CN109507409A - A kind of test strips and its preparation method and application based on the amplification of molybdenum disulfide composite nano materials signal - Google Patents
A kind of test strips and its preparation method and application based on the amplification of molybdenum disulfide composite nano materials signal Download PDFInfo
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- CN109507409A CN109507409A CN201811368699.1A CN201811368699A CN109507409A CN 109507409 A CN109507409 A CN 109507409A CN 201811368699 A CN201811368699 A CN 201811368699A CN 109507409 A CN109507409 A CN 109507409A
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- G—PHYSICS
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- 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
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Abstract
The invention belongs to reagent detection fields, and in particular to a kind of test strips and its preparation method and application based on the amplification of molybdenum disulfide composite nano materials signal.The test strips are with MoS2@Au nanocomposite is marker material.The test strips include coated antibody or aptamers, immunological probe, developing solution and cellulose nitrate film；The immunological probe is by MoS2@Au nanocomposite and antibody or aptamers coupling are made.The present invention utilizes MoS2The catalysis of nano material itself and light thermal property are prepared for uniform, stable novel test paper marker material MoS2@Au nanocomposite；Using it as marker material, on the one hand there is very strong suction-operated to antibody or aptamers due to nanogold, considerably increase marker material to the adsorbance and absorption stability of antibody or aptamers, be conducive to the detection sensitivity and specificity that improve object.
Description
Technical field
The invention belongs to reagent detection fields, and in particular to it is a kind of based on molybdenum disulfide composite nano materials signal amplification
Test strips and its preparation method and application.
Background technique
The rapid detection method for establishing hazardous material in a kind of easy, sensitive food is the effective way to ensure food safety
Diameter.It is easy, quickly and cost is relatively low is established at present for endangering in food due to immunity test strip rapid detection method
Most wide one of the method for application range in the rapid detection method of evil object.Test strips method can be used range estimation and carry out qualitative determination,
It can be quickly obtained testing result, but sensitivity is to be improved.Therefore property (color and the photo-thermal of test strips marker material are utilized
Deng) realize that the sensitive of target determinand, half-quantitative detection become the research hotspot of researcher.
The marker material of test strips mainly has following three classes at present, and all has respective advantage and disadvantage: enzymatic reaction material,
The catalyzed coloration judgment experiment result of material can be passed through；Dye marker material (colloidal gold, latex microsphere etc.), passes through coloring material
The power of colour developing has indefiniteness or sxemiquantitative judgment experiment result；Fluorescence or magnetic Nano material (quantum dot, ultra paramagnetic particle
Deng), the quantitative analysis of result can be realized by instrument.Wherein enzymatic reaction is limited by cost and biological enzyme stability of molecule
That applies is less.Colloidal gold strip is current most widely used fast inspection technology, but its sensitivity, stability and repeatability
Raising is the hot spot studied at present.Fluorescence or magnetic Nano material are expensive, and the water solubility and biocompatibility of material need
It improves and detection needs to limit the development and application of such Lateral Flow Strip by large-scale instrument etc..Therefore synthesis is above
The advantage of marker material, searching is cheap, and water-soluble and biocompatibility is good, and there is color and catalytic performance can pass through letter
Just the marker material that instrument improves detection sensitivity becomes research tendency.
Summary of the invention
To solve the problems, such as that above-mentioned detection exists, the present invention provides one kind to be based on molybdenum disulfide composite nano materials signal
Test strips of amplification and its preparation method and application.Rapid detection test strip of the present invention is with molybdenum disulfide-nanogold
(MoS2@Au) nanocomposite is marker material, overcome the single molybdenum disulfide connection technology that amount of antibody is small, stability is poor
Problem, and efficiently use MoS2The excellent catalysis of@Au nanocomposite and photothermal conversion performance are realized to target determinand
Quickly, Sensitive Detection.
To achieve the above object, the invention adopts the following technical scheme:
First aspect of the present invention, provide molybdenum disulfide nano-composite material as marker material answering in testing product
With.
The second aspect of the present invention provides a kind of test strips based on the amplification of molybdenum disulfide composite nano materials signal, institute
Test strips are stated with MoS2@Au nanocomposite is marker material.
Third aspect of the present invention provides the test strips described above based on the amplification of molybdenum disulfide composite nano materials signal
Preparation method, comprising the following steps:
(1) it after nitrocellulose filter dropwise addition antibody or aptamers, coated antibody, closed, cleaned；Obtain antibody or suitable
The coated test strips of ligand；
(2)MoS2The preparation of@Au nanocomposite: mixed with chlorauric acid solution after the processing of molybdenum disulfide solution microwave ultrasound
After closing 3~10min of reaction, supernatant is abandoned in centrifugation, and precipitating is redissolved in ultrapure water, be centrifuged again, redissolve to get；
(3) by MoS2After@Au nanocomposite and antibody or aptamers coupling, immunological probe is formed, target is to be measured
Object, immunological probe, developing solution be added dropwise in the antibody coupling test strips that step (1) is prepared to get.
The 4th aspect of the present invention, provides the above-described test paper based on the amplification of molybdenum disulfide composite nano materials signal
Item is applied in detection microorganism, albumen and biological micromolecule.
The present invention obtains beneficial technical effect:
(1) present invention utilizes MoS2The catalysis of nano material itself and light thermal property are prepared for uniform, stable novel examination
Paper slip marker material MoS2@Au nanocomposite；Using it as marker material, on the one hand since nanogold is to antibody or adaptation
Body has very strong suction-operated, considerably increases marker material to the adsorbance and absorption stability of antibody or aptamers, has
Conducive to the detection sensitivity and specificity for improving object；
(2) present invention utilizes MoS2@Au nanocomposite divides greatly substrate catalyzed coloration performance instead of traditional biological
Sub- enzymatic reaction substantially increases the stability of catalytic performance, reduces testing cost, at the same using the catalysis of nano material and
The sensitivity that photothermy will test improves about 1 order of magnitude compared with vision-based detection limit；
(3) present invention utilizes MoS2The excellent photothermal conversion performance of@Au nanocomposite is, it can be achieved that the fast of determinand
Speed detection, the entire detection time of Rapid detection test strip of the present invention is about 30min, hence it is evident that is higher than enzyme linked immunological (detection about 3h)
Detect speed；
(4)MoS2Nano material it is cheap, water-soluble and biocompatibility is excellent, does not need low-temperature storage, can show
The cost and storage stability for reducing test strips are write, convenient for the large-scale production of test strips of the present invention.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
Fig. 1 Rapid detection test strip building block principle figure of the present invention；
Fig. 2 Rapid detection test strip detection limit canonical plotting of the present invention.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, find cheap, the water-soluble and good marker material of biocompatibility is
It is needed at present.Based on this, the present invention provides molybdenum disulfide nano-composite materials as marker material in testing product
Application.
Further, molybdenum disulfide nano-composite material can appoint for molybdenum disulfide and graphene, colloidal gold and nanoscale magnetic bead
It is a kind of compound obtained.The present inventor has found for the first time: molybdenum disulfide and graphene, colloidal gold and the compound obtained nanometer of nanoscale magnetic bead
Material can be used as quick detection of the marker material for substance.
The second aspect of the present invention provides a kind of test strips based on the amplification of molybdenum disulfide composite nano materials signal, institute
Test strips are stated with MoS2@Au nanocomposite is marker material.
Further, the MoS2The preparation method of@Au nanocomposite is will be at molybdenum disulfide solution microwave ultrasound
After reason and after 3~10min of chlorauric acid solution hybrid reaction, supernatant is abandoned in centrifugation, and precipitating is redissolved in ultrapure water, is centrifuged again, is multiple
It is molten to get.This method step is simple, MoS2@Au nanocomposite yield is high, stability is good.
Further, the test strips include coated antibody or aptamers, immunological probe, developing solution and nitrocellulose
Film；The immunological probe is by MoS2@Au nanocomposite and antibody or aptamers coupling are made.
Further, the developing solution is made of substrate buffer solution, 3,3', 5,5'- tetramethyl benzidines and hydrogen peroxide.
The above test strips ingredient is cooperated, is mutually cooperateed with, and can realize quick, Sensitive Detection to substance.
The substrate buffer solution is disodium hydrogen phosphate-citrate buffer solution, pH5.0.
Third aspect of the present invention provides the test strips described above based on the amplification of molybdenum disulfide composite nano materials signal
Preparation method, comprising the following steps:
(1) it after nitrocellulose filter dropwise addition antibody or aptamers, coated antibody, closed, cleaned；Obtain antibody or suitable
Ligand is coated with test strips；
(2)MoS2The preparation of@Au nanocomposite: mixed with chlorauric acid solution after the processing of molybdenum disulfide solution microwave ultrasound
After closing 3~10min of reaction, supernatant is abandoned in centrifugation, and precipitating is redissolved in ultrapure water, be centrifuged again, redissolve to get；
(3) by MoS2After@Au nanocomposite and antibody or aptamers coupling, immunological probe is formed, target is to be measured
Object, immunological probe, developing solution be added dropwise in the antibody coupling test strips that step (1) is prepared to get.
Further, step (3) MoS2@Au nanocomposite pH value is 6.0~8.0.In the pH value range, MoS2@
Au nanocomposite catalytic performance and photoelectric properties are all good；When pH value is lower than 6.0, MoS2The catalysis of@Au nanocomposite
Reduced performance, when pH value is higher than 8.0, MoS2@Au nanocomposite photoelectric properties are influenced significantly by pH value.
The 4th aspect of the present invention, provides the above-described test paper based on the amplification of molybdenum disulfide composite nano materials signal
Application of the item in detection microorganism, albumen and biological micromolecule.
Further, the microorganism is salmonella.The present invention is based on the amplifications of molybdenum disulfide composite nano materials signal
Test strips to Salmeterol fluticasone propionate high sensitivity, minimum detection limit is up to 102CFU/mL。
The present invention is based on the test strips testing principles of molybdenum disulfide composite nano materials signal amplification: by MoS2Au nanometers of@
Composite material is coupled with antibody or aptamers, and preparing, there is the detection of peroxide catalyzed performance and photothermal conversion performance to visit
Needle.
Nitrocellulose filter is activated, certain density target determinand is added dropwise in cellulose nitrate film point sample area
Antibody is placed in 37 DEG C of constant incubator 15min, is then closed with skimmed milk power solution.It washs and dries after closing
Success prepares test strips.Determinand is mixed into incubation with above-mentioned detection probe, then mixed liquor dropwise addition is taken to be regarded in test strips
Detection is felt, by adding MoS2Substrate carries out catalyzed coloration to improve sensitivity.The detection zone of laser irradiation test strips utilizes
MoS2Excellent photothermal conversion performance establishes the quantitative relation of the temperature difference Yu target determinand, realizes the quick, clever of target determinand
Quick half-quantitative detection.
In order to enable those skilled in the art can clearly understand technical solution of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
A kind of test strips based on the amplification of molybdenum disulfide composite nano materials signal of embodiment 1
The Rapid detection test strip is with MoS2@Au nanocomposite is marker material.
The MoS2@Au nanocomposite the preparation method comprises the following steps:
(1) sample molybdenum disulfide 6mg is weighed with electronic balance, molybdenum disulfide is placed in amber glass bottle, it is super that 2mL is added
Pure water, microwave ultrasound handle 2h, and the molybdenum disulfide solution that concentration is 3mg/mL is made.
(2) taking concentration is that 1% 600 μ L of chlorauric acid solution is diluted to 6mL with ultrapure water in the EP pipe of 10mL, then inhales
The above-mentioned molybdenum disulfide solution 2mL being ultrasonically treated is taken to be added in EP pipe, molybdenum disulfide and chlorauric acid solution hybrid reaction 5min.
(3) reaction solution is dispensed into centrifuge tube, setting centrifuge speed is 8000rpm, 0.5h is centrifuged, with shifting after centrifugation
Liquid rifle exhausts supernatant, then is redissolved in 3mL ultrapure water with Vltrasonic device, and the same terms are centrifuged again, finally that precipitating is multiple
It is dissolved in 200 μ L ultrapure waters, MoS is made2@Au nanocomposite, and measure material pH value.
The test strips include coated antibody or aptamers, immunological probe, developing solution and cellulose nitrate film；It is described
Immunological probe is by MoS2@Au nanocomposite and antibody or aptamers coupling are made.
The developing solution is made of substrate buffer solution, 3,3', 5,5'- tetramethyl benzidine and hydrogen peroxide.Specific dosage
And composition are as follows: the substrate buffer solution (pH=5) of 3940 μ L, 3,3', 5, the 5'- tetramethyl benzidines (TMB, 10mg/ml) of 40 μ L
With the system of 20 μ L hydrogen peroxide (30%) composition.
The preparation method of the test strips:
(1) antibody or aptamers is added dropwise in nitrocellulose filter, coated antibody is placed on 37 DEG C of constant temperature trainings after coated antibody
Support case 15min；The skimmed milk power that compound concentration is 4% is added dropwise 15 μ L 0.2%Tween-20 and confining liquid is made, by coated antibody
Film put into 37 DEG C of closing 1.5h of confining liquid, wash 1min with the PBS flushing liquor of 0.1mol/L after closing and dry；Obtain antibody
It is coupled test strips；
(2)MoS2The preparation of@Au nanocomposite: specific preparation method is same as above；With the NaOH or HCl of 1mol/L
The pH value of MoS2@Au nanocomposite is adjusted respectively to 7.0 stand-by；
(3)MoS2The 2.5 μ L of antibody that 2.5 μ L of@Au and concentration are 20 μ g/mL is coupled 5min, is closed with 1% skimmed milk power
MoS2@Au- antibody coupling matter 5min, 7000rpm is centrifuged 5min after closing, and supernatant is sucked, and precipitating is redissolved with 5 μ L PBS；Shape
At immunological probe, by immunological probe, developing solution be added dropwise in the antibody coupling test strips that step (1) is prepared to get.
Application of the Rapid detection test strip described in embodiment 2 in detection salmonella
The application specifically:
1) processing of salmonella
1. in superclean bench, by the oese picking of salmonella typhimurium bacterium colony sterilizing in test tube to liquid LB
In culture medium, 37 DEG C of shaken cultivation 12h.
2. the 1mL bacterium solution of collection is diluted to the 10 of original bacteria liquid, 10 respectively2、103、104、105、106、107、108After times,
So that bacterial concentration gradient is successively about 108、107、106、105、104、103、102、101CFU/mL, each 25 μ L bacterium solution of self-priming coating
To beef-protein medium, it is placed in culture 16h in 37 DEG C of incubators and is counted.
3. the above-mentioned salmonella bacterium solution 1mL 1. cultivated is taken to be placed in EP pipe, 5min is centrifuged with 4000rpm, after abandoning supernatant
It is cleaned 1 time with 1mL PBS buffer solution, redissolution is placed in 4 DEG C of low-temperature storages in PBS buffer solution.
2) preparation of antibodies toward salmonella coupling test strips
The 2 μ L of salmonella typhimurium antibody of 0.2mg/mL, coated antibody postposition is added dropwise in nitrocellulose filter point sample area
In 37 DEG C of constant incubator 15min.The skimmed milk power that compound concentration is 4% is added dropwise 15 μ L 0.2%Tween-20 and closing is made
Liquid.The film of coated antibody is immersed into 37 DEG C of closing 1.5h of confining liquid, washs 1min with the PBS flushing liquor of 0.1mol/L after closing
And it dries.
3) preparation of immunological probe
The MoS for being 8.0 by pH value2The antibody coupling 5min that@Au and concentration are 20 μ g/mL, is closed with 1% skimmed milk power
MoS2@Au- antibody coupling matter 5min, 7000rpm is centrifuged 5min after closing, and after abandoning supernatant, precipitating is redissolved with PBS, is formed immune
Probe
4) detection of salmonella
By salmonella bacterium solution and MoS210 μ L of@Au- antibody coupling matter mixed liquor uses PBS after reacting 5min in point sample area
1min is rinsed, 20 μ L of developing solution is added dropwise after drying, is immediately placed in 37 DEG C of insulating boxs and is protected from light dry 15min, finally observe test strips
Develop the color result: the color development area color of the test strips containing salmonella by no discoloration indigo plant colour developing.
5) using photo-thermal effect to salmonella quantitative detection
Infrared thermal imagery is used with the laser irradiation point sample area 1min of 808nm wavelength using the best duration of laser irradiation and wavelength
The temperature value of irradiation front and back and the temperature difference of blank space at instrument measurement record test strips point sample, calculate Δ T (formula 1).Pass through
9 detections are carried out to blank film, calculate the average value and standard deviation of △ T, determine that the sensitivity of this method is 3 times of blank
Standard deviation i.e. 1.84.Using the logarithm of salmonella quantity as abscissa, standard curve is drawn by ordinate of Δ T, is obtained
Equation of linear regression is Y=2.31X+0.823, R2=0.949, and the number and the temperature difference for illustrating salmonella are 102-107CFU/mL
Between be in good linear relationship, therefore, using the photo-thermal effect of nano material, the minimum detection limit of salmonella in the method
It is 102CFU/mL。
Δ T=Δ T1-ΔT0Formula 1
Δ T, temperature increase.
ΔT0, temperature increase before and after blank (being not added with bacterium) sample laser irradiation.
ΔT1, add the temperature increase after sample before and after laser irradiation.
While test, with MoS2It is the test strips of material building as compareing, individually by MoS2After antibody coupling,
It is detected for marking, detection is limited to 106CFU/mL。
Test example
Test strips assembling condition is optimized, optimal conditions include: confining liquid concentration (skimmed milk power, Tween-
20), the pH value and antibody concentration of nanocomposite.
(1) confining liquid concentration:
Compound concentration is followed successively by 1%, 2%, 3%, 4% skimmed milk power；
Compound concentration is followed successively by 0%, 0.2%, 0.4%, 0.6%, 0.8% Tween-20.
(2) pH value of nanocomposite
MoS is adjusted respectively with the NaOH of 1mol/L or HCl2The pH value of@Au nanocomposite is to 6.0,7.0,8.0.
(3) antibody concentration
Antibody concentration is diluted to the degreasing that 1 μ g/mL, 5 μ g/mL, 20 μ g/mL, 50 μ g/mL are stand-by, and compound concentration is 4%
Milk powder and be added dropwise 15 μ L 0.2%Tween-20 confining liquid is made.
Detected by 2 method of above-described embodiment, test result shows: skimmed milk power optium concentration is 4% in confining liquid
(w/v), Tween-20 concentration is 0.2% (w/v), and nanocomposite pH value is 8, and antibody degree concentration is 20 μ g/mL
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. application of the molybdenum disulfide nano-composite material as marker material in testing product.
2. application according to claim 1, which is characterized in that molybdenum disulfide nano-composite material can be molybdenum disulfide and stone
Black alkene, colloidal gold and nanoscale magnetic bead are any compound obtained.
3. it is a kind of based on molybdenum disulfide composite nano materials signal amplification test strips, which is characterized in that the test strips with
MoS2@Au nanocomposite is marker material.
4. test strips according to claim 3, which is characterized in that the MoS2The preparation method of@Au nanocomposite is
By, with after 3~10min of chlorauric acid solution hybrid reaction, supernatant is abandoned in centrifugation, will be precipitated after the processing of molybdenum disulfide solution microwave ultrasound
Redissolve in ultrapure water, be centrifuged again, redissolve to get.
5. test strips according to claim 3, which is characterized in that the test strips include coated antibody or aptamers, exempt from
Epidemic disease probe, developing solution and cellulose nitrate film；The immunological probe is by MoS2@Au nanocomposite and antibody are adapted to
Body coupling is made.
6. test strips according to claim 5, which is characterized in that the developing solution is by substrate buffer solution, 3,3', and 5,5'-
Tetramethyl benzidine and hydrogen peroxide composition.
7. the preparation method of any test strips based on the amplification of molybdenum disulfide composite nano materials signal of claim 3~6,
Characterized by comprising the following steps:
(1) it after nitrocellulose filter dropwise addition antibody or aptamers, coated antibody, closed, cleaned；Obtain antibody or aptamers
Coated test strips；
(2)MoS2The preparation of@Au nanocomposite: it is mixed with chlorauric acid solution instead after the processing of molybdenum disulfide solution microwave ultrasound
After answering 3~10min, supernatant is abandoned in centrifugation, and precipitating is redissolved in ultrapure water, be centrifuged again, redissolve to get；
(3) by MoS2After@Au nanocomposite and antibody or aptamers coupling, immunological probe is formed, by target determinand, is exempted from
Epidemic disease probe, developing solution be added dropwise in the antibody coupling test strips that step (1) is prepared to get.
8. preparation method according to claim 7, which is characterized in that step (3) MoS2@Au nanocomposite pH value is
6.0~8.0.
9. application of any test strips of claim 3~5 in detection microorganism, albumen and biological micromolecule.
10. application according to claim 9, which is characterized in that the microorganism is salmonella.
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| CN110887964A (en) * | 2019-11-07 | 2020-03-17 | 西北农林科技大学 | Sensitive probe, method for detecting tetracycline and application |
| CN111505284A (en) * | 2020-04-23 | 2020-08-07 | 中国科学院重庆绿色智能技术研究院 | Test paper strip and sensor for detecting novel coronavirus SARS-CoV-2, preparation and application thereof |
| CN113912123A (en) * | 2021-09-23 | 2022-01-11 | 山东师范大学 | Salmonella typhimurium multimode test strip based on magnetic molybdenum disulfide catalysis and photothermal effect, and detection method and application thereof |
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