CN105241872B - Detect the method and agents useful for same of half lactadherin -1 in blood - Google Patents
Detect the method and agents useful for same of half lactadherin -1 in blood Download PDFInfo
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- CN105241872B CN105241872B CN201510551551.1A CN201510551551A CN105241872B CN 105241872 B CN105241872 B CN 105241872B CN 201510551551 A CN201510551551 A CN 201510551551A CN 105241872 B CN105241872 B CN 105241872B
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Abstract
The present invention provides a kind of method and agents useful for same of detection sample to be tested half lactadherin 1 especially in blood, the method for the present invention includes：Using the gold nanorods of Lactose-modified as probe, it is made to contact mixing with sample to be tested, 20~37 DEG C of maintenance system temperature stands 10 minutes to 2 hours, half lactadherin 1 is detected by detecting the color of mixed system.The present invention is with the gold nanorods (Lac GNRs) of Lactose-modified for probe, using ultraviolet-visible near infrared spectrometer as detection means, efficient and special detection is carried out to gelactin 1 in molecular level and cellular level, and the galectin 1 in blood of human body is accurately detected, make a kind of means of cancer metastasis early monitoring.
Description
Technical field
The present invention relates to the methods and agents useful for same of half lactadherin -1 (galectin-1) in detection blood.
Background technology
Cancer is a kind of disease seriously threatening global human life and health, and there are about more than 700 ten thousand in the annual whole world according to statistics
People die of cancer (Ahmedin, J., Rebecca S., Elizabeth W., Hao, Y.P., Xu, J.Q., Taylor, M.,
Michael,J.T.Cancer Statistics,CA:Cancer J.Clin.2008,58,71-96).China's cancer morbidity
Linear ascendant trend, and middle and advanced stage has been in when a large amount of patient assessments, the possibility of healing is lost substantially.Middle and terminal cancer
The refractory major reason more of patient is that cancer easily shifts.Primary tumo(u)r can carry out operation excision or radiotherapy, but
The cancer disseminated but often is difficult to the treatment of above-mentioned means without damaging normal structure.The transfer of tumour is along with undesirable pre-
Effect and low survival rate afterwards contribute to failover events if can research and develop effective detection means carries out early monitoring to it
It finds and intervenes as early as possible in time, be conducive to extend patient's service life and improve the quality of living (Liao Zijun, thunder flare, champion, Liu Wen
It is super, Li Hongsheng, remaining international politics, metastases, Shaanxi science tech publishing house, 2007, p322-p331).
Metastases are a complicated cascade process, and during this complexity, tumour cell generally requires largely
Express certain specific proteins (such as integrin, cadherins, immunoglobulin, matrix metalloproteinase, the plasmin activation factor,
Angiogenesis factor etc.) it realizes degradation of cell epimatrix, invasion adjacent cells layer, induction cancer cell motility, escape siberian crabapple
System attack promotes functions (Zeng Yixin, Lv Youyong, Zhu Minghua, Chen Guoqiang, the Gong such as new vascular generation and transfer tumor cell proliferation
Jianping, Guo Zhuming, oncology (third edition), People's Health Publisher, 2012, p241-p252).Certain secretory proteins may be into
Enter the circulatory system, be detected in blood, become effective biomarker of metastases, these biomarkers are carried out
Detection can help to realize the diagnosis early of metastases, become metastases prediction important means (Xue, H., Li, B.J.,
Zhang,J.,Wu,M.,Huang,Q.,Wu,Q.,Sheng,H.,Wu,D.,Hu,J.,Lai,M.,Identification of
Serum Biomarkers for Colorectal Cancer Metastasis Using a Differential
Secretome Approach.J.Proteome.Res.,2010,9,545-555)。
Half lactadherin -1 (galectin-1) is a kind of important serum markers, the proliferation of tumour cell, invasion,
Transfer, angiogenesis and immunologic escape etc. play a significant role (Liu, F.T., Rabinovich, G.A.Galectins
as Modulators of Tumor Progression.Nature Reviews Cancer.2005,5,29-41).Largely grind
Study carefully and shows galectin-1 in kinds of tumors (lung cancer, liver cancer, gastric cancer, cervical carcinoma, breast cancer, glioma, melanoma
Deng) in have an overexpression, expression compared with normal person apparent raising (Kuo, P.L., Hung, J.Y., Huang,
S.K.,Chou,S.H.,Cheng,D.E.,Jong,Y.J.,Hung,C.H.,Yang,C.J.,Tsai,Y.M.,Hsu,Y.L.,
Huang,M.S.,Lung Cancer-Derived Galectin-1Mediates Dendritic Cell Anergy
through Inhibitor of DNA Binding 3/IL-10Signaling Pathway.J.Immunol.2011,186,
1521-1530；Chen,J.,Tang,d.,Wang,S.,Li,Q.G.,Zhang,J.R.,Li,P.,Lu,Q.,Niu,G.,Gao,
J.,Ye,N.Y.,Wang,D.R.,High Expressions of Galectin-1and VEGF are Associated
with Poor Prognosis in Gastric Cancer Patients,Tumor Biol.2014,35,2513-2519)。
Meanwhile as a kind of substance of overexpression, galectin-1 can be secreted into tumor stroma and blood of human body its effect that plays,
Therefore galectin-1 can be as a serum markers of metastases.Galectin-1 in blood is detected,
It is likely to become a kind of novel means of metastases early monitoring.However, although the blood galectin-1 of cancer patient is dense
Degree is higher than normal person, but its absolute content is still very low (ng/ml ranks), still lacks efficiently and conveniently detection side at present
Method, early detection and therapeutic intervention to cancer metastasis help little.
Invention content
It is an object of the present invention to be directed to lack in the prior art that half lactadherin -1 in blood is effectively detected
(galectin-1) present situation provides a kind of method efficiently and conveniently detecting galectin-1 in blood, improves detection spirit
Sensitivity and specificity, with for cancer metastasis early detection and therapeutic intervention help is provided.
Another object of the present invention is to provide one kind for realize efficiently and conveniently detect blood in galectin-1 and
The detection reagent specially designed is applied to galectin-1 in detection blood, has higher sensitivity and specificity.
In order to achieve the above object, on the one hand, the present invention provides half lactadherins -1 in a kind of detection sample to be tested
(galectin-1) method, this method mainly include with the gold nanorods of Lactose-modified (Lac-GNRs) be probe, with purple
Outside-visible light-near infrared spectrometer is detection means, to carry out efficient and special detection to gelactin-1.
Specifically, the present invention provides a kind of method of half lactadherin -1 in detection sample to be tested, this method includes：
Using the gold nanorods of Lactose-modified as probe, it is made to contact mixing, maintenance system temperature 20~37 with sample to be tested
DEG C 10 minutes to 2 hours are stood, the color by detecting mixed system detects half lactadherin -1.
The method of the present invention can be in molecular level cellular level or directly to the galectin-1 in blood of human body
It is accurately detected, since the transfer of galectin-1 and tumour is closely related, the method for the present invention is likely to become
A kind of novel means of cancer metastasis early monitoring.
Specific embodiment according to the present invention, -1 (galectin- of half lactadherin in detection sample to be tested of the invention
1) in method, the sample to be tested can be the aqueous solution of galectin-1, cancer cell culture solution or test individual
Blood or serum etc., method of the invention have higher detection sensitivity and specificity.
Specific embodiment according to the present invention, -1 (galectin- of half lactadherin in detection sample to be tested of the invention
1) method in the specific implementation, can be first by sample to be tested (such as the aqueous solution of galectin-1, cancer cell culture medium or human body
Serum etc.) it is added in gold nanorods (Lac-GNRs) aqueous solution of institute's Lactose-modified, 10 minutes to 2 hours are stood after shaking up,
Optics is carried out to system using ultraviolet-visible light-near infrared spectrometer (UV-Vis-NIS spectrophotometer)
Detection, judges whether contain galectin-1 in object to be checked, or can also visually observe system by the variation of absorption curve
Color change is to detect half lactadherin -1 in measuring samples.
Specific embodiment according to the present invention, of the invention detects in sample to be tested in the method for half lactadherin -1,
In the gold nanorods aqueous solution of the Lactose-modified, a concentration of 10nM to the 100nM of gold nanorods of Lactose-modified.
Specific embodiment according to the present invention, of the invention detects in sample to be tested in the method for half lactadherin -1,
The volume ratio of the gold nanorods aqueous solution of the sample to be tested and Lactose-modified is 1：1~100.
The method of half lactadherin -1, high sensitivity, Galectin- in sample to be tested in the detection sample to be tested of the present invention
1 concentration can also be detected down to 1pM.
On the other hand, the present invention has also been devised a kind of special for galectin-1 in realization efficiently and conveniently detection blood
The detection reagent of door design, mainly includes the gold nanorods (Lac-GNRs) of Lactose-modified.Specific implementation according to the present invention
Scheme, the gold nanorods of the Lactose-modified be the lactose containing sulfydryl connect with gold nanorods by gold-sulfide linkage to be formed it is compound
Object.
Gold nanorods are one extremely important in minimal feeding due to its unique surface plasma resonance effect
Tool.Surface plasma resonance effect makes it to absorb the incident light on its resonant frequency, causes light energy in the frequency
Loss, is easily detected by fluorescence detector.This surface plasma resonance is extremely sensitive to the surface environment of gold, gold surface
Reunite caused by micro-variations, including surface modification, extraneous factor or de-agglomerated can cause clearly optical absorption
Change (Cao, J., Sun, T., Grattan, K.T.V.Gold Nanorod-Based Localized Surface Plasmon
Resonance Biosensors:A Review,Sensors and Actuators B,2014,195,332–351).Simultaneously
Gold nanorods have special space anisotropic, rod-like morphology with surface of horizontal and vertical both direction etc. from
Sub-resonance peak, be allowed to response to trace materials it is especially sensitive (Huang, H.W.Qu, C.T., Liu, X.Y., Huang, S.W.,
Xu,Z,J.,Zhu,Y.J.,Chu,P.K.,Amplification of Localized Surface Plasmon
Resonance Signals by a Gold Nanorod Assembly and Ultra-Sensitive Detection of
Mercury,Chem.Commun.,2011,47,6897-6899).Just because of these characteristics, gold nanorods are likely to become
The tool of galectin-1 detections.
But to reach the efficient and specific detections of galectin-1, other than making base material with gold nanorods,
Also need to load can specific recognition galectin-1 functional materials.In the present invention, selection can be carried out with galectin-1
The chemical small molecule of specific binding --- lactose carries out functionalization as identification substance to gold nanorods.It is born on gold nanorods
Lactose in load, identification function is realized with lactose, realizes optic response function with gold nanorods, it is efficient that galectin-1 may be implemented
And the detection of specificity, and the substance that can avoid traditional performance recognition reaction can for example be specifically bound with object to be checked
Of high cost, complicated for operation, mutability caused by antibody or small-molecule substance easily causes gold nanorods from the defects of reunion.
Specific embodiment according to the present invention, the gold nanorods (Lac-GNRs) of Lactose-modified of the invention be according to
Lower section method is prepared：
Gold nanorods and the lactose containing sulfydryl are mixed, 8~15 hours is stood at room temperature, is made for detecting tumor marker
The bioprobe of half lactadherin -1 of object.The bioprobe is that the lactose containing sulfydryl passes through gold-sulfide linkage and the Jenner
Rice stick connects the compound to be formed.
It is prepared by the gold nanorods (Lac-GNRs) of specific embodiment according to the present invention, Lactose-modified of the invention
When, the length of the gold nanorods is about 60~80nm, wide about 20~25nm.Preferably, the gold nanorods surface is coated with
Cetyl trimethylammonium bromide molecule, the method that Seed inducement in aqueous phase solution may be used are prepared：First use boron hydrogen
Change sodium reduction gold chloride, prepare the gold seeds of 1~2nm, then gold nano seed is added to gold chloride (HAuCl4), silver nitrate
(AgNO3), dilute hydrochloric acid (HCl), ascorbic acid, Dodecydimethylammonium bronides (CTAB) mixed aqueous solution in, generate wide
About 20~25nm, it is about 60~80nm, the gold nanorods that surface is covered by CTAB.The preparation-obtained gold nanorods of the present invention
The peak position at lateral plasmon absorption peak is 510nm or so, the peak position at longitudinal plasmon absorption peak is 700~
800nm。
It is prepared by the gold nanorods (Lac-GNRs) of specific embodiment according to the present invention, Lactose-modified of the invention
When, the lactose containing sulfydryl has structural formulaIt can profit
With full acetyl group lactose, (full acetyl group lactose is commercially available or is voluntarily prepared with reference to the prior art, typically acetic anhydride, acetic acid
Sodium and lactose react acquisition in a heated condition) through reaction steps such as glycosylation, sulfhydrylation and deacetylations, in the end group of lactose
It introduces at carbon atom and is prepared containing the linking arm of sulfydryl.Specifically, the lactose containing sulfydryl can be according to such as lower section
Method is prepared：
By full acetyl group lactose (compound 1, structural formula) contract with chloro three
Ethylene glycol (compound 2, structural formula) under the effect of the catalyst, it is stirred to react 36~72 at room temperature
Hour, after purification, obtained structural formula isCompound 3；Describedization
The molar ratio for closing object 1 and the compound 2 is 1：1.2~2；
(b) reaction 3-5 hours is stirred at room temperature in the compound 3 and thioacetic acid potassium, after purification, structural formula is made
ForCompound 4；The compound 3 and the thioacetic acid potassium
Molar ratio is 1：1.2~2；
(c) compound 4 is dissolved in methanol, metallic sodium is then added, is stirred overnight at room temperature, after purification, be made
Lactose (compound 5, structural formula containing sulfydryl)。
It is prepared by the gold nanorods (Lac-GNRs) of specific embodiment according to the present invention, Lactose-modified of the invention
When, the molar ratio of the lactose containing sulfydryl and the gold nanorods is preferably 1 × 104:1-3×104:1.It is preparation-obtained
The gold nanorods of Lactose-modified are coated with cetyl trimethylammonium bromide bilayer on gold nanorods surface, contain sulfydryl
Lactose have some or all of cetyl trimethylammonium bromide bilayer region the gold nanorods surface is uncoated
It connect to form compound with the gold nanorods by gold-sulfide linkage.
Specific embodiment according to the present invention, method of the invention can utilize the gold nanorods of the Lactose-modified
(Lac-GNRs) to the detection of galectin-1 in molecular level, cellular level or blood level.
In the specific embodiment of the present invention, the present invention on a molecular scale carries out the detection of galectin-1
Assessment finds that Lac-GNRs can and its delicately detect that the presence of galectin-1, detectable limit can reach 1pM, and
And response changes with the variation of galectin-1 concentration.The combination of Galectin-1 and lactose can induce gold nano
Stick is reunited, to which the variation of light absorption occur in ultraviolet-visible light-near infrared region (UV-Vis-NIS).When past system
When the middle galectin-1 that 1nM is added, the absorption peak at 770nm drastically reduces, and occurs a new absorption at 1100nm
Peak shows that gold nanorods are seriously reunited, and the color of Lac-GNR solution becomes purple from orange at this time；And work as
When the concentration of galectin-1 reduces most 100pM, the absorption peak at 770nm also has apparent reduction, also occurs at 900nm
One new peak, shows that the reunion of gold nanorods is also obvious.The concentration of Galectin-1 can drop to always 1pM, at this time
Still there is obvious reduction at the peak of 770nm, shows that Lac-GNRs is especially sensitive to the response of galectin-1, may be implemented extremely low
The detection of the galectin-1 of concentration.
Lac-GNRs cannot identify other albumen (such as ox of lactose other than it can be responded to galectin-1
Haemocyanin BSA) response it is excessively poor.Such as it is the apparent change that can induce absorption peak that the galectin-1 of 1nm is added into system
Change, even and if the much bigger BSA of concentration is added, or the galactolipin close with galectin-1 structures being added with concentration coagulates
Collect -3 (galectin-3) of element, cannot significantly change the absorption of gold nanorods, shows the specificity of Lac-GNRs very well.
In another embodiment of the present invention, the present invention carries out the detection of galectin-1 in cellular level
Research.Since galectin-1 is a kind of secretory protein, it can be secreted into tumour epimatrix and play a role.Utilize cell culture
Method cancer cell is cultivated, take its culture solution to be detected, should be able to detect the presence of galectin-1.The present invention is total
HUH-7 (human liver cancer cell), MCF-7 (human breast cancer cell), JEG-3 (human chorionic cancer cell), (the people palaces HeLa are co-cultured
Neck cancer cell), Hep-G2 (human liver cancer cell), 6 kinds of tumour cells such as C6 (mouse brain glioblastoma cell), and their cell is trained
Nutrient solution is detected, it is found that the culture solution of this 6 kinds of tumour cells can cause the significant decrease of Lac-GNRs absorption curves,
Illustrate that Lac-GNRs can accurately detect galectin-1 therein.
In another embodiment of the present invention, the present invention human blood levels to the detection of galectin-1 into
Research is gone.Since in the cancer metastasis stage, galectin-1 can be expressed largely, and it is secreted into play in blood and makees
With, therefore the galectin-1 concentration in cancer metastasis blood samples of patients will be apparently higher than normal person.The present invention acquires 5 normally
The blood of people and 15 blood for having been confirmed the cancer patient for transfer, detect it, find Lac-GNRs energy
Significantly distinguish the blood of normal person and cancer patient：The blood of normal person is only capable of causing the micro red shift of Lac-GNRs absorption curves
(moving right), but the blood of cancer patient can not only cause a large amount of red shifts of Lac-GNRs absorption curves, moreover it is possible to it causes to inhale
Receive the significant decrease of curve., the result shows that Lac-GNRs accurately detects the galectin-1 in blood, having can for this
A kind of means of useful clinically monitoring cancer metastasis can be become.
And other than the light absorption to the regions UV-Vis-NIS measures, also directly with the naked eye testing result can be carried out
Judge：It is added the Lac-GNR solution of normal human serum, color and is not added before serum consistent；And cancer patient's serum is added
Lac-GNR solution, color is obviously thin out.The significant change of this color greatly strengthens the visuality and conveniently of detection
Property.
In conclusion the method for detecting half lactadherin -1 in sample to be tested of the present invention, it can be to swollen in blood of human body
Tumor prognostic markers object galectin-1 carries out vitro detection, with the gold nanorods (Lac-GNRs) of Lactose-modified for probe, with purple
Outside-visible light-near infrared spectrometer is detection means, is carried out to gelactin-1 in molecular level and cellular level efficient
And special detection, and the galectin-1 in blood of human body is accurately detected, using the method for the present invention to coming from
After half lactadherin -1 is detected in the sample to be tested of the test individuals such as human body, the method for the present invention can be utilized to coming from simultaneously
Half lactadherin -1 in the same sample of healthy individuals is detected, and compares the sample of sample to be tested testing result and healthy individuals
The testing result of product judges test individual according to absorption curves difference condition or color distortion situation between the two
Health condition makes a kind of means of cancer metastasis early monitoring.The present invention main advantageous effects include：
The modification that the present invention successfully stablizes gold nanorods using carbohydrate small molecule-lactose constructs a kind of new
Can be detecting the molecular probe of tumor prognosis marker galectin-1；Using the molecular probe in blood
Tumor prognosis marker galectin-1 has carried out accurate detection, can not only realize the efficient and specific inspections of galectin-1
Go out, and the blood of normal person and cancer metastasis patient can be distinguished, it is possible to have real Clinical practice value, become tumour
Shift a kind of novel means of early monitoring.In addition to this, since Lac-GNRs avoids the large biological molecules such as enzyme and antibody
It uses, greatly reduces testing cost, simplify operating procedure.Since detection means is to use common optical detection work
Tool --- ultraviolet-visible light-near infrared spectrometer, technology of the invention also have many advantages, such as simple, convenient easy.
Description of the drawings
Fig. 1 is that the gold nanorods of Seed inducement in the embodiment of the present invention 1 synthesize schematic diagram.
Fig. 2A is the absorption curve figure of gold nanorods prepared in the embodiment of the present invention 1.
Fig. 2 B are the micro-structure diagram of gold nanorods prepared in the embodiment of the present invention 1.
Fig. 3 is the synthetic route chart of lactose ligand in the embodiment of the present invention 1.
Fig. 4 is the Lactose-modified schematic diagram on gold nanorods surface in the embodiment of the present invention 1.
Fig. 5 A are that the element of gold nanorods before and after Lactose-modified in the embodiment of the present invention 1 constitutes variation spectrogram (XPS).
Fig. 5 B are that the surface charge (zeta potential) of gold nanorods before and after Lactose-modified in the embodiment of the present invention 1 becomes
Block diagram is compared in change.
Fig. 5 C are the light of ultraviolet-visible light-near infrared region of gold nanorods before and after Lactose-modified in the embodiment of the present invention 1
Change of absorption compares figure.
Fig. 5 D are the micro-structure diagram of gold nanorods after Lactose-modified in the embodiment of the present invention 1.
Fig. 6 A are that Lac-GNRs closes the detection absorption curve of galectin-1 with the variation of concentration in the embodiment of the present invention 2
System's figure.
Fig. 6 B are the application Lac-GNRs of the embodiment of the present invention 2 to 1nM galectin-1 are added in the detection of galectin-1
When Lac-GNRs reunion degree photo.
Fig. 6 C are the application of the embodiment of the present invention 2 Lac-GNRs to solution colour variation diagram in the detection of galectin-1.
Fig. 7 is to detect the specific detection that galectin-1 is shown using Lac-GNRs in the embodiment of the present invention 2 to compare
Collection of illustrative plates.
Fig. 8 is the detection knot using Lac-GNRs to galectin-1 in tumor cell culture liquid in the embodiment of the present invention 3
Fruit.
Fig. 9 is the testing result using Lac-GNRs to galectin-1 in blood of human body in the embodiment of the present invention 4.
Figure 10 is using Lac-GNRs in the embodiment of the present invention 4 to solution face when the detection of galectin-1 in blood of human body
Color change result.
Specific implementation mode
Essence for a clearer understanding of the present invention below by specific embodiment and coordinates attached drawing further specifically
The bright present invention, but the present invention is not therefore subject to any restriction.In the following examples, the experimental methods for specific conditions are not specified,
Usually carried out according to the routine operation of fields or according to the normal condition proposed by manufacturer.
The preparation of embodiment 1, the gold nanorods detection probe of Lactose-modified
In the present embodiment, using the gold nanorods synthetic method of Seed inducement, prepare dispersion performance is good, grain size is single, tool
The gold nanorods that has suitable axial ratio, can be stabilized；Synthesize suitable lactose ligand；Made by the strong coordination of gold-sulfide linkage
With lactose ligand is loaded to gold nanorods surface, realizes to the surface lactosylation of gold nanorods, prepares the Jenner of Lactose-modified
Rice stick detection probe, and its structure is confirmed.
1, the preparation of gold nanorods
Golden stick is prepared using the gold nanorods synthetic method of Seed inducement in aqueous phase solution.First use sodium borohydride reduction chlorine gold
Acid prepares the gold seeds of 1-2nm, then gold nano seed is added to gold chloride (HAuCl4), silver nitrate (AgNO3), dilute hydrochloric acid
(HCl), it in the mixed aqueous solution of ascorbic acid, Dodecydimethylammonium bronides (CTAB), generates wide about 20~25nm, be about
The gold nanorods that 60~80nm, surface are covered by CTAB.Specific experimental implementation is following (in combination with shown in Figure 1)：
By the HAuCl of 0.5mM4It is dissolved in the CTAB solution of 0.2M, is vigorously stirred, the cold NaHB of 600 microlitres of 10mM is added4It is molten
Liquid stirs 2 hours at 37 DEG C, obtains gold nano seed solution；
Then toward the HAuCl of 5mM40.2M CTAB, 0.1M AgNO are added in solution3, 1.2M HCl and 10mM Vitamin Cs
Acid, when solution by it is dark red become colourless after, be added at 50 microlitres, 37 DEG C of the gold nano seed solution of above-mentioned preparation and hatch 12 hours
10000rpm is centrifuged 10 minutes afterwards, and residue carries out redisperse with ultra-pure water after removing supernatant, obtains surface and is covered by CTAB
Gold nanorods aqueous solution (CTAB-GNRs).
Optical absorption map shows the gold nanorods prepared respectively and at 510nm and 770nm has respectively to have weak and strong
It absorbs (short axle for corresponding to gold nanorods respectively absorbs and long axis absorbs, as shown in Figure 2 A).Transmission electron microscope collection of illustrative plates (TEM) shows gold
The width of nanometer rods is about 20nm~25nm, and length is about 60nm~80nm (as shown in Figure 2 B).
2, the synthesis of lactose ligand
From full acetyl group lactose, through three-step reactions such as glycosylation, sulfhydrylation and deacetylations, in the end group carbon of lactose
The linking arm containing sulfydryl is introduced at atom, prepares lactose ligand (the synthetic route such as Fig. 3 institutes that can be had an effect with gold nanorods
Show).Specific experimentation is as follows：
(1) system of -2,3,6,2 ', 3 ', 4 ', 6 '-seven acetyl group lactose (compound 3) of 1- β-(chloro triethylene Glycol base)
It is standby：
Sodium acetate is added in acetic anhydride, is heated to 130 DEG C, is slowly added to lactose and obtains reaction solution, acetic anhydride, sodium acetate
Molar ratio with lactose is 12:12:1, temperature is then reduced to 110 DEG C, and the reaction was continued 5 hours, and reaction solution is poured into ice water
It places 12 hours, solid is precipitated, filtering, obtained filter cake ethyl alcohol recrystallization is to get to full acetyl group lactose, and structural formula is such as
Under：
By full acetyl group lactose (1,3.5 grams of compound) and chloro triethylene Glycol (Change
Close 2,1.3 grams of object) it is dissolved in dichloromethane (CH2Cl2, 5 milliliters), it is added 500 milligramsMolecular sieve, stirring are added after twenty minutes
Boron trifluoride ether (BF3-Et2O, 7.8 milliliters), stir 72 hours at room temperature.After reaction, boron trifluoride ether is filtered out,
Filtrate is diluted with dichloromethane, and successively saturated sodium bicarbonate solution and saturated common salt aqueous solution wash dilution, anhydrous slufuric acid
Sodium is dried, and is then removed by filtration sodium sulphate, filtrate is evaporated with Rotary Evaporators, and remaining residue chromatography post separation is changed
Close object 3.The structural formula of compound 3 is as follows：
The nuclear magnetic data of compound 3 is as follows：1HNMR(400MHz,CDCl3,25℃):δ=1.97 (s, 3H), 2.05 (m,
12H),2.12(s,3H)2.16(s,3H),3.6-3.8(m,12H),3.8-3.95(m,2H),4.05-4.15(m,4H),4.47-
4.58 (m, 2H), 4.56 (d, 2H, J=7.8), 4.89 (t, 1H), 4.95 (dd, 1H, J=10.5,3.3Hz, CH), 5.1 (dd,
1H, J=10.5,7.8), 5.2 (t, 1H, J=9.3Hz) 5.34 (d, 1H, J=3.3Hz, CH).
(2) -2,3,6,2 ', 3 ', 4 ', 6 '-seven acetyl group lactose (compounds of 1- β-(ethanethioyl triethylene Glycol base)
4) preparation：
Compound 3 (770 milligrams) is dissolved in n,N-Dimethylformamide (DMF, 3 milliliters), thioacetyl potassium is added
(KSAc, 550 milligrams) is stirred 4 hours at room temperature.After reaction stops, ethyl acetate is added into reaction solution, uses water successively, satisfy
(each 2 times) are washed with sodium bicarbonate solution and saturated common salt aqueous solution, then anhydrous sodium sulfate drying is removed by filtration sodium sulphate,
Filtrate is evaporated with Rotary Evaporators, obtains compound 4.The structural formula of compound 4 is as follows：
The nuclear magnetic data of compound 4 is as follows：1HNMR(400MHz,CDCl3,25℃):δ=1.855 (s, 3H), 1.935 (s,
3H), 1.941 (m, 6H), 1.955 (s, 3H), 2.014 (s, 3H), 2.046 (s, 3H), 2.23 (s, 3H), 2.92 (tr, J=
6.3Hz, 2H), 3.6-3.8 (m, 12H), 3.85 (m, 2H), 4.01 (m, 2H), 4.38 (dd, 1H, J=1.8Hz, 12.6Hz),
4.42 (d, 1H, J=7.8Hz), 4.49 (d, 1H, J=7.8Hz), 4.78 (dd, 1H, J=7.8Hz, 9.3Hz), 4.86 (dd,
1H, J=10.5,3.3Hz), 4.9 (dd, 1H, J=10.5,7.8Hz), 5.1 (t, 1H, JH=9.3Hz), 5.24 (d, 1H, J=
3.6Hz).13C NMR(400MHz,CDCl3):19.920,20.049,20.049,20.079,20.130,20.22,20.27,
28.212,29.982,60.298,61.526,66.131,68.497,68.580,69.139,69.72,70.28,70.07,
70.409,70.77,71.1,72.0,72.271,75.75,100.01,100.458,168.452,168.99,169.12,
169.37,169.51,169.68,169.7,194.7.
(3) preparation of 1- β-(sulfydryl triethylene Glycol base) lactose (compound 5)：
Compound 4 (600 milligrams) is dissolved in 2 ml methanols, suitable metallic sodium, the weight of metallic sodium are added into solution
It is the 5% of 4 weight of compound, is stirred overnight at room temperature.After reaction, suitable acidic cationic resin is added, by solution
PH modulation 7.Filtering, filtrate are evaporated with Rotary Evaporators, and residue is dissolved in suitable quantity of water, and freeze-drying obtains compound 5.Compound 5
Structural formula is as follows：
The nuclear magnetic data of compound 5 is as follows:1HNMR(400MHz,MeOD,25℃):δ=2.8 (t, 2H, J=6.6Hz),
3.35 (2H, s), 3.5-3.8 (20H, m), 3.9 (d, 2H, J=3.3Hz), 4.1 (2H, m), 4.45 (d, 1H, J=7.8Hz)),
4.51 (1H, d, J=7.8Hz)13C NMR(400MHz,MeOD,25℃):δ=102.54,101.65,77.82,74.88,
73.89,72.29,70.41,69.25,68.27,60.6,59.61,48.51,42.79。
3, the Lactose-modified of gold nanorods
Ligand exchange reaction is carried out with the aqueous solution of above-mentioned lactose ligand and gold nanorods solution, passes through Au-S coordinations
Lactose ligand is loaded into gold nanorods surface (as shown in Figure 4), passes through-x-ray photoelectron spectroscopy (XPS), surface charge
(Zeta potential), ultraviolet-visible light-near-infrared absorption and microstructure (TEM) characterize modification result.
Specific experiment operation is as follows：
The aqueous solution of 100 microlitres of sulfydryl lactose ligand (compound 5) is added to the gold nanorods of 5 milliliters of above-mentioned preparations
In solution, the molar ratio of lactose and gold nanorods containing sulfydryl is 1 × 104：1,15 hours are stood at room temperature, without stirring for,
Stop reaction, the bioprobe (being indicated with Lac-GNRs) for detecting tumor marker galectin-1 is made, which visits
Needle is that the lactose containing sulfydryl connect the compound to be formed with gold nanorods by gold-sulfide linkage.Reaction product is obtained by the experiment
Lac-GNR aqueous solutions.
As shown in Fig. 5 A~Fig. 5 D, XPS atlas analysis shows gold nanorods (Lac-GNRs) the ratio modification after Lactose-modified
Carbon (C) content of preceding gold nanorods (CTAB-GNRs) reduces, and the content of oxygen element (O) increases (Fig. 5 A), shows gold
The part CTAB of nanorod surfaces is replaced (additional oxygen comes from lactose) by lactose；Zeta tests show the surface of Lac-GNRs
Charge ratio CTAB-GNRs low (Fig. 5 B) further demonstrates part CTAB and is replaced that (CTAB is positively charged, and lactose by lactose
Neutral)；The light absorption of ultraviolet-visible light-near infrared region shows that the lateral absorption peak of gold nanorods after Lactose-modified does not have
It changes, and a small amount of red shift (Fig. 5 C) then has occurred in longitudinal Absorption Spectroscopy；Fig. 5 D then show the microstructure of Lac-GNRs.
The above result shows that gold nanorods success is by Lactose-modified, the gold nanorods detection probe of Lactose-modified is able to success
Structure.
The detection of embodiment 2, molecular level gold nanorods (Lac-GNRs) to galectin-1
The present embodiment on a molecular scale assesses the detection of galectin-1.
Detect method used in galectin-1：Operationally, by 100 microlitres of objects to be checked, (galectin-1's is water-soluble
Liquid) be added in the Lac-GNR aqueous solutions prepared by 1 milliliter of embodiment 1, be stored at room temperature after shaking up one hour, using it is ultraviolet-can
Light-exposed-near infrared spectrometer (UV-Vis-NIS spectrophotometer) carries out optical detection to system, measures
The light absorption of 250nm to 1100nm judges whether contain galectin-1 in object to be checked by the variation of absorption curve, or even can
With the naked eye to be judged by variation in color.
The testing result of the present embodiment finds that Lac-GNRs can and its delicately detect the presence of galectin-1, inspection
1pM can be reached by surveying the limit, and response changes (Fig. 6 A) with the variation of galectin-1 concentration.Galectin-1
Gold nanorods are can induce with the combination of lactose to reunite, in ultraviolet-visible light-near infrared region (UV-Vis-
NIS the variation of light absorption) occurs.When the galectin-1 of 1nM is added into system, the absorption peak at 770nm drastically reduces,
And occur a new absorption peak at 1100nm, show that gold nanorods seriously occur to reunite (Fig. 6 B), Lac-GNR is molten at this time
The color of liquid becomes purple (Fig. 6 C) from orange；And when the concentration of galectin-1 reduces most 100pM, the suction at 770nm
Receiving peak also has apparent reduction, also occurs a new peak at 900nm, shows that the reunion of gold nanorods is also obvious.
The concentration of Galectin-1 can drop to always 1pM (Fig. 6 A), and still there is obvious reduction at the peak of 770nm at this time, shows Lac-
GNRs is especially sensitive to the response of galectin-1, and the detection of the galectin-1 of extremely low concentration may be implemented.
Lac-GNRs cannot identify other albumen (such as ox of lactose other than it can be responded to galectin-1
Haemocyanin BSA) response it is excessively poor.Such as it is added 1nM's into the Lac-GNR water solution systems prepared by embodiment 1
Galectin-1 be can induce the significant change of absorption peak, even and if the much bigger BSA of concentration is added, or be added with concentration
The galectin-3 (galectin-3) close with galectin-1 structures, cannot significantly change the suction of gold nanorods
It receives (as shown in Figure 7), shows the specificity of Lac-GNRs very well.
The detection of embodiment 3, cellular level Lac-GNRs to galectin-1
The present embodiment studies the detection of galectin-1 in cellular level, since galectin-1 is a kind of point
Albumen is secreted, can be secreted into tumour epimatrix and play a role.Cancer cell is cultivated using the method for cell culture, takes it
Culture solution is detected, and should be able to detect the presence of galectin-1.
The present embodiment has always co-cultured HUH-7 (human liver cancer cell), MCF-7 (human breast cancer cell), JEG-3 (human chorionics
Film cancer cell), HeLa (human cervical carcinoma cell), Hep-G2 (human liver cancer cell), 6 kinds of tumours such as C6 (mouse brain glioblastoma cell) it is thin
Born of the same parents, and their cell culture fluid is detected.The condition of culture of cell is：By cell seeding to containing 10% fetal calf serum
H-DMEM culture solutions in (the primary section's minimum essential medium of high glycoform Dole, cell concentration are about 2x 106It is a) in, at 37 DEG C
It is middle hatching 24 hours, then remove culture solution, the new culture solution of equivalent be added, again hatch 48 hours, take out culture solution into
Row measures.
Detect method used in galectin-1：Operationally, by 100 microlitres of objects to be checked, (cancer of galectin-1 is thin
Born of the same parents' culture medium) it is added in the Lac-GNR aqueous solutions prepared by 1 milliliter of embodiment 1, it is stored at room temperature one hour, uses after shaking up
Ultraviolet-visible light-near infrared spectrometer (UV-Vis-NIS spectrophotometer) carries out optical detection to system,
Judge whether contain galectin-1 in object to be checked by the variation of absorption curve
Testing result finds that the culture solution of six kinds of tumour cells used in the present embodiment can cause Lac-GNRs light absorptions bent
The significant decrease (Fig. 8) of line illustrates that Lac-GNRs can accurately detect galectin-1 therein.
The detection of embodiment 4, blood level Lac-GNRs to galectin-1
The present embodiment studies the detection of galectin-1 in human blood levels, due in cancer metastasis rank
Section, galectin-1 can be expressed largely, and be secreted into blood and played a role, therefore in cancer metastasis blood samples of patients
Galectin-1 concentration will be apparently higher than normal person.
Detect method used in galectin-1：Operationally, by 100 microlitres of object (human bodies of galectin-1 to be checked
Serum) be added in the Lac-GNR aqueous solutions prepared by 1 milliliter of embodiment 1, be stored at room temperature after shaking up one hour, using it is ultraviolet-
Visible light-near infrared spectrometer (UV-Vis-NIS spectrophotometer) carries out optical detection to system, measures
The light absorption of 250nm to 1100nm judges whether contain galectin-1 in object to be checked by the variation of absorption curve, or even can
With the naked eye to be judged by variation in color.
The present embodiment acquires the blood and 15 blood for having been confirmed the cancer patient for transfer of 5 normal persons,
It is detected, it is found that Lac-GNRs can significantly distinguish the blood of normal person and cancer patient：The blood of normal person is only capable of
The micro red shift (referring to Fig. 9, moving right) of Lac-GNRs absorption curves is caused, but the blood of cancer patient can not only cause
A large amount of red shifts of Lac-GNRs absorption curves, moreover it is possible to cause the significant decrease of absorption curve.It is somebody's turn to do the result shows that Lac-GNRs is to blood
Galectin-1 in liquid is accurately detected, it is possible to become a kind of means of useful clinically monitoring cancer metastasis.
And other than the light absorption to the regions UV-Vis-NIS measures, also directly with the naked eye testing result can be carried out
Judge：It is added the Lac-GNR solution of normal human serum, color and is not added before serum consistent (as shown in the 1st row in Figure 10)；
And the Lac-GNR solution of cancer patient's serum is added, color is apparent thin out (as shown in the 2nd~4 row in Figure 10).This color
Significant change greatly strengthen the visuality and convenience of detection.
Claims (9)
1. the method for half lactadherin -1, this method include in a kind of detection sample to be tested：
Using the gold nanorods of Lactose-modified as probe, it is made to contact mixing with sample to be tested, 20~37 DEG C of maintenance system temperature is quiet
It sets 10 minutes to 2 hours, half lactadherin -1 is detected by detecting the color of mixed system；
Wherein, a length of 60~80nm of the gold nanorods, width are 20~25nm；
Wherein, in the gold nanorods aqueous solution of the Lactose-modified, a concentration of 10nM to the 100nM of gold nanorods of Lactose-modified；
The detectable limit of half lactadherin -1 is 1pM in sample to be tested.
2. according to the method described in claim 1, wherein, the sample to be tested is thin for the aqueous solution of half lactadherin -1, cancer
The blood or serum of born of the same parents' culture solution or test individual.
3. according to the method described in claim 1, this method includes：
Sample to be tested is added in the gold nanorods aqueous solution of institute's Lactose-modified, 10 minutes to 2 hours are stood after shaking up, used
Ultraviolet-visible light-near infrared spectrometer carries out optical detection to system or visually observes system color change to detect
Half lactadherin -1 in measuring samples.
4. method according to claim 1 or 3, wherein the gold nanorods aqueous solution of the sample to be tested and Lactose-modified
Volume ratio be 1：1~100.
5. according to the method described in claim 1, wherein, the gold nanorods of the Lactose-modified are that the lactose containing sulfydryl passes through
Gold-sulfide linkage connect the compound to be formed with gold nanorods.
6. according to the method described in claim 1, wherein, the gold nanorods of the Lactose-modified are to be prepared into accordance with the following methods
It arrives：
Gold nanorods and the lactose containing sulfydryl are mixed, stand 8~15 hours at room temperature, are made for detecting tumor marker half
The bioprobe of lactadherin -1.
7. according to the method described in claim 6, wherein, the gold nanorods are long 60~80nm, wide 20~25nm, surface quilt
The gold nanorods of CTAB coverings；The lactose containing sulfydryl be at the terminal carbon of lactose introduce the linking arm containing sulfydryl and
It is prepared.
8. according to the method described in claim 6, wherein, the molar ratio of the lactose containing sulfydryl and the gold nanorods is 1
×104:1~3 × 104:1。
9. according to the method described in claim 1, it is double of curdling collection in molecular level, cellular level or blood level
Element -1 is detected.
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| 一维单分散性纳米金的制备及在传感器中应用研究;黄颖娟;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》;20150115(第01期);第一章 * |
| 应用糖量子点-糖金纳米粒子体系检测微生物;柴金凤;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》;20140815(第08期);B014-482 * |
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