CN105158467A - Biological probe used for detecting tumour marker galectin-1 and preparation method thereof - Google Patents
Biological probe used for detecting tumour marker galectin-1 and preparation method thereof Download PDFInfo
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Abstract
The invention provides a biological probe used for detecting tumour marker galectin-1, and the biological probe is a compound formed by connecting sulfhydryl-containing lactose with a gold nanorod via a gold-sulfur bond. The sulfhydryl-containing lactose is firstly used for modifying the gold nanorod, the sulfhydryl-containing lactose is connected with the gold nanorod via the gold-sulfur bond, and the combination capability is strong. The gold nanorod is loaded with the carbohydrate micromolecule lactose and is subjected to stable specific modification, the lactose part in the obtained probe possesses specific recognition effect on galectin-1, and additionally because of the precise optical response effect of the gold nanorod, finally the biological probe is capable of efficiently specifically detecting galectin-1. Furthermore, the invention also provides a preparation method for the biological probe used for detecting the tumour marker galectin-1, and the preparation method for the biological probe is simple and practicable, mild in conditions and relatively low in cost.
Description
Technical field
The present invention relates to technological field of biochemistry, being specifically related to a kind of bioprobe for detecting tumor marker half lactadherin-1 and preparation method thereof.
Background technology
Cancer is a kind of disease of serious threat global human life and health, and the major reason that patient with advanced cancer refractory is healed is that cancer easily shifts.Primary tumo(u)r can carry out excision or radiation therapy, but the cancer of having disseminated often is difficult to treat by above-mentioned means, and easy damaging normal tissue in therapeutic process.The transfer of tumour, along with bad outcome and low survival rate, if can develop effective detection means to carry out early monitoring to it, then contributes to the Timeliness coverage of transfer and intervenes as early as possible, is conducive to extending patient's life-span and improving the quality of living.
The tumor marker detected in blood can help the diagnosis early realizing metastases, becomes the important means of metastases prediction.Half lactadherin-1 (galectin-1, gal-1) plays a significant role in the propagation of tumour cell, invasion and attack, transfer, Angiogenesis and immunologic escape etc., and and there is close relationship between the prognosis of cancer patient and survival rate.What is more important, galectin-1 can be secreted in blood of human body and play a role, and can be detected as a kind of serum markers.Although but the blood galectin-1 concentration ratio normal person of cancer patient wants high, its absolute value is very low (generally in ng/ml rank) still, therefore needs the very high bioprobe of sensitivity and specificity to detect.
Prior art generally adopts biomacromolecule such as antibody etc. to modify gold nanorods, but antibody modification cost is higher, complicated operation, antibody protein changeableness, in addition, prior art adopts antibody modification gold nanorods easily to cause self reuniting and making it lose the problems such as surface plasma resonance effect of gold nanorods.
The present invention adopts Small molecular lactose as the modified ligand of gold nanorods, avoid adopt as when the biomacromolecule such as antibody is modified the modified ligand that meets with expensive, albumen changeableness, the gold nanorods problem of easily reuniting, bioprobe provided by the invention is cheap, stability better and gold nanorods is not easily reunited.
In bioprobe, lactose weight accounts for 5% of bioprobe weight.
Gold nanorods, due to the surface plasma resonance effect of its uniqueness, is a very important instrument in minimal feeding.Surface plasma resonance effect makes it to absorb the incident light on its resonant frequency, causes the loss of luminous energy in this frequency, is detected optically device easily and detects.Gold nanorods has special space anisotropic simultaneously, its rod-like morphology makes it the surface plasma resonance peak with horizontal and vertical both direction, make it responsive especially to the response of trace materials, the present invention adopts gold nanorods accurately can detect galectin-1 as a part for bioprobe.
Galectin-1, due to its structure containing special beta galactose glycosidic bond identified region, therefore can carry out specific recognition to lactose.After lactose containing sulfydryl is connected with gold nanorods by gold-sulfide linkage by the present invention, carbohydrate Small molecular-lactose in load on gold nanorods, stable and specific modification can have been carried out to gold nanorods, in the probe obtained, lactose fraction has the effect to galectin-1 specific recognition, add that gold nanorods has the effect of accurate optic response, final bioprobe can realize to galectin-1 efficiently and detect specifically.
The bioprobe of the detection tumor marker galectin-1 that first aspect present invention provides, the lactose containing sulfydryl is adopted to modify gold nanorods first, lactose containing sulfydryl is connected by gold-sulfide linkage with gold nanorods, binding ability is strong, finally construct a kind of newly can in order to detect the bioprobe of tumor marker galectin-1, this bioprobe can realize to galectin-1 efficiently and detect specifically.
Second aspect present invention provides a kind of preparation method of the bioprobe for detecting tumor marker galectin-1, comprises the following steps:
(1) gold nanorods is provided;
(2) provide lactose, lactose is the obtained lactose containing sulfydryl after mercaptolation;
(3) mix by gold nanorods with containing the lactose of sulfydryl, at room temperature leave standstill 8-15 hour, obtain the bioprobe for detecting tumor marker galectin-1, bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Lactose containing sulfydryl is
Be 1 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1-3 × 10
4: 1.
Containing the lactose of sulfydryl
preparation method be:
A structural formula is by ()
compound 1 and structure be
compound 2 under the effect of catalyzer, stirred at ambient temperature reaction 36-72 hour, after purifying, obtained structural formula is
compound 3; The mol ratio of compound 1 and compound 2 is 1:1.2-1:2; Chemical equation is as follows:
B () (chemical formula is by compound 3 and thioacetic acid potassium
kSAc) at room temperature stirring reaction 3-5 hour, after purifying, obtained structural formula is
compound 4; The mol ratio of compound 3 and thioacetic acid potassium is 1:1.2-1:2, and chemical equation is as follows:
C compound 4 is dissolved in methyl alcohol by (), then add sodium metal, room temperature for overnight, and after purifying, obtained structural formula is
containing the lactose of sulfydryl; Chemical equation is as follows:
Step (1) adopts Seed inducement legal system for gold nanorods, comprises the following steps:
Adopt sodium borohydride reduction gold chloride, prepare the gold nano seed that particle diameter is 1-2nm, then gold nano seed is joined containing gold chloride (HAuCl
4), silver nitrate (AgNO
3), in the mixed aqueous solution of watery hydrochloric acid (HCl), ascorbic acid, cetyl trimethyl ammonium bromide (CTAB), prepare wide 20-25nm, long 60-80nm, gold nanorods that surface is covered by CTAB bilayer.
Step (1) adopts Seed inducement legal system for gold nanorods, comprises the following steps:
By the HAuCl of 0.5mM
4be dissolved in the CTAB solution of 0.2M, vigorous stirring, add the cold NaHB of appropriate 10mM
4solution, stirs 2 hours with preparation containing gold nano seed solution at 37 DEG C; Then at the HAuCl of 5mM
40.2MCTAB, 0.1MAgNO is added in solution
3, 1.2MHCl and 10mM ascorbic acid, when solution by dark red become colourless after, add gold nano seed solution, hatch after 12 hours centrifugal at 37 DEG C, after removing supernatant, residue ultrapure water disperses again, obtains the gold nanorods aqueous solution (representing with CTAB-GNRs) that surface is covered by CTAB bilayer.
In step (a), compound 1 is the lactose of full acetyl group protection.
In step (a), the preparation method of compound 1 is:
Sodium acetate is added in acetic anhydride, be heated to 120-130 DEG C, slowly add lactose and obtain reactant liquor, the mol ratio of acetic anhydride, sodium acetate and lactose is 12:12:1, then temperature is reduced to 110 DEG C and continues reaction 5 hours, poured into by reactant liquor in frozen water and place 12 hours, by the solid filtering of separating out, the filter cake ethyl alcohol recrystallization obtained obtains compound 1.
In step (a), compound 2 is 2-chloroethoxy-2-ethoxy diethanol.
In step (a), compound 1 and compound 2 are dissolved in methylene chloride (CH
2cl
2) in, then add appropriate molecular sieve, stir and add catalyzer after 20 minutes, stirred at ambient temperature reacts.
In step (a), catalyzer is boron trifluoride diethyl etherate (BF
3-Et
2o), the mol ratio of catalyzer and compound 1 is 4:1-6:1.
In step (a), being operating as of purifying: after reaction terminates, filter out catalyzer, obtain filtrate, filtrate dchloromethane, after dilution, use saturated sodium bicarbonate solution and saturated common salt solution washing respectively, anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate obtains residue with Rotary Evaporators evaporate to dryness, and residue chromatographic column is separated, and obtains compound 3.
In step (a), compound 3 is 1-β-(chloro triethylene Glycol base)-2,3,6,2 ', 3 ', 4 ', 6 '-seven acetyl group lactose.
In step (b), compound 3 is first dissolved in DMF (DMF), then add thioacetic acid potassium (KSAc), stirred at ambient temperature reacts.
In step (b), the step of purifying is:
Reaction obtains reactant liquor, in reactant liquor, adds ethyl acetate after stopping, then water, saturated sodium bicarbonate solution and saturated common salt solution washing 2 times are used successively, anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, obtains compound 4.
Compound 4 is 1-β-(ethanethioyl triethylene Glycol base)-2,3,6,2 ', 3 ', 4 ', 6 '-seven acetyl group lactose.
In step (c), the sodium methoxide adopting sodium metal and methyl alcohol reaction to obtain sloughs the Acetyl Protecting Groups in compound 4.
Be dissolved in methyl alcohol by compound 4 and obtain mixed solution, the volumetric molar concentration of compound 4 in mixed solution is 0.4-0.6M, and the weight of sodium metal is the 4%-5% of compound 4 weight.
In step (c), being operating as of purifying:
After reaction terminates, obtain reactant liquor, add appropriate acidic cationic resin in reactant liquor, the pH of solution is adjusted to 7, then filters, filtrate uses Rotary Evaporators evaporate to dryness, and residue is dissolved in suitable quantity of water, and freeze-drying obtains the lactose containing sulfydryl.
Lactose containing sulfydryl is 1-β-(sulfydryl triethylene Glycol base) lactose.
Be 1 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1-3 × 10
4: 1.
The present invention is the obtained lactose containing sulfydryl
from the lactose of the full acetyl group protection of compound 1, to replace through end group successively, sulfhydrylation and deacetylation three-step reaction, introduce the linking arm containing sulfydryl at the terminal carbon place of lactose, prepare can and the lactose containing sulfydryl that reacts of gold nanorods.
Carry out ligand exchange reaction containing the lactose of sulfydryl and gold nanorods, by Au-S coordination, the lactose containing sulfydryl being loaded to gold nanorods surface, obtaining a kind of bioprobe for detecting tumor marker galectin-1 newly.
The preparation method for the bioprobe detecting tumor marker galectin-1 that second aspect present invention provides is simple, mild condition, and cost is lower, obtained bioprobe can realize galectin-1 efficiently and detect specifically.
Summary of the invention
For solving the problem, the invention provides a kind of bioprobe for detecting tumor marker half lactadherin-1 and preparation method thereof.Bioprobe of the present invention is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.Described bioprobe can realize double lactadherin-1 efficiently and detect specifically.The invention also discloses the preparation method of described bioprobe, preparation method is simple.
First aspect present invention provides a kind of for detecting the bioprobe of tumor marker half lactadherin-1 (galectin-1), and described bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Preferably, the described lactose containing sulfydryl is
Preferably, described is 1 × 10 containing the lactose of sulfydryl and the mol ratio of described gold nanorods
4: 1-3 × 10
4: 1.
Preferably, the length of described gold nanorods is 60-80nm, wide for 20-25nm.
Preferably, the peak position of the horizontal plasmon absorption peak (SPR) of described gold nanorods is 510nm, and the peak position of longitudinal plasmon absorption peak (SPR) is 700-800nm.
Preferably, described gold nanorods Surface coating has cetyl trimethyl ammonium bromide (CTAB) bilayer, and the described lactose containing sulfydryl is connected to form compound in the surperficial part or all of region not being coated with CTAB of described gold nanorods by gold-sulfide linkage and gold nanorods.
In the present invention, gold nanorods, due to the surface plasma resonance effect of its uniqueness, can absorb the incident light on its resonant frequency, causes the loss of luminous energy in this frequency, is detected optically device easily and detects.Gold nanorods has special space anisotropic simultaneously, its rod-like morphology makes it the surface plasma resonance peak with horizontal and vertical both direction, make it responsive especially to the response of trace materials, the present invention adopts gold nanorods accurately can detect galectin-1 as a part for bioprobe.
Galectin-1, due to its structure containing special beta galactose glycosidic bond identified region, therefore can carry out specific recognition to lactose.After the described lactose containing sulfydryl is connected with gold nanorods by gold-sulfide linkage by the present invention, carbohydrate Small molecular-lactose in load on gold nanorods, stable and specific modification can have been carried out to gold nanorods, in the described probe obtained, lactose fraction has the effect to galectin-1 specific recognition, add that gold nanorods has the effect of accurate optic response, final described bioprobe can realize to galectin-1 efficiently and detect specifically.
The bioprobe of the detection tumor marker galectin-1 that first aspect present invention provides, the lactose containing sulfydryl is adopted to modify gold nanorods first, the described lactose containing sulfydryl is connected by gold-sulfide linkage with described gold nanorods, binding ability is strong, finally construct a kind of newly can in order to detect the bioprobe of tumor marker galectin-1, described bioprobe can realize to galectin-1 efficiently and detect specifically.
Second aspect present invention provides a kind of preparation method of the bioprobe for detecting tumor marker galectin-1, comprises the following steps:
(1) gold nanorods is provided;
(2) provide lactose, described lactose is the obtained lactose containing sulfydryl after mercaptolation;
(3) described gold nanorods and the described lactose containing sulfydryl are mixed, left at room temperature 8-15 hour, obtain the bioprobe for detecting tumor marker galectin-1, described bioprobe is the described compound be connected to form by gold-sulfide linkage and described gold nanorods containing the lactose of sulfydryl.
Preferably, the described lactose containing sulfydryl is
Preferably, described is 1 × 10 containing the lactose of sulfydryl and the mol ratio of described gold nanorods
4: 1-3 × 10
4: 1.
More preferably, the described lactose containing sulfydryl
preparation method be:
A structural formula is by ()
compound 1 and structure be
compound 2 under the effect of catalyzer, stirred at ambient temperature reaction 36-72 hour, after purifying, obtained structural formula is
compound 3; The mol ratio of described compound 1 and described compound 2 is 1:1.2-1:2;
B () (chemical formula is by described compound 3 and thioacetic acid potassium
kSAc) at room temperature stirring reaction 3-5 hour, after purifying, obtained structural formula is
compound 4; The mol ratio of described compound 3 and described thioacetic acid potassium (KSAc) is 1:1.2-1:2;
C described compound 4 is dissolved in methyl alcohol by (), then add sodium metal, room temperature for overnight, and after purifying, obtained structural formula is
containing the lactose of sulfydryl.
The present invention is the obtained lactose containing sulfydryl
from the lactose of the full acetyl group protection of compound 1, successively through end group replacement, sulfhydrylation and deacetylation three-step reaction, introduce the linking arm containing sulfydryl at the terminal carbon place of lactose, prepare the lactose containing sulfydryl described in reacting with gold nanorods.
The preparation method of the bioprobe of the described detection tumor marker galectin-1 that second aspect present invention provides is simple, and reaction conditions is gentle, and cost is lower, obtained described bioprobe can realize galectin-1 efficiently and detect specifically.
To sum up, beneficial effect of the present invention comprises the following aspects:
The invention provides a kind of bioprobe for detecting tumor marker galectin-1, described bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.Described bioprobe can realize to galectin-1 efficiently and detect specifically.Present invention also offers the preparation method of described bioprobe, preparation method is simple, and reaction conditions is gentle, and cost is lower.
Accompanying drawing explanation
Fig. 1 is the synthesis schematic diagram of embodiment 1 gold nanorods;
Fig. 2 is absorption curve (A) and the transmission electron microscope picture (B) of the gold nanorods that embodiment 1 obtains;
Fig. 3 is that embodiment 1 is for detecting the synthesis schematic diagram of the bioprobe of tumor marker galectin-1;
Fig. 4 is the change of properties figure of gold nanorods before and after reacting with the lactose containing sulfydryl in embodiment 1; Element formation (representing with x-ray photoelectron power spectrum (XPS) figure) change that (A) is gold nanorods before and after reaction is schemed in Fig. 4; Figure (B) is the change of gold nanorods surface charge (Zeta potential) before and after reaction; Figure (C) is the light absorption change of the ultraviolet-visible light-near infrared region of gold nanorods before and after reaction; Figure (D) is the micromechanism (representing with transmission electron microscope picture) of gold nanorods after reaction;
Fig. 5 be the obtained bioprobe (representing with Lac-GNRs) of embodiment 1 to the Detection results figure of galectin-1, in Fig. 5, Fig. 5 (A) is for bioprobe absorption curve is with the variation relation of concentration; The reunion degree of gold nanorods in bioprobe when Fig. 5 (B) adds 1nMgalectin-1; The change of Fig. 5 (C) bioprobe solution colour;
Fig. 6 is the specific effect figure of embodiment 1 obtained bioprobe (representing with Lac-GNRs) when detecting galectin-1.
Embodiment
The following stated is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
First aspect present invention provides a kind of bioprobe for detecting tumor marker galectin-1, and bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Lactose containing sulfydryl is
Be 1 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1-3 × 10
4: 1.
The length of gold nanorods be 60-80nm, wide be 20-25nm.
The peak position of the horizontal plasmon absorption peak (SPR) of gold nanorods is 510nm, and the peak position of longitudinal plasmon absorption peak (SPR) is 700-800nm.
Gold nanorods Surface coating has cetyl trimethyl ammonium bromide (CTAB) bilayer, and the part or all of region that the lactose containing sulfydryl is not coated with CTAB on gold nanorods surface is connected to form compound by gold-sulfide linkage and gold nanorods.
Gold nanorods Surface coating has cetyl trimethyl ammonium bromide (CTAB) bilayer, containing the lactose of sulfenyl when reacting with gold nanorods, the part CTAB bilayer on gold nanorods surface is substituted for by ligand exchange reaction, gold nanorods surface in the bioprobe finally obtained is except being modified with lactose, subregion is also coated with CTAB bilayer, and the part or all of region not namely being coated with cetyl trimethyl ammonium bromide bilayer containing the lactose of sulfydryl on gold nanorods surface is connected to form compound by gold-sulfide linkage and gold nanorods.CTAB bilayer is protective agent and the stabilizing agent of gold nanorods, can avoid the reunion of gold nanorods.
Embodiment 1:
For detecting a preparation method for the bioprobe of tumor marker galectin-1, comprise the following steps:
(1) adopt Seed inducement legal system for gold nanorods:
By the HAuCl of 0.5mM
4be dissolved in the CTAB solution of 0.2M, vigorous stirring, add the cold NaHB of appropriate 10mM
4solution, stirs 2 hours to prepare gold nano seed solution at 37 DEG C.Then at the HAuCl of 5mM
40.2MCTAB, 0.1MAgNO is added in solution
3, 1.2MHCl and 10mM ascorbic acid (AA), when solution by dark red become colourless after, add the gold nano seed solution of above-mentioned preparation, hatch after 12 hours centrifugal at 37 DEG C, after removing supernatant, residue ultrapure water disperses again, obtains the gold nanorods aqueous solution (CTAB-GNRs) that surface is covered by CTAB.
(2) with compound 1 for raw material preparation is containing the lactose of sulfydryl, comprise the following steps:
Wherein the preparation method of compound 1 is:
Sodium acetate is added in acetic anhydride, be heated to 130 DEG C, slowly add lactose and obtain reactant liquor, the mol ratio of acetic anhydride, sodium acetate and lactose is 12:12:1, then temperature is reduced to 110 DEG C and continues reaction 5 hours, reactant liquor is poured in frozen water and place 12 hours, by the solid filtering of separating out, the filter cake ethyl alcohol recrystallization obtained obtains compound 1, and the structural formula of compound 1 is
(a) 1-β-(chloro triethylene Glycol base)-2; 3; 6; 2 '; 3 '; the preparation of 4 ', 6 '-seven acetyl group lactose (compound 3): the lactose (compound 1) protected by full acetyl group and chloro triethylene Glycol (compound 2) are dissolved in methylene chloride (CH
2cl
2), the mol ratio of compound 1 and compound 2 is 1:1.2, adds appropriate molecular sieve, stirs and adds boron trifluoride diethyl etherate (BF after 20 minutes
3-Et
2o), the mol ratio of boron trifluoride diethyl etherate and compound 1 is 4:1.Stirred at ambient temperature 72 hours.After reaction terminates, filter out boron trifluoride diethyl etherate, filtrate dchloromethane, dilution priority saturated sodium bicarbonate solution and saturated common salt solution washing, anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, remaining residue chromatographic column is separated, and obtains compound 3.
The nuclear magnetic data of compound 3 is as follows:
1hNMR (400MHz, CDCl
3, 25 DEG C): 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).
(b) 1-β-(ethanethioyl triethylene Glycol base)-2, 3, 6, 2 ', 3 ', 4 ', the preparation of 6 '-seven acetyl group lactose (compound 4): compound 3 is dissolved in N, dinethylformamide (DMF), add thioacetyl potassium (KSAc), stirred at ambient temperature 4 hours, the mol ratio of compound 3 and thioacetic acid potassium is 1:1.2, after reaction stops, ethyl acetate is added in reactant liquor, use water successively, saturated sodium bicarbonate solution and saturated common salt solution washing (each 2 times), anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, obtain compound 4.
The nuclear magnetic data of compound 4 is as follows:
1hNMR (400MHz, CDCl
3, 25 DEG C): 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).
13cNMR (400MHz, CDCl
3): 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.
The preparation of (c) 1-β-(sulfydryl triethylene Glycol base) lactose (lactose containing sulfydryl): compound 4 is dissolved in methyl alcohol, obtain mixed solution, in mixed solution, the volumetric molar concentration of compound 4 is 0.5M, in solution, add appropriate sodium metal, the weight of sodium metal is 5% of compound 4 weight.Room temperature for overnight.After reaction terminates, add appropriate acidic cationic resin, the pH of solution is adjusted to 7.Filter, filtrate uses Rotary Evaporators evaporate to dryness, and residue is dissolved in suitable quantity of water, and freeze-drying obtains the lactose containing sulfydryl.
Nuclear magnetic data containing the lactose of sulfydryl is as follows:
1hNMR (400MHz, MeOD, 25 DEG C): 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).
13cNMR (400MHz, MeOD, 25 DEG C): 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) aqueous solution of the lactose containing sulfydryl obtained for the step (2) of 20mM being joined in the obtained gold nanorods solution of step (1), is 1 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1, at room temperature leave standstill 15 hours, do not need to stir, stop reaction, obtain the bioprobe (representing with Lac-GNRs) for detecting tumor marker galectin-1, bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Fig. 1 is the synthesis schematic diagram of gold nanorods; As can be seen from Figure 1, gold chloride (HAuCl
4) by gold nano seed (representing with 1 in figure) obtained after sodium borohydride reduction, then gold nano seed is added have gold chloride (HAuCl
4), silver nitrate (AgNO
3), in the mixed aqueous solution of watery hydrochloric acid (HCl), ascorbic acid (AA), cetyl trimethyl ammonium bromide (CTAB), final obtained gold nanorods (representing with 3 in figure), this gold nanorods surface coverage has CTAB (representing with 2 in figure) bilayer.
Fig. 2 is absorption curve (A) and the transmission electron microscope picture (B) of the gold nanorods that embodiment 1 obtains; The optical absorption map of Fig. 2 (A) show the gold nanorods prepared have at 510nm and 770nm place respectively have respectively weak with strong absorption (minor axis of corresponding gold nanorods absorbs and major axis absorption respectively).The transmission electron microscope collection of illustrative plates (TEM) of Fig. 2 (B) shows that the width of gold nanorods is about 20nm, and length is about 70nm.Gold nanorods size uniformity, the dispersiveness of the present embodiment synthesis are better.
Fig. 3 is the synthesis schematic diagram of the bioprobe for detecting tumor marker galectin-1, as can be seen from Figure 3, surface coverage has the gold nanorods (representing with 5 in figure) of CTAB (representing with 4 in figure) bilayer and obtains the bioprobe (representing with 7 in figure) for detecting tumor marker galectin-1 after reacting containing the lactose (representing with 6 in figure) of sulfydryl, gold nanorods is connected by gold-sulfide linkage with the lactose containing sulfydryl, finally connect lactose fraction (representing with 8 in figure) on gold nanorods surface, containing the lactose of sulfenyl when reacting with gold nanorods, the part CTAB bilayer on gold nanorods surface is substituted for by ligand exchange reaction.
Fig. 4 is the change of properties figure of gold nanorods before and after reacting with the lactose containing sulfydryl in embodiment 1;
Element formation (representing with x-ray photoelectron power spectrum (XPS) figure) change that (A) is gold nanorods before and after reaction is schemed in Fig. 4; Figure (B) is the change of gold nanorods surface charge (Zeta potential) before and after reaction; Figure (C) is the light absorption change of the ultraviolet-visible light-near infrared region of gold nanorods before and after reaction; Figure (D) is the micromechanism (representing with transmission electron microscope picture) of gold nanorods after reaction.
As shown in Figure 4 A, XPS atlas analysis shows the compound that is connected to form by gold-sulfide linkage and gold nanorods containing the lactose of sulfydryl and bioprobe (representing with Lac-GNRs) than carbon (C) content of the gold nanorods (representing with CTAB-GNRs) before the Lactose-modified containing sulfydryl to be reduced, and the content of oxygen element (O) increases, show that the part CTAB on gold nanorods surface is replaced (additional oxygen is from lactose) by lactose; As shown in Figure 4 B, the surface charge of Zeta potential test display Lac-GNRs is lower than CTAB-GNRs, demonstrates part CTAB further and be instead of (CTAB is positively charged, and lactose neutral) by lactose; As shown in Figure 4 C, after the light absorption of ultraviolet-visible light-near infrared region shows the Lactose-modified containing sulfydryl, the horizontal absorption peak of gold nanorods does not change, and longitudinal Absorption Spectroscopy then there occurs a small amount of red shift; Illustrate that modification is containing after the lactose of sulfydryl on gold nanorods, the optical characteristics of gold nanorods does not change.Fig. 4 D then shows the micromechanism of Lac-GNRs.As can be seen from Fig. 4 D, in the bioprobe that the present embodiment is obtained, gold nanorods is not reunited.Above result shows gold nanorods successfully by the Lactose-modified containing sulfydryl, and lactose on finally connecting on gold nanorods, has successfully obtained the bioprobe for detecting tumor marker galectin-1.
Embodiment 2:
For detecting a preparation method for the bioprobe of tumor marker galectin-1, comprise the following steps:
(1) adopt Seed inducement legal system for gold nanorods:
By the HAuCl of 0.5mM
4be dissolved in the CTAB solution of 0.2M, vigorous stirring, add the cold NaHB of appropriate 10mM
4solution, stirs 2 hours to prepare gold nano seed solution at 37 DEG C.Then at the HAuCl of 5mM
40.2MCTAB, 0.1MAgNO is added in solution
3, 1.2MHCl and 10mM ascorbic acid, when solution by dark red become colourless after, add the gold nano seed solution of above-mentioned preparation, hatch after 12 hours centrifugal at 37 DEG C, after removing supernatant, residue ultrapure water disperses again, obtains the gold nanorods aqueous solution (CTAB-GNRs) that surface is covered by CTAB.
(2) with compound 1 for raw material preparation is containing the lactose of sulfydryl, comprise the following steps:
Wherein the preparation method of compound 1 is:
Sodium acetate is added in acetic anhydride, be heated to 130 DEG C, slowly add lactose and obtain reactant liquor, the mol ratio of acetic anhydride, sodium acetate and lactose is 12:12:1, then temperature is reduced to 110 DEG C and continues reaction 5 hours, poured into by reactant liquor in frozen water and place 12 hours, by the solid filtering of separating out, the filter cake ethyl alcohol recrystallization obtained obtains compound 1;
(a) 1-β-(chloro triethylene Glycol base)-2; 3; 6; 2 '; 3 '; the preparation of 4 ', 6 '-seven acetyl group lactose (compound 3): the lactose (compound 1) protected by full acetyl group and chloro triethylene Glycol (compound 2) are dissolved in methylene chloride (CH
2cl
2), the mol ratio of compound 1 and compound 2 is 1:2, adds appropriate molecular sieve, stirs and adds boron trifluoride diethyl etherate (BF after 20 minutes
3-Et
2o), the mol ratio of boron trifluoride diethyl etherate and compound 1 is 6:1.Stirred at ambient temperature 36 hours.After reaction terminates, filter out boron trifluoride diethyl etherate, filtrate dchloromethane, dilution priority saturated sodium bicarbonate solution and saturated common salt solution washing, anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, remaining residue chromatographic column is separated, and obtains compound 3.
(b) 1-β-(ethanethioyl triethylene Glycol base)-2, 3, 6, 2 ', 3 ', 4 ', the preparation of 6 '-seven acetyl group lactose (compound 4): compound 3 is dissolved in N, dinethylformamide (DMF), add thioacetyl potassium (KSAc), stirred at ambient temperature 3 hours, the mol ratio of compound 3 and thioacetic acid potassium is 1:2, after reaction stops, ethyl acetate is added in reactant liquor, use water successively, saturated sodium bicarbonate solution and saturated common salt solution washing (each 2 times), anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, obtain compound 4.
The preparation of (c) 1-β-(sulfydryl triethylene Glycol base) lactose (lactose containing sulfydryl): compound 4 is dissolved in methyl alcohol, obtain mixed solution, in mixed solution, the volumetric molar concentration of compound 4 is 0.4M, in solution, add appropriate sodium metal, the weight of sodium metal is 4% of compound 4 weight.Room temperature for overnight.After reaction terminates, add appropriate acidic cationic resin, the pH of solution is adjusted to 7.Filter, filtrate uses Rotary Evaporators evaporate to dryness, and residue is dissolved in suitable quantity of water, and freeze-drying obtains the lactose containing sulfydryl.
(3) aqueous solution of the lactose containing sulfydryl obtained for the step (2) of 20mM being joined in the obtained gold nanorods solution of step (1), is 3 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1, at room temperature leave standstill 8 hours, do not need to stir, stopping reaction, obtaining the bioprobe for detecting tumor marker galectin-1, bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Embodiment 3
For detecting a preparation method for the bioprobe of tumor marker galectin-1, comprise the following steps:
(1) adopt Seed inducement legal system for gold nanorods:
By the HAuCl of 0.5mM
4be dissolved in the CTAB solution of 0.2M, vigorous stirring, add the cold NaHB of appropriate 10mM
4solution, stirs 2 hours to prepare gold nano seed solution at 37 DEG C.Then at the HAuCl of 5mM
40.2MCTAB, 0.1MAgNO is added in solution
3, 1.2MHCl and 10mM ascorbic acid, when solution by dark red become colourless after, add the gold nano seed solution of above-mentioned preparation, hatch after 12 hours centrifugal at 37 DEG C, after removing supernatant, residue ultrapure water disperses again, obtains the gold nanorods aqueous solution (CTAB-GNRs) that surface is covered by CTAB.
(2) with compound 1 for raw material preparation is containing the lactose of sulfydryl, comprise the following steps:
Wherein the preparation method of compound 1 is:
Sodium acetate is added in acetic anhydride, be heated to 130 DEG C, slowly add lactose and obtain reactant liquor, the mol ratio of acetic anhydride, sodium acetate and lactose is 12:12:1, then temperature is reduced to 110 DEG C and continues reaction 5 hours, poured into by reactant liquor in frozen water and place 12 hours, by the solid filtering of separating out, the filter cake ethyl alcohol recrystallization obtained obtains compound 1;
(a) 1-β-(chloro triethylene Glycol base)-2; 3; 6; 2 '; 3 '; the preparation of 4 ', 6 '-seven acetyl group lactose (compound 3): the lactose (compound 1) protected by full acetyl group and chloro triethylene Glycol (compound 2) are dissolved in methylene chloride (CH
2cl
2), the mol ratio of compound 1 and compound 2 is 1:1.5, adds appropriate molecular sieve, stirs and adds boron trifluoride diethyl etherate (BF after 20 minutes
3-Et
2o), the mol ratio of boron trifluoride diethyl etherate and compound 1 is 5:1.Stirred at ambient temperature 48 hours.After reaction terminates, filter out boron trifluoride diethyl etherate, filtrate dchloromethane, dilution priority saturated sodium bicarbonate solution and saturated common salt solution washing, anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, remaining residue chromatographic column is separated, and obtains compound 3.
(b) 1-β-(ethanethioyl triethylene Glycol base)-2, 3, 6, 2 ', 3 ', 4 ', the preparation of 6 '-seven acetyl group lactose (compound 4): compound 3 is dissolved in N, dinethylformamide (DMF), add thioacetyl potassium (KSAc), stirred at ambient temperature 5 hours, the mol ratio of compound 3 and thioacetic acid potassium is 1:1.5, after reaction stops, ethyl acetate is added in reactant liquor, use water successively, saturated sodium bicarbonate solution and saturated common salt solution washing (each 2 times), anhydrous sodium sulfate drying, then removed by filtration sodium sulphate, filtrate uses Rotary Evaporators evaporate to dryness, obtain compound 4.
The preparation of (c) 1-β-(sulfydryl triethylene Glycol base) lactose (lactose containing sulfydryl): compound 4 is dissolved in methyl alcohol, obtain mixed solution, in mixed solution, the volumetric molar concentration of compound 4 is 0.6M, in solution, add appropriate sodium metal, the weight of sodium metal is 5% of compound 4 weight.Room temperature for overnight.After reaction terminates, add appropriate acidic cationic resin, the pH of solution is adjusted to 7.Filter, filtrate uses Rotary Evaporators evaporate to dryness, and residue is dissolved in suitable quantity of water, and freeze-drying obtains the lactose containing sulfydryl.
(3) aqueous solution of the lactose containing sulfydryl obtained for 20mM step (2) being joined in the obtained gold nanorods solution of step (1), is 2 × 10 containing the lactose of sulfydryl and the mol ratio of gold nanorods
4: 1, at room temperature leave standstill 10 hours, do not need to stir, stopping reaction, obtaining the bioprobe for detecting tumor marker galectin-1, bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
Application Example
Lac-GNRs is on a molecular scale to the testing result of galectin-1:
The present invention assesses the detection of galectin-1 on a molecular scale, find that Lac-GNRs (bioprobe prepared by such as embodiment 1) can detect the existence of galectin-1 extremely delicately, detection limit can reach 1pM (picomole often rises), and response changes along with the change of galectin-1 concentration.
Fig. 5 is the Detection results figure of Lac-GNRs to galectin-1, and in Fig. 5, Fig. 5 (A) is for bioprobe absorption curve is with the variation relation of concentration; The reunion degree of Lac-GNRs when Fig. 5 (B) is for adding 1nMgalectin-1; Fig. 5 (C) is for adding the change of galectin-1 artifact probe solution color; The combination of galectin-1 and lactose can induce gold nanorods to reunite, thus at ultraviolet-visible light-near infrared region (UV-Vis-NIS), the change of light absorption occurs.As can be seen from Fig. 5 (A), when adding the galectin-1 of 1nM (nanomole often rises) in system, the absorption peak at 770nm place sharply reduces, and at the new absorption peak of 1100nm place appearance one, show that gold nanorods is seriously reunited (as shown in Figure 5 B), now the color of Lac-GNR solution becomes purple (the right centrifuge tube in 5C) from orange (left side centrifuge tube in 5C); And when the concentration of galectin-1 reduce most 100pM time, the absorption peak at 770nm place also has obvious reduction, has also occurred a new peak at 900nm place, shows that the reunion of gold nanorods is also obvious.The concentration of Galectin-1 can drop to 1pM (Fig. 5 A) always, and now the peak of 770nm still has obvious reduction, shows that Lac-GNRs is responsive especially to the response of galectin-1, can realize the detection of the galectin-1 to extremely low concentration.
To other, Lac-GNRs, except responding galectin-1, can not identify that the albumen (as bovine serum albumin BSA) of lactose responds non-constant.Specific effect figure when Fig. 6 is Lac-GNRs detection galectin-1.As can be seen from Figure 6, the galectin-1 adding 1nM in system can induce the significant change of absorption peak, even and if add the much bigger BSA of concentration (15nM), or add the CBP-35 (galectin-3 akin with galectin-1 structure of same concentration, 1nM), all significantly can not change the absorption of gold nanorods, show that the Lac-GNRs's that the present invention obtains is fine to the detection specificity of galectin-1.
To sum up, the present invention has successfully prepared a kind of bioprobe for detecting tumor marker galectin-1, and this bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.This bioprobe can realize to galectin-1 efficiently and detect specifically.The preparation method of bioprobe of the present invention is simple, and reaction conditions is gentle, and cost is lower.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. for detecting a bioprobe for tumor marker half lactadherin-1, it is characterized in that, described bioprobe is the compound that the lactose containing sulfydryl is connected to form by gold-sulfide linkage and gold nanorods.
2. as claimed in claim 1 for detecting the bioprobe of tumor marker half lactadherin-1, it is characterized in that, the described lactose containing sulfydryl is
3. as claimed in claim 1 or 2 for detecting the bioprobe of tumor marker half lactadherin-1, it is characterized in that, described is 1 × 10 containing the lactose of sulfydryl and the mol ratio of described gold nanorods
4: 1-3 × 10
4: 1.
4. as claimed in claim 1 or 2 for detecting the bioprobe of tumor marker half lactadherin-1, it is characterized in that, the length of described gold nanorods be 60-80nm, wide be 20-25nm.
5. as claimed in claim 1 or 2 for detecting the bioprobe of tumor marker half lactadherin-1, it is characterized in that, the peak position at the horizontal plasmon absorption peak of described gold nanorods is 510nm, and the peak position at longitudinal plasmon absorption peak is 700-800nm.
6. as claimed in claim 1 or 2 for detecting the bioprobe of tumor marker half lactadherin-1, it is characterized in that, described gold nanorods Surface coating has cetyl trimethyl ammonium bromide bilayer, and the described lactose containing sulfydryl is connected to form compound in the surperficial part or all of region not being coated with cetyl trimethyl ammonium bromide bilayer of described gold nanorods by gold-sulfide linkage and described gold nanorods.
7., for detecting a preparation method for the bioprobe of tumor marker half lactadherin-1, it is characterized in that, comprise the following steps:
(1) gold nanorods is provided;
(2) provide lactose, described lactose is the obtained lactose containing sulfydryl after mercaptolation;
(3) described gold nanorods and the described lactose containing sulfydryl are mixed, left at room temperature 8-15 hour, obtain the bioprobe for detecting tumor marker half lactadherin-1, described bioprobe is the described compound be connected to form by gold-sulfide linkage and described gold nanorods containing the lactose of sulfydryl.
8. as claimed in claim 7 for detecting the preparation method of the bioprobe of tumor marker half lactadherin-1, it is characterized in that, the described lactose containing sulfydryl is
9. as claimed in claim 7 or 8 for detecting the preparation method of the bioprobe of tumor marker half lactadherin-1, it is characterized in that, described is 1 × 10 containing the lactose of sulfydryl and the mol ratio of described gold nanorods
4: 1-3 × 10
4: 1.
10. as claimed in claim 8 for detecting the preparation method of the bioprobe of tumor marker half lactadherin-1, it is characterized in that, the described lactose containing sulfydryl
preparation method be:
A structural formula is by ()
compound 1 and structure be
compound 2 under the effect of catalyzer, stirred at ambient temperature reaction 36-72 hour, after purifying, obtained structural formula is
compound 3; The mol ratio of described compound 1 and described compound 2 is 1:1.2-1:2;
B (), by described compound 3 and thioacetic acid potassium at room temperature stirring reaction 3-5 hour, after purifying, obtained structural formula is
compound 4; The mol ratio of described compound 3 and described thioacetic acid potassium is 1:1.2-1:2;
C described compound 4 is dissolved in methyl alcohol by (), then add sodium metal, room temperature for overnight, and after purifying, obtained structural formula is
containing the lactose of sulfydryl.
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| CN108709884A (en) * | 2018-05-24 | 2018-10-26 | 南京工业大学 | Method for preparing glycosylated nano-gold by one-step method and application thereof |
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