screening using a plurality of biomarkers and multi-spectral imaging | |
JP2011209101A (en) | PROGNOSIS PREDICTION INSPECTION METHOD OF MALIGNANT TUMOR WITH CapG AS MARKER | |
CN109060756A (en) | Rare cell detection method in a kind of blood based on surface enhanced effect |
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 | ||
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: 20200728 |