CN104502614B - A kind of based on gold nanoclusters analogue enztme kit and preparation method thereof and application - Google Patents
A kind of based on gold nanoclusters analogue enztme kit and preparation method thereof and application Download PDFInfo
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- CN104502614B CN104502614B CN201510037912.0A CN201510037912A CN104502614B CN 104502614 B CN104502614 B CN 104502614B CN 201510037912 A CN201510037912 A CN 201510037912A CN 104502614 B CN104502614 B CN 104502614B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
Abstract
The invention discloses a kind of based on gold nanoclusters analogue enztme kit and preparation method thereof and application. The present invention is based on gold nanoclusters analogue enztme kit and comprise following material: the T3 thyroid hormone antibody of 96 hole polystyrene microwell plates, T3 thyroid hormone antibody or breast cancer antibody, gold nanoclusters mark or the breast cancer antibody of gold nanoclusters mark, sample diluting liquid, PBS cushioning liquid, termination cushioning liquid, chromogenic substrate. Pass through the inventive method, can obtain the gold nano grain solution of different colours, the change color of this nano particle can regulate and control according to the concentration of hydrogen peroxide, reach the ultra trace of T3 thyroid hormone and breast cancer antigen has been detected by the variation of color and absorbance, and have very high sensitivity, the inventive method is simple, and cost is lower, highly sensitive, can realize fast and accurately and detecting.
Description
Technical field
The invention belongs to nano-biosensing and technical field of biological, be specifically related to a kind of based on JennerThe preparation of rice bunch analogue enztme kit and be applied to the fast detecting of T3 thyroid hormone and breast cancer antigenMethod.
Background technology
At present, breast cancer is one of modal malignant tumour of women, and the incidence of disease accounts for the various malignant tumours of whole body7-10%, become and threatened the Etiological of WomanHealth. Because cancer early symptom is not obvious, be difficult toFound early, and serious harm WomanHealth, thereby the early diagnosis and therapy of cancer is still urgently solutionOne of most important medical problem certainly. T3 thyroid hormone is to maintain body eubolism, promotes growthGrow necessary important hormone. Thyroid hormone is mainly to promote protein synthetic, particularly makes bone, boneThe protein such as bone flesh, liver are synthetic obviously to be increased, this to growth in petticoats, grow significant. AndThyroid hormone too much or very fewly all can directly be detrimental to health, impels the generation of disease.
Fluorescence intensity is high owing to having for gold nanoclusters, the ability of strong anti-light bleaching, good biological compatibility, lowAdvantages such as the fluorescent stability that toxicity is become reconciled and obtain extensive concern. It is certain that research shows that gold nanoclusters hasCatalytic capability, under the existence of gold nanoclusters, H2O2Energy Quick Oxidation 3,3', 5,5'-tetramethyl benzidine(TMB) make it become blue, based on this, we set up the new method that a kind of bimodulus detects, and both can utilize colorChange hydrogen peroxide is carried out to visual half-quantitative detection, the standard of size that again can be by absorbance to hydrogen peroxideReally content quantitatively detects. Equally, gold nano grain is because preparation is simple and convenient, character is stable, rawThe advantages such as thing compatibility is good are widely used in Analytical Chemistry in Life Science. Can be facilitated by reducing process by gold chloridePrepare the nanogold particle of various different-grain diameters, its color takes on a red color to blueness according to diameter. WithTime, gold nano grain presents different colors with the variation of diameter, and its ultraviolet absorption peak also can correspondingly occurChange. In the preparation of gold nano grain, hydrogen peroxide solution is one of important reactant, and gold nanoclustersHydrogen peroxide in energy catalytic gold nano particle preparation condition decomposes it, and the concentration of hydrogen peroxide changes afterwardsBecome, gold nano grain will present different colors, from red, extremely blueness of aubergine.
Summary of the invention
It is a kind of based on gold nanoclusters analogue enztme kit that one of object of the present invention is to provide, and it quick and preciselyDetection T3 thyroid hormone and breast cancer antigen, make gold nanoclusters successfully replace traditional enzyme linked immunologicalBiology enzyme in adsorption reaction, significantly improves sensitivity and the stability of detection.
The present invention is based on gold nanoclusters analogue enztme kit, it comprises following material: 96 hole polystyrene microporesThe T3 thyroid hormone antibody of plate, T3 thyroid hormone antibody or breast cancer antibody, gold nanoclusters markOr the breast cancer antibody of gold nanoclusters mark, sample diluting liquid, PBS cushioning liquid, stop cushioning liquid,Chromogenic substrate.
Concrete, the T3 thyroid hormone antibody of described gold nanoclusters mark is: use activator EDC andAfter NHS activates gold nanoclusters, T3 thyroid hormone antibody is joined to the gold nano after activationIn bunch solution, react 24 hours gained of dialysis treatment 2 hours; The breast cancer of described gold nanoclusters markAntibody is: after use activator EDC and NHS activate gold nanoclusters, breast cancer antibody is addedEnter to activation after gold nanoclusters solution in, react 24 hours gained of dialysis treatment 2 hours.
Concrete, described sample diluting liquid is: first T3 thyroid hormone solution entered with NaOH solutionAfter row dilution, then use the PBS cushioning liquid that pH=7.2-7.4, concentration are 0.01mol/L to be diluted to denseDegree is 2 × 10-12To 2 × 10-16The solution of mg/mL; Or: by pH=7.2-7.4 for breast cancer antigen,Concentration is that to be diluted to concentration be 7.52 × 10 to the PBS cushioning liquid of 0.01mol/L-9To 7.52 × 10-14U/mLSolution.
Concrete, described chromogenic substrate be citric acid three sodium solution taking concentration as 4mmol/L, concentration asThe hydrogen peroxide solution of 320 μ mol/L and concentration are the prepared gold nano of the chlorauric acid solution of 2mmol/LGrain solution, the volume ratio of three kinds of solution is 1:1:1.
Concrete, described termination cushioning liquid is that concentration is the BSA solution of 1mg/mL.
Two of object of the present invention is to provide the above-mentioned preparation method based on gold nanoclusters analogue enztme kit,It comprises the steps:
(1) T3 thyroid hormone antibody or breast cancer antibody are received on 96 hole polystyrene microwell plates,At 4 DEG C, reaction is spent the night, and after three times PBS cushioning liquid rinses, adding termination cushioning liquid is that concentration is 1The BSA solution reaction of mg/mL 1 hour;
After (2) three PBS cushioning liquid rinses, then in different microwell plates, add the sample of various concentrationDilution, being diluted to concentration is 2 × 10-12To 2 × 10-16The T3 thyroid hormone solution of mg/mL orBeing diluted to concentration is 7.52 × 10-9To 7.52 × 10-14The breast cancer antigen solution of U/mL, reacts 3 hours;
After (3) three times PBS cushioning liquid rinses, the more corresponding T3 thyroid gland that adds gold nanoclusters markThe breast cancer antibody of hormone antibody or gold nanoclusters mark, reacts 3 hours;
(4) rinse three times with PBS cushioning liquid again, then add citric acid three sodium solution and hydrogen peroxideSolution, reacted after 40 minutes, added chlorauric acid solution; After 30 minutes, observe solution in different microwell platesThe variation of color, with the chromogenic substrate gold nano grain solution generating in contrast experiment be that trisodium citrate is moltenThe color of the prepared gold nano grain solution of liquid, hydrogen peroxide solution and chlorauric acid solution contrasts; And useUltraviolet specrophotometer is surveyed the variation of its spectrogram in the scope of wavelength 450nm-700nm, and record is differentThe sample diluting liquid of concentration is at the absorbance at wavelength 550nm place, at the bottom of the colour developing generating in check experimentThe absorbance of thing gold nano grain solution contrasts, and can obtain testing result, realizes T3 thyroid glandThe fast detecting of hormone and breast cancer antigen.
Concrete, the T3 thyroid hormone antibody of the described gold nanoclusters mark of step (3) is: use activationAfter agent EDC and NHS activate gold nanoclusters, T3 thyroid hormone antibody is joined to activationAfter gold nanoclusters solution in, react 24 hours gained of dialysis treatment 2 hours; The described gold of step (3)The breast cancer antibody of nano-cluster mark is: use activator EDC and NHS to activate it to gold nanoclustersAfter, breast cancer antibody is joined in the gold nanoclusters solution after activation, react dialysis treatment 24 2 hoursHour gained.
Concrete, in step, described in (4), add citric acid three sodium solution, hydrogen peroxide solution and gold chlorideIn solution, citric acid three sodium solution concentration is 4mmol/L, and hydrogen peroxide solution concentration is 320 μ mol/L, chlorineAuric acid solution concentration is 2mmol/L, and the volume ratio of three kinds of solution is 1:1:1; Described in step (4)The chromogenic substrate gold nano grain solution generating in contrast experiment is: the citric acid three taking concentration as 4mmol/LThe chlorauric acid solution institute that the hydrogen peroxide solution that sodium solution, concentration are 320 μ mol/L and concentration are 2mmol/LThe gold nano grain solution of preparation, the volume ratio of three kinds of solution is 1:1:1.
Three of object of the present invention is to provide the above-mentioned preparation method based on gold nanoclusters analogue enztme kitApplication, be applied to the breast in T3 thyroid hormone content detection or the human serum in human serumGland cancer antigenic content detects.
Concrete, be 2 × 10 to the detection limit value of T3 thyroid hormone content-15Mg/mL; Anti-to breast cancerFormer detection limit value is 7.52 × 10-13U/mL。
The characteristic that the present invention utilizes gold nanoclusters catalysis and decomposes hydrogen peroxide, gold nanoclusters and gold nano grainThese two kinds of nano materials organically combine, and utilize the life in gold nanoclusters substituted enzyme connection imnnoadsorptionThing enzyme, adds the specific binding of antibody and antigen, has formed the sandwich of " sandwich ", successPrepare the analogue enztme kit that detects T3 thyroid hormone and breast cancer antigen, and phenomenon is obvious, behaviourDo easy, highly sensitive, can determine effectively accurately T3 thyroid hormone in human serum content andThe concentration of breast cancer antigen.
By method processing of the present invention, can obtain the gold nano grain solution of different colours, this nanometerThe change color of grain can regulate and control according to the concentration of hydrogen peroxide, reaches by the variation of color and absorbanceArrive the ultra trace of T3 thyroid hormone and breast cancer antigen has been detected, and had very high sensitiveDegree, compared with prior art, the inventive method is simple, and cost is lower, highly sensitive, can realize fastDetect accurately, and all there is very important reference for the detection of other large biological molecule and diseaseBe worth, there is very wide application prospect.
Brief description of the drawings
Fig. 1 is the gold nano grain under the hydrogen peroxide condition of variable concentrations prepared of the embodiment of the present invention 1Ultraviolet spectrogram. Wherein, along with the increase of hydrogen peroxide concentration, it is large that the absorbance of gold nano grain becomes gradually.
Fig. 2 is that the gold nano grain under the hydrogen peroxide condition of variable concentrations prepared by the embodiment of the present invention 1 existsThe absorbance change curve at 550nm place. In the time that the concentration of hydrogen peroxide is less than 320 μ mol/L, absorbanceValue raises gradually along with the increase of hydrogen peroxide concentration, but in the time that the concentration of hydrogen peroxide is greater than 320 μ mol/L,No longer there is obvious variation in its absorbance. Therefore when the concentration that we choose hydrogen peroxide is 320 μ mol/LOptimum detection concentration when preparing kit.
Fig. 3 is the gold nano grain under the hydrogen peroxide condition of variable concentrations prepared of the embodiment of the present invention 1Change color is to photograph and picture. In the time that the concentration of hydrogen peroxide is less than 320 μ mol/L, the gold nano grain of preparation isBlueness, there is reunion in gold nanoclusters particle now, but be greater than 320 μ mol/L when the concentration of hydrogen peroxideTime, the gold nano grain of preparation is red, now gained is finely dispersed gold nano grain solution; Fig. 3In, from left to right, the color of 4 solution in the left side gradually becomes blue from colourless, after the 5th, becomesRedness, and color is deepened gradually.
Fig. 4 be the concentration at hydrogen peroxide prepared by the embodiment of the present invention 1 prepare while being greater than 320 μ mol/L redThe transmission electron microscope picture of the gold nano grain of look solution. As can be seen from Figure 4, the gold nano grain of preparation dividesEvenly loose.
Fig. 5 is the indigo plant that the concentration at hydrogen peroxide prepared by the embodiment of the present invention 1 is prepared while being less than 320 μ mol/LThe transmission electron microscope picture of the gold nano grain of look solution. As can be seen from Figure 5, the gold nano grain of preparationThere is obvious agglomeration.
Fig. 6 is the chromogenic substrate during to the detection of T3 thyroid hormone standard liquid in the embodiment of the present invention 4And contrast experiment's chromogenic substrate is at the absorbance block diagram at 550nm place. In Fig. 6, every group of columnMiddle left side black histogram graph representation contrast experiment's chromogenic substrate is in the absorbance size at 550nm place.
Fig. 7 is the chromogenic substrate during to the detection of T3 thyroid hormone standard liquid in the embodiment of the present invention 4And the change color of contrast experiment's chromogenic substrate is to photograph and picture. In Fig. 7, a upper row's solution is real for contrastingTest chromogenic substrate.
Fig. 8 is the chromogenic substrate during to the detection of T3 thyroid hormone standard liquid in the embodiment of the present invention 4And contrast experiment's chromogenic substrate is at the canonical plotting of 550nm place absorbance variation.
Fig. 9 will be applied to 10 differences based on gold nanoclusters analogue enztme kit in the embodiment of the present invention 5In human serum, the color of chromogenic substrate and contrast experiment's chromogenic substrate becomes when T3 thyroid hormone content detectionChange photograph and picture. In Fig. 9, a upper row's solution is contrast experiment's chromogenic substrate.
Figure 10 be chromogenic substrate when breast cancer antigen standard liquid is detected in the embodiment of the present invention 6 andContrast experiment's chromogenic substrate is at the absorbance block diagram at 550nm place. In Figure 10, left in every group of columnLimit black histogram graph representation contrast experiment's chromogenic substrate is in the absorbance size at 550nm place.
Figure 11 be in the embodiment of the present invention 6 chromogenic substrate during to the detection of breast cancer antigen standard liquid withAnd the change color of contrast experiment's chromogenic substrate is to photograph and picture. In Figure 11, a upper row's solution is real for contrastingTest chromogenic substrate.
Figure 12 be in the embodiment of the present invention 6 chromogenic substrate during to the detection of breast cancer antigen standard liquid withAnd contrast experiment's chromogenic substrate is at the canonical plotting of 550nm place absorbance variation.
Figure 13 will be applied to 5 differences based on gold nanoclusters analogue enztme kit in the embodiment of the present invention 7The change color of chromogenic substrate and contrast experiment's chromogenic substrate when breast cancer antigen content detection in human serumTo photograph and picture. A upper row's solution is contrast experiment's chromogenic substrate.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail.
Embodiment 1:
Get 9 centrifuge tubes, add wherein respectively 100 μ L citric acid three sodium solutions as stabilizing agent, afterwardsAdd respectively 100 μ L variable concentrations hydrogen peroxide solution (concentration is 120,160,200,240,280,320,360,400,440 μ mol/L), 100 μ L chlorauric acid solutions (concentration is 2mmol/L). AddThe volume ratio of citric acid three sodium solution, chlorauric acid solution, hydrogen peroxide solution is 1:1:1. Be placed under room temperatureReaction, after 15 minutes, observes solution colour and changes, and to be now gold nano grain molten for obtained solutionLiquid, prepared gold nano grain has obvious absworption peak in wavelength 550nm left and right. Use ultraviolet spectrometry lightDegree meter detects the variation of its ultraviolet spectrogram in the scope of wavelength 450nm-700nm, and is recorded in differenceThe absorbance at 550nm wavelength place under the condition of the hydrogen peroxide of concentration. Fig. 1, Fig. 2, Fig. 3, Fig. 4,Fig. 5 is the experimental features figure of the gold nano grain under variable concentrations prepared of the present embodiment.
From figure, can draw, when the concentration of hydrogen peroxide is less than 320 μ mol/L, along with the increase of hydrogen peroxide concentration,The absorbance of the gold nano grain generating increases gradually, and now solution is blue, has occurred significantlyReunite, when the increase along with hydrogen peroxide concentration during to 320 μ mol/L of its concentration, its absorbance no longer occursSignificantly change, now solution is red, and gained solution is finely dispersed gold nano grain.
Embodiment 2:
Get 5 centrifuge tubes, add wherein respectively concentration be 10mmol/L, 8mmol/L, 4mmol/L,The citric acid three sodium solution of 2mmol/L, 1mmol/L, adding afterwards concentration is the two of 320 μ mol/L againThe oxygen aqueous solution and concentration are the chlorauric acid solution of 2mmol/L. The citric acid three sodium solution, the gold chloride that addThe volume ratio of solution, hydrogen peroxide solution is 1:1:1. After 15 minutes, observe solution colour and change. WithThe variation of citric acid three sodium solution concentration, the gold nano grain color of preparation is not identical yet. When citric acid threeWhen the concentration of sodium solution is 2mmol/L, solution is colourless, and during for 4mmol/L, solution is red, because ofThis can say, in the time that the concentration of trisodium citrate is 4mmol/L, be optium concentration.
Embodiment 3:
Get 5 centrifuge tubes, add wherein respectively concentration be 0.004mol/L citric acid three sodium solution andConcentration is the hydrogen peroxide solution of 320 μ mol/L, add wherein respectively more afterwards concentration be 1mmol/L,The chlorauric acid solution of 1.5mmol/L, 2mmol/L, 2.5mmol/L, 3.0mmol/L. The lemon addingThe volume ratio of acid three sodium solutions, chlorauric acid solution, hydrogen peroxide solution is 1:1:1. After 15 minutes, seeExamining solution colour changes. In the time that the concentration of chlorauric acid solution is less than 2mmol/L, the color of solution is purplish redLook, in the time that the concentration of chlorauric acid solution is greater than 2mmol/L, solution becomes blueness, therefore, works as gold chlorideWhen the concentration of solution is 2mmol/L, it is optium concentration.
Embodiment 4:
Referring to Fig. 6, Fig. 7, Fig. 8, for the detection of T3 thyroid hormone. First at 96 hole polystyrenesIn microwell plate, add T3 thyroid hormone antibody, after 4 DEG C of reactions are spent the night, with PBS cushioning liquid flushing three times,Add BSA solution reaction after 1 hour, use PBS cushioning liquid to rinse three times, then add the T3 of variable concentrationsThyroid hormone (2 × 10-12～2×10-16Mg/mL) reaction 3 hours, PBS cushioning liquid rinses three againAfter inferior, add the T3 thyroid hormone antibody response 3 hours that connects gold nanoclusters, use equallyPBS cushioning liquid rinses three times, adds citric acid three sodium solution and 100 μ L that 100 μ L concentration are 4mMConcentration is the hydrogen peroxide solution reaction 40 minutes of 320 μ mol/L, then to add 100 μ L concentration be 2mmol/LChlorauric acid solution. After 30 minutes, its UV absorption spectrogram and change color contrast figure are as Fig. 6, Fig. 7Shown in. In the time that the concentration of T3 thyroid hormone is 0, in microwell plate, the color of gold nano grain is red,Now in microwell plate, do not form the sandwich structure of sandwich, do not connect the gold that has catalytic actionNano-cluster decomposes hydrogen peroxide, has generated red solution, and at the T3 of variable concentrations thyroid hormoneCondition under, due to the specific binding effect of T3 thyroid hormone antibody and antigen, shape in microwell plateBecome sandwich structure, in the time generating gold nano grain, gold nanoclusters has played to hydrogen peroxide solution the work decomposingWith, therefore form blue solution. When the concentration of T3 thyroid hormone is 2 × 10-16When mg/mL, nowThe concentration of T3 thyroid hormone is very little, and the amount of the gold nanoclusters being connected is little, Wu FafenSeparate hydrogen peroxide, reached the detectability to T3 thyroid hormone, its detection is limited to 2 × 10-15mg/mL。
Embodiment 5:
For the detection of human serum T3 thyroid hormone content. First in 96 hole polystyrene microwell platesAdd T3 thyroid hormone antibody, after 4 DEG C of reactions are spent the night, with PBS cushioning liquid flushing three times, addAfter BSA solution reaction 1 hour, rinse and add again dilution 10 for three times with PBS cushioning liquid6Human serum doubly.React 3 hours, PBS cushioning liquid rinses after three times again, adds and connects gold nanoclustersT3 thyroid hormone antibody response 3 hours, rinses three times with PBS cushioning liquid equally, adds 100 μ LConcentration is that the citric acid three sodium solution of 4mM and 100 μ L concentration are that the hydrogen peroxide solution of 320 μ mol/L is anti-Answer 40 minutes, then add the chlorauric acid solution that 100 μ L concentration are 2mmol/L. After 30 minutes, itsChange color as shown in Figure 9, in the time that the concentration of T3 thyroid hormone is 0, gold nano grain in microwell plateColor be red, and in microwell plate adding different people serum after, solution colour changes difference,T3 thyroid hormone content difference in different people serum. Detect by experiment 10 different human bloods that obtainIn clear, the content of thyroid hormone is 3.06 × 10-7mg/mL、7.47×10-6mg/mL、1.8×10-7mg/mL、1.47×10-6mg/mL、2.32×10-6mg/mL、1.59×10-6mg/mL、5.12×10-7mg/mL、4.87×10-7mg/mL、2.55×10-7mg/mL、7.44×10-7mg/mL。
Embodiment 6:
Referring to Figure 10, Figure 11, Figure 12, for the detection of breast cancer antigen. First at 96 hole polystyrenesIn microwell plate, add breast cancer antibody, after 4 DEG C of reactions are spent the night, with PBS cushioning liquid flushing three times, addAfter BSA solution reaction 1 hour, rinse and add again for three times the breast cancer of variable concentrations anti-with PBS cushioning liquidFormer (7.52 × 10-9—7.52×10-14U/mL) reaction 3 hours, PBS cushioning liquid flushing three is taken second place againAfter, add the breast cancer antibody response 3 hours that connects gold nanoclusters, molten by PBS buffering equallyLiquid rinses three times, adds citric acid three sodium solution and the 100 μ L concentration that 100 μ L concentration are 4mM to beThe hydrogen peroxide solution reaction of 320 μ mol/L 40 minutes, then to add 100 μ L concentration be 2mmol/LChlorauric acid solution. After 30 minutes, its uv absorption spectra and change color contrast as Figure 10, Figure 11Shown in, in the time that the concentration of breast cancer antigen is 0, in microwell plate, the color of gold nano grain is red, nowIn microwell plate, do not form the sandwich structure of sandwich, do not connect the gold nano that has catalytic actionBunch hydrogen peroxide is decomposed, generated red solution, and under the condition of the breast cancer antigen of variable concentrations,Due to the specific binding effect of breast cancer antibody and antigen, in microwell plate, form sandwich structure, giving birth toWhile becoming gold nano grain, gold nanoclusters has played the effect of decomposing to hydrogen peroxide solution, has therefore formed bluenessSolution. When the concentration of breast cancer antigen is 7.52 × 10-14When U/mL, now the concentration of breast cancer antigenVery little, the amount of the gold nanoclusters being connected is little, cannot decompose hydrogen peroxide, has reached breast cancerThe detectability of antigen, its detection is limited to 7.52 × 10-13U/mL。
Embodiment 7:
For the detection of human serum breast cancer antigenic content. First at 5 96 hole polystyrene micropore plate holesThe middle breast cancer antibody that adds respectively, after 4 DEG C of reactions are spent the night, with PBS cushioning liquid flushing three times, adds BSAAfter solution reaction 1 hour, rinse and add again dilution 10 for three times with PBS cushioning liquid10Human serum doubly.React 3 hours, PBS cushioning liquid rinses after three times again, adds and connects gold nanoclustersBreast cancer antibody response 3 hours, rinses three times with PBS cushioning liquid equally, adds 100 μ L concentration to beThe citric acid three sodium solution of 4mM and 100 μ L concentration are the hydrogen peroxide solution reaction 40 of 320 μ mol/LMinute, then add the chlorauric acid solution that 100 μ L concentration are 2mmol/L. After 30 minutes, its color becomesChange as shown in figure 13, in the time that the concentration of breast cancer antigen is 0, in microwell plate, the color of gold nano grain isRedness, and in microwell plate after adding different people serum, it is different that solution colour changes, i.e. different human bloodsBreast cancer antigen content difference in clear. Detecting by experiment breast cancer in 5 different people serum that obtain resistsFormer content is 2.7 × 10-10U/Ml、1.08×10-10U/mL、3.79×10-10U/mL、 3.24×10-10U/mL、5.19×10-10U/mL。
Claims (4)
1. based on a gold nanoclusters analogue enztme kit, it is characterized in that comprising following material: 96 holes are poly-The T3 first shape of styrene microwell plate, T3 thyroid hormone antibody or breast cancer antibody, gold nanoclusters markThe breast cancer antibody of glandular hormone antibody or gold nanoclusters mark, sample diluting liquid, PBS cushioning liquid, endOnly cushioning liquid, chromogenic substrate;
The T3 thyroid hormone antibody of described gold nanoclusters mark is: use activator EDC and NHS coupleAfter gold nanoclusters activates, T3 thyroid hormone antibody is joined to the gold nanoclusters solution after activationIn, react 24 hours gained of dialysis treatment 2 hours; The breast cancer antibody of described gold nanoclusters mark is:After use activator EDC and NHS activate gold nanoclusters, breast cancer antibody is joined to activationAfter gold nanoclusters solution in, react 24 hours gained of dialysis treatment 2 hours;
Described sample diluting liquid is: first T3 thyroid hormone solution diluted to it with NaOH solutionAfter, then to use PBS cushioning liquid that pH=7.2-7.4, concentration are 0.01mol/L to be diluted to concentration be 2 × 10-12To 2 × 10-16The solution of mg/mL; Or: by pH=7.2-7.4, concentration for breast cancer antigen beIt is 7.52 × 10 that the PBS cushioning liquid of 0.01mol/L is diluted to concentration-9To 7.52 × 10-14The solution of U/mL;
Described chromogenic substrate is that citric acid three sodium solution, the concentration taking concentration as 4mmol/L is 320 μ mol/LHydrogen peroxide solution and concentration be 2mmol/L the prepared gold nano grain solution of chlorauric acid solution, threeThe volume ratio of planting solution is 1:1:1;
Described termination cushioning liquid is that concentration is the BSA solution of 1mg/mL.
2. the preparation method based on gold nanoclusters analogue enztme kit as claimed in claim 1, its spyLevy and be to comprise the steps:
(1) T3 thyroid hormone antibody or breast cancer antibody are received on 96 hole polystyrene microwell plates,At 4 DEG C, reaction is spent the night, and after three times PBS cushioning liquid rinses, adding termination cushioning liquid is that concentration is 1The BSA solution reaction of mg/mL 1 hour;
After (2) three PBS cushioning liquid rinses, then in different microwell plates, add the sample of various concentrationDilution, being diluted to concentration is 2 × 10-12To 2 × 10-16The T3 thyroid hormone solution of mg/mL orBeing diluted to concentration is 7.52 × 10-9To 7.52 × 10-14The breast cancer antigen solution of U/mL, reacts 3 hours;
After (3) three times PBS cushioning liquid rinses, the more corresponding T3 thyroid gland that adds gold nanoclusters markThe breast cancer antibody of hormone antibody or gold nanoclusters mark, reacts 3 hours;
(4) rinse three times with PBS cushioning liquid again, then add citric acid three sodium solution and hydrogen peroxideSolution, reacted after 40 minutes, added chlorauric acid solution; After 30 minutes, observe solution in different microwell platesThe variation of color, with the chromogenic substrate gold nano grain solution generating in contrast experiment be that trisodium citrate is moltenThe color of the prepared gold nano grain solution of liquid, hydrogen peroxide solution and chlorauric acid solution contrasts; And useUltraviolet specrophotometer is surveyed the variation of its spectrogram in the scope of wavelength 450nm-700nm, and record is differentThe sample diluting liquid of concentration is at the absorbance at wavelength 550nm place, at the bottom of the colour developing generating in contrast experimentThe absorbance of thing gold nano grain solution contrasts, and can obtain testing result, realizes T3 thyroid glandThe fast detecting of hormone and breast cancer antigen.
3. the preparation method based on gold nanoclusters analogue enztme kit according to claim 2, its featureBe: the T3 thyroid hormone antibody of the described gold nanoclusters mark of step (3) is: use activator EDCAfter gold nanoclusters being activated with NHS, T3 thyroid hormone antibody is joined to the Jenner after activationIn rice bunch solution, react 24 hours gained of dialysis treatment 2 hours; The described gold nanoclusters mark of step (3)The breast cancer antibody of note is: after use activator EDC and NHS activate gold nanoclusters, by breastGland cancer antibody joins in the gold nanoclusters solution after activation, reacts 24 hours institutes of dialysis treatment 2 hours.
4. the preparation method based on gold nanoclusters analogue enztme kit according to claim 2, its featureBe: described in step (4), add in citric acid three sodium solution, hydrogen peroxide solution and chlorauric acid solution,Citric acid three sodium solution concentration is 4mmol/L, and hydrogen peroxide solution concentration is 320 μ mol/L, chlorauric acid solutionConcentration is 2mmol/L, and the volume ratio of three kinds of solution is 1:1:1; Contrast experiment described in step (4)The chromogenic substrate gold nano grain solution of middle generation is: citric acid three sodium solution taking concentration as 4mmol/L,Concentration is the hydrogen peroxide solution of 320 μ mol/L and the prepared gold of chlorauric acid solution that concentration is 2mmol/LNanoparticles solution, the volume ratio of three kinds of solution is 1:1:1.
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| CN105798324B (en) * | 2016-03-21 | 2017-11-10 | 中山大学 | One kind is based on self-assembled structures analogue enztme and preparation method and application |
| CN107525930B (en) * | 2017-08-23 | 2019-02-15 | 湖南科技大学 | Detect the preparation method and application of staphylococcus aureus and enterotoxin B kit |
| CN107748257A (en) * | 2017-10-25 | 2018-03-02 | 湖南科技大学 | Preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box |
| CN108008123B (en) * | 2017-11-23 | 2020-06-30 | 国家纳米科学中心 | Full-plasmon enzyme-linked immunosorbent assay kit and application thereof |
| CN108031859A (en) * | 2017-11-29 | 2018-05-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for synthesizing gold nanoclusters and products thereof and application |
| CN109358195B (en) * | 2018-11-06 | 2022-04-26 | 枣庄学院 | Hollow gold platinum alloy nanocage core-porous silica shell nanoenzyme and preparation method and application thereof |
| CN110508828B (en) * | 2019-08-28 | 2022-05-03 | 淮北师范大学 | Preparation method of orange-red fluorescent copper nanocluster based on L-methionine |
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| CN101706504A (en) * | 2009-11-27 | 2010-05-12 | 东南大学 | Method for applying gold nanoparticles mimetic enzyme in biological detection |
| CN103267850A (en) * | 2013-04-27 | 2013-08-28 | 深圳先进技术研究院 | Probe for pathological diagnosis of tumor, and preparation method and application thereof |
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