CN107543809A - A kind of glutathione molecules detection test strips and detection method - Google Patents
A kind of glutathione molecules detection test strips and detection method Download PDFInfo
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Abstract
A kind of glutathione molecules detection test strips, the pad must be through steps of processing：1）By the polysuccinimated biotin aqueous solution hybrid reaction 30min at 37 DEG C of amidized nano particle, methylol made from nano particle amination, precipitation separation, gained sediment is through washing to obtain biotinylated nano particle；2）Add moisture to dissipate above-mentioned biotinylated nano particle, then the mixing of nanoscale twins solution is added into the system, precipitation separation, gained sediment is through washing to obtain biotinylation nano particle/nanoscale twins compound；3）Biotinylation nano particle/nanoscale twins compound is mixed with the aqueous solution of polysorbas20, and is applied on pad and dries.The present invention is simple and convenient, preferable to GSH selectivity；Detection time is short, it is only necessary to 10min, and detection accuracy is high.
Description
Technical field
The invention belongs to nano material technology, Sidestream chromatography technology, bioassay technique field, and in particular to a kind of paddy Guang
Sweet peptide molecule detection test strips and detection method.
Background technology
GSH (glutathione) is non-protein class compounds containing thiol groups important in human body, be widely present in mammal and
In eukaryotic.Glutathione provides critical function in biosystem, such as：Reduce intracellular Free Radical Level, remain new
Old metabolic process, remove caused noxious material in metabolic processes, statocyte internal oxidition reduction pressure.Research shows,
Horizontal and many diseases of human body GSH-PX activity, such as：The diseases such as Apoptosis, aging, heart disease, Parkinson, senile dementia
Shape is closely related.Therefore, to meet clinical and medical science needs, detecting glutathione content rapidly and sensitively turns into increasingly urgent
Research topic.Up to the present, many glutathione detection methods are reported out.The detection side of currently used GSH contents
Method has high performance liquid chromatography (HPLC), SERS method (SERS), capillary electrophoresis, Enzyme-linked Immunosorbent Assay to survey
Fixed (ELISA) method etc..These methods established have important advantage in quantitative detection glutathione, but these are detected
Method is cumbersome time-consuming, and cost is higher, and needs the human users of specialty.
The content of the invention
Present invention aims at providing a kind of glutathione molecules detection test strips, while provide glutathione molecules
Detection method is the another goal of the invention of the present invention.
Based on above-mentioned purpose, this invention takes following technical scheme：
A kind of glutathione molecules detection test strips, the test strips include substrate, sample are set gradually on the substrate
Product pad, pad, nitrocellulose filter and adsorptive pads, labelled streptavidin detection line on the nitrocellulose filter are described
Pad must be through steps of processing：
1) by the polysuccinimated biotin water of amidized nano particle, methylol made from nano particle amination
Solution hybrid reaction 30min at 37 DEG C, precipitation separation, gained sediment is through washing to obtain biotinylated nano particle；
2) add moisture to dissipate above-mentioned biotinylated nano particle, then the mixing of nanoscale twins solution added into the system,
Precipitation separation, gained sediment is through washing to obtain biotinylation nano particle/nanoscale twins compound；
3) biotinylation nano particle/nanoscale twins compound is mixed with the aqueous solution of polysorbas20, and is applied to pad
Upper drying.
The sample pad and pad fibreglass film are made；The substrate is PVC board.
The concentration of the Streptavidin is 0.1~5mg/mL；Sample pad, pad, nitrocellulose filter and adsorptive pads
Length be respectively 26mm, 6mm, 20mm and 33mm；The width of test strips is 3.9mm.
In step 1), when precipitation separates, isopropanol mixing is first added, then centrifuge.
In step 1), the concentration of the amidized nano particle is 0.01~2 μM, the polysuccinimated life of methylol
The concentration of the thing element aqueous solution is 2~1000 μM；The amidized nano particle biotin polysuccinimated with methylol is water-soluble
The volume ratio of liquid is 200uL：(200~300) uL.
In step 2), the concentration of nanoscale twins solution is 0.3~0.4mg/mL；Nanoscale twins solution and amidized nanometer
The volume ratio of particle is 200uL：(60~80) uL.
In step 3), using vacuum drying, drying temperature is 37 DEG C.
In step 3), the volume percent content of polysorbas20 is 1%~5% in the polysorbas20 aqueous solution.
The nano particle is water-soluble gold nano cluster, water-soluble silver nano-cluster, water-soluble copper zinc indium/ZnS quantum dots
(CuZnIn/ZnS), water-soluble cadmium selenide/ZnS quantum dots (CdSe/ZnS), carbon quantum dot (Carbon dots) and graphite
One kind in alkene quantum dot (Graphene Quantum dots)；And the fluorescence emission wavelengths of the nano particle be 400nm~
750nm；The nanoscale twins are manganese dioxide nano-plates layer (MnO2) or hydroxide cobalt oxide nanoscale twins nanosheets
(CoOOH nanoflakes)。
A kind of glutathione molecules detection method, comprises the following steps：
I) the glutathione titer of various concentrations is added drop-wise in described test strips and runs 10~30min of sample, measure is not
With the fluorescence intensity level A of titer under concentration；
Ii it is) ordinate mapping by abscissa, fluorescence intensity level A of the concentration of glutathione titer, in finite concentration
In the range of linear be fitted to obtain glutathione standard curve and fit equation；
Iii the testing sample of unknown concentration) is added drop-wise to race 10~30min of sample, fluorescence intensity in the test strips
Value A1, fluorescence intensity level A1 substitutions fit equation is calculated to the concentration of testing sample.
In the present invention, the biotinylated nano particle with fluorescent effect is prepared first, it uses nano particle
Fluorescence emission wavelengths are 400nm~750nm, recycle manganese dioxide nano-plates layer to load biotinylated nano particle, are prepared
Into biotinylation nano particle/nanoscale twins compound, the fluorescence of biotinylated nano particle is quenched, in combination with chromatography
Lateral Flow Strip, the biotinylated nano particle of nanoscale twins load is fixed to the conversion zone (pad) of test strips.
When containing glutathione (GSH) in prepare liquid, GSH can clear up nanoscale twins and discharge biotinylated nano particle, biology
The fluorescence of elementization nano particle recovers.Under capillary action, biotinylated quantum dot moves forward, and utilizes the chain in detection line
Mould Avidin (Streptavidin) and the specific binding of biotin (Biotin), so as to capture nano particle.Therefore pass through
The fluorescence signal power of nano particle in measurement detection line detects GSH purpose so as to reach.To detect the GSH of various concentrations
Based on the fluorescent value of standard liquid, the titer of various concentrations and the standard curve of fluorescent value are established, is realized to testing sample
The measure of middle GSH contents.
Compared with prior art, the present invention has following technique effect：
1st, instant invention overcomes the deficiency of traditional detection, there is provided a kind of convenient, quick biology and methods for clinical diagnosis,
Simple to operate, adaptability is stronger, preferable to GSH selectivity；Detection time is short, it is only necessary to 10min, and detection accuracy
It is high；
2nd, the present invention is to the less demanding of instrument, it is not necessary to and the instrument of complex and expensive, cost is relatively low, and process is very easy,
The scale for being advantageous to method is promoted, significant in molecule diagnosis, medical science detection and clinical practice direction.
Brief description of the drawings
Fig. 1 is the composition detection principle diagram of test strips；
Fig. 2 is the assembling process schematic diagram of test strips；
Fig. 3 is ELISA test strip signal-obtaining process schematic；
Fig. 4 is the fluorescence intensity block diagram of various chaff interferences；
Fig. 5 is the structure enlargement diagram to step 2 in Fig. 2.
Embodiment
The present invention is described in further details with reference to embodiment, but protection scope of the present invention is not limited to
This.
In the present invention, sample pad, pad, adsorptive pads and PVC bottom plates are all from Shanghai Jin Biao companies；Nitrocellulose filter
Purchased from Millipore companies.
Embodiment 1
A kind of glutathione molecules detection test strips, as shown in figure 1, the test strips include PVC substrates, the substrate
On set gradually sample pad, pad, nitrocellulose filter and adsorptive pads, on the nitrocellulose filter mark have for
0.3mg/mL Streptavidin detection line, the sample pad and pad fibreglass film are made；Sample pad, combination
The length of pad, nitrocellulose filter and adsorptive pads is respectively 26mm, 6mm, 20mm and 33mm；The width of test strips is 3.9mm.
The pad must be through steps of processing：
1) nano particle amination is obtained into amidized nano particle, by 2 μM of amidized nano particles of 200uL concentration,
800 μM of methylols of 200uL concentration polysuccinimated biotin aqueous solution hybrid reaction 30min at 37 DEG C, add 1mL
Isopropanol centrifuge, gained sediment washing three times biotinylated nano particle；
2) add 200uL moisture to dissipate above-mentioned biotinylated nano particle, then add 60uL concentration into the system and be
0.277 mg/mL nanoscale twins solution mixing, centrifugation, gained sediment through wash three times after biotinylation nano particle/
Nanoscale twins compound；
3) by biotinylation nano particle/nanoscale twins compound, (percent by volume of polysorbas20 contains with 200uL polysorbas20s
Measure and mixed for the aqueous solution 3%), and be applied to 37 DEG C of vacuum drying on pad.
Nano particle is water-soluble copper zinc indium/ZnS quantum dots (CuZnIn/ZnS)；The nanoscale twins solution is two
Manganese oxide nanoscale twins (MnO2Nanosheets) and water mixed liquor.
A kind of glutathione molecules detection method, comprises the following steps：
I) concentration 0.05mM, 0.1mM, 0.2mM, 0.5mM, 0.8mM, 1mM, 1.5mM glutathione standard is respectively configured
Liquid, it is added drop-wise in above-mentioned test strips and runs sample 10min, the fluorescence intensity A of titer under measure various concentrations, respectively 237.12,
267.56th, 314.42,395.61,454.83,545.83 and 649.43；, can be by the test paper of the present invention for convenience of detecting during detection
Bar is placed in the paper box fastened by upper plate 1 and lower plate 2, liquid filling hole 6 and detection hole 7 is set respectively on the upper plate 1, under described
The fixture block 5 of fixed test strips, support bar 4 and the button block 3 fastened with upper plate are provided with plate 2, button block 3 is arranged on upper plate 1
Corresponding catching groove, by setting liquid filling hole 6 that detection liquid is added dropwise on upper plate 1 during detection, fluorescence detection equipment is placed on after fastening
In, detection liquid is detected by the detection hole 7 set on upper plate 1, specifically as shown in Figure 2 and Figure 4；
Ii it is) ordinate mapping by abscissa, fluorescence intensity A of the concentration of glutathione titer, in finite concentration model
Enclose and interior linear be fitted to obtain glutathione standard curve and fit equation Y=282.23X+244.79, R2=0.9872；
Iii prepare liquid A) is added drop-wise to race sample 10min in the test strips, fluorescence intensity 355.95 is recorded, further according to paddy
The GSH contents that testing sample is calculated in the sweet peptide standard curve of Guang and fit equation are 0.394mM.
Embodiment 2
Glutathione molecules detection test strips in the present embodiment are the same as embodiment 1.
Prepare liquid B is added drop-wise to race sample 20min in test strips, fluorescence intensity level 420.27 is recorded, further according to glutathione
The GSH contents that testing sample is calculated in standard curve and fit equation are 0.622mM.
Embodiment 3
Test strips embodiment 1 is used in glutathione molecules detection in the present embodiment.
Prepare liquid C is added drop-wise to race sample 30min in test strips, fluorescence intensity level 521.03 is recorded, further according to glutathione
The GSH contents that testing sample is calculated in standard curve and fit equation are 0.979mM.In the present embodiment, glutathione standard
Curve and fit equation are the same as embodiment 1.
Embodiment 4
Test strips embodiment 1 is used in glutathione molecules detection in the present embodiment, and difference is, in step 1), ammonia
The concentration of the nano particle of base is 0.01 μM, and the concentration of the polysuccinimated biotin aqueous solution of methylol is 2 μM；Step
2) in, the concentration of nanoscale twins solution is 0.4mg/mL；In step 3), the volume percent content of polysorbas20 is 5%.
Embodiment 5
Test strips embodiment 1 is used in glutathione molecules detection in the present embodiment, and difference is, amino in step 1)
The concentration of the nano particle of change is 0.01 μM, and the concentration of the polysuccinimated biotin aqueous solution of methylol is 2 μM；Step 2)
In, the concentration of nanoscale twins solution is 0.3g/mL, and the volume ratio of nanoscale twins solution and amidized nano particle is 200uL：
80uL；The volume ratio of amidized nano particle and the polysuccinimated biotin aqueous solution of methylol is 200uL：300uL；
In step 3), the volume percent content of polysorbas20 is 1%.
The nano particle is water-soluble gold nano cluster；The nanoscale twins are hydroxide cobalt oxide nanoscale twins (CoOOH
nanoflakes)。
Embodiment 6
Test strips embodiment 1 is used in glutathione molecules detection in the present embodiment, and difference is, in step 1), hydroxyl
The concentration of the biotin aqueous solution of methyl succinimide is 1000 μM；In step 3), the volume percent content of polysorbas20
For 5%.The nano particle is water-soluble silver nano-cluster.Nano particle is also an option that water soluble selenium in other embodiments
It is a kind of in cadmium/ZnS quantum dots, carbon quantum dot and graphene quantum dot.
1st, interference is tested：
To investigate selectivity of the inventive method to GSH, a series of interference experiments are devised.Concrete scheme is：Will not
Chaff interference with concentration is added drop-wise in test strips, and wherein the concentration of chaff interference is higher than the concentration of glutathione 10 times.Run sample 10
After~30min, quantum dot fluorescence intensity level on ELISA test strip line is recorded.The fluorescence intensity level of each chaff interference is sat to be vertical
Mark, the block diagram of the fluorescence intensity level of various chaff interferences is drawn, as shown in Figure 4.From Fig. 1 result, detection of the invention
Method is preferable to GSH selectivity.
2nd, evaluation test-recovery testu of adaptability and reliability
In order to verify the adaptability of the inventive method and reliability, the mark-on reclaims of actual sample have been designed and carried out below
Experiment.Recovery testu refers to add a certain amount of standard sample in sample to be measured, then detected with this detection method
The content of sample, reach the checking to this experimental method, as the evaluation of adaptability and reliability to this method, specific implementation
It is as follows：
After three parts of human serum samples (sample 1, sample 1, sample 1) are diluted into 20 times, with the detection method of the invention established
The content of GSH in three parts of human serum samples is measured, is 0.076mM, 0.117mM, 0.065mM respectively, then each addition is isometric
0.2mM GSH standard liquids, are detected by method provided by the invention, the results are shown in Table shown in 1：
System detects glutathione mark-on reclaims result in the present invention of table 1
The testing result of table 1 shows that method provided by the invention has preferable reappearance and accuracy, is especially suitable for examining
The GSH contents surveyed in biological sample.
Claims (10)
- A kind of 1. glutathione molecules detection test strips, it is characterised in that the test strips include substrate, on the substrate according to Secondary setting sample pad, pad, nitrocellulose filter and adsorptive pads, labelled streptavidin detection on the nitrocellulose filter Line, the pad must be through steps of processing：1）By the polysuccinimated biotin aqueous solution of amidized nano particle, methylol made from nano particle amination The hybrid reaction 30min at 37 DEG C, precipitation separation, gained sediment is through washing to obtain biotinylated nano particle；2）Add moisture to dissipate above-mentioned biotinylated nano particle, then the mixing of nanoscale twins solution, precipitation are added into the system Separation, gained sediment is through washing to obtain biotinylation nano particle/nanoscale twins compound；3）Biotinylation nano particle/nanoscale twins compound is mixed with the aqueous solution of polysorbas20, and is applied on pad and does It is dry.
- 2. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that the sample pad and pad Fibreglass film is made；The substrate is PVC board.
- 3. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that the Streptavidin it is dense Spend for 0.1~5mg/mL；Sample pad, pad, the length of nitrocellulose filter and adsorptive pads be respectively 26mm, 6mm, 20mm and 33mm；The width of test strips is 3.9 mm.
- 4. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that step 1）In, the amino The volume ratio of the polysuccinimated biotin aqueous solution of nano particle, the methylol of change, during precipitation separation, first add isopropanol Mixing, then centrifuge.
- 5. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that step 1）In, the amino The concentration of the nano particle of change is 0.01~2 μM, and the concentration of the polysuccinimated biotin aqueous solution of methylol is 2~1000 μM；The volume ratio of amidized nano particle and the polysuccinimated biotin aqueous solution of methylol is 200 uL：（200~ 300）uL.
- 6. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that step 2）In, nanoscale twins The concentration of solution is 0.3~0.4mg/mL；The volume ratio of nanoscale twins solution and amidized nano particle is 200uL：60uL ~80uL.
- 7. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that step 3）In, using vacuum Dry, drying temperature is 37 DEG C.
- 8. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that step 3）In, polysorbas20 water The volume percent content of polysorbas20 is 1%~5% in solution.
- 9. glutathione molecules detection test strips as claimed in claim 1, it is characterised in that the nano particle is water-soluble Property gold nanoclusters, water-soluble silver nano-cluster, water-soluble copper zinc indium/ZnS quantum dots, water-soluble cadmium selenide/zinc sulphide quantum One kind in point, carbon quantum dot and graphene quantum dot；And the fluorescence emission wavelengths of the nano particle are 400 nm~750 nm；The nanoscale twins are manganese dioxide nano-plates layer or hydroxide cobalt oxide nanoscale twins.
- 10. a kind of glutathione molecules detection method, it is characterised in that comprise the following steps：i）The glutathione titer of various concentrations is added drop-wise in any described test strips of claim 1-9 run sample 10~ 30min, determine the fluorescence intensity level A of titer under various concentrations；ii）It is ordinate mapping by abscissa, fluorescence intensity level A of the concentration of glutathione titer, in finite concentration scope It is interior linear to be fitted to obtain glutathione standard curve and fit equation；iii）The testing sample of unknown concentration is added drop-wise to race 10~30min of sample, fluorescence intensity value A1 in the test strips, Fluorescence intensity level A1 substitutions fit equation is calculated to the concentration of testing sample.
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| CN109187935A (en) * | 2018-09-11 | 2019-01-11 | 浙江理工大学 | A method of Ancient Silk Textile is detected based on microwave |
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| CN108660185A (en) * | 2018-03-09 | 2018-10-16 | 山东师范大学 | A kind of biosensor and preparation method based on golden selenium key |
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| CN110873793A (en) * | 2018-08-29 | 2020-03-10 | 中国农业大学 | High-flux ultrasensitive fluorescent gold nano-cluster immunochromatographic test strip and application thereof |
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| CN110702914A (en) * | 2019-09-17 | 2020-01-17 | 江苏大学 | Preparation method of fluorescence biosensor for detecting ochratoxin A |
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