CN108982853A - Paper-based device for detecting monoamine oxidase content and preparation method and application thereof - Google Patents
Paper-based device for detecting monoamine oxidase content and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a paper-based device for detecting monoamine oxidase content and a preparation method and application thereof. The paper-based device comprises chromatographic paper and a microfluidic detection area printed on the chromatographic paper, wherein the microfluidic detection area contains an antibody of monoamine oxidase A or an antibody of monoamine oxidase B. Selecting chromatographic paper with a clean surface; printing a microfluidic detection area on the surface of the chromatographic paper by using wax, and placing and baking the printed chromatographic paper to enable the wax to permeate the back to form a hydrophilic and hydrophobic microfluidic channel; washing, dripping an antibody of monoamine oxidase in the microfluidic detection area, and washing with PBST; and finally, dropwise adding bovine serum albumin in the microfluidic detection area, and washing by using PBST. The paper-based device obtained by the invention has simple and rapid structure, clear boundary between detection areas, and can design a plurality of detection areas and realize high-flux detection at the same time; can be fully applied to early diagnosis products of brain glioma and Parkinson disease.
Description
Technical field
The invention belongs to test paper rapid quantitative detection technical fields, and in particular to a kind of paper for detecting monoamine oxidase content
Base device and the preparation method and application thereof.
Background technique
Paper base device is to replace the materials such as traditional quartz, glass, silicon, high polymer with paper, is added by the surface in paper
The advantages of work provides the micro-analysis device of the microfluidic channel of certain structure, combines microflow control technique and paper].With tradition
Micro-fluidic chip compare, paper base device has the advantage that (1) paper abundance, can be produced in batches；(2) it does not need
External pump, the main component of paper are celluloses, and fluid flows through capillary action on paper；(3) sample consumption is lower；(4)
It is low to detect background, is conducive to photometry detection；(5) bio-compatibility is good, can change the property of paper by chemical modification；(6) one
Secondary property portable analysis, it is easy to operate, in this embodiment it is not even necessary to professional operator.Paper base device is clinical diagnosis, environmental monitoring
And portable inspectiont and real time monitoring needed for Food Safety Analysis provide a wide platform.In addition, right
In the low developed area that medical staff and Medical Devices are in short supply, paper base device is the effective tool for realizing inexpensive care diagnostic,
Because of its unique advantage, the context of detection in cancer, infectious disease and other physiological maladies is applied, causes more and more researchs
The concern of person.
Monoamine oxidase (MAO) is naturally occurring a kind of enzyme in human body, catalysis Monoamines oxidative deamination
Enzyme.Human body is included there are two types of monoamine oxidase: monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B).MAO-A is main
It is distributed in catecholaminergic neuron；Monoamine oxidase B be mainly distributed on serotoninergic neuron, histaminergic neuron and
In Deiter's cells, both hypotypes all can be such that monoamine neurotransmitter inactivates.And the overexpression of MAO-B can cause
The diseases such as a series of disease, such as Parkinson disease, depression, helicobacter pylori or glioma.
Inside glioma under inheritance susceptible factor and the interaction of external environment pathogenic factor, in the inhereditary material of cell
(DNA) and epigenetic levels of substance it, has occurred and is enough carcinogenic mutation (and combination of mutation)；These mutation, driving are thin
Born of the same parents it is lasting into the cell cycle carry out mitosis, escape apoptosis, the growth contact inhibition for hiding cell, protected from immune inhibit
Deng, and cell is made to obtain the abnormal energy metabolism being adapted with sustainable growth, the growth of induction tumor neogenetic blood vessels, anoxic and necrosis
Deng change.In its research process, researcher has found that the overexpression of MAO-A albumen and treating brain glioma disease are closely bound up, therefore
Physiological condition of the MAO-A albumen as treating brain glioma disease, the important means as diagnosis early period.
Parkinson's disease (PD) is to take place mostly in one kind of person in middle and old age with substantia nigra compacta dopaminergic neuron loss, line
Shape body dopamine reduces the central nervous system retrograde degeneration's disease being characterized, and the cause of disease and pathogenesis are still unclear at present
Chu, causes dopamine secretion to reduce, acetylcholine is opposite increases related with the reduction of substantia nigra dopaminergic neuron, is most common
One of extrapyramidal disease.The symptom of PD mainly shown are as follows: static tremor, hypermyotonia, bradykinesia, appearance
Gesture reflecting barrier etc..MAO-B enzyme is overexpressed in its disease surveillance index, therefore clinically detection MAO-B enzyme content can be examined tentatively
Disconnected PD disease.
SHSY5Y cell line, HepG-2 cell line and Hela cell are the classical cell line for studying monoamine oxidase,
Containing only MAO-A albumen in SHSY5Y cell line, containing only containing in MAO-B albumen and Hela cell line in HepG-2 cell line
MAO-A albumen and MAO-B albumen.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of paper base devices for detecting monoamine oxidase content
And the preparation method and application thereof.
A kind of paper base device detecting monoamine oxidase content, including chromatographic paper and prints on the micro-fluidic inspection on chromatographic paper
Region is surveyed, the micro-fluidic detection region is printed by wax spray printer, contains monoamine on the micro-fluidic detection region
The antibody of oxidizing ferment, the monoamine oxidase are monoamine oxidase A or oxidase B；Monoamine oxidase is a kind of containing FAD catalysis oxygen
The enzyme for changing monoamine deamination reaction is mainly used for the metabolism of catalysis oxidation endogenous and exogenous Monoamines, adjusts and maintains
Intracellular biological amine balance.There are mainly two types of homology monoamine oxidase, monoamine oxidase A and monoamine oxidase Bs in vivo.
It is that the detection zone shape is the circle that internal diameter is 8mm as improved, edge is close and distant aquaporin.
A kind of preparation method of above-mentioned paper base device for detecting monoamine oxidase content, comprising the following steps:
Step 1, the clean chromatographic paper of surface cleaning is chosen, it is spare；
Step 2, on chromatographic paper surface, wax prints micro-fluidic detection region, and the chromatographic paper after printing is placed baking so that wax
Back is penetrated into, the microfluidic channel of hydrophobe is formed；
Step 3, will through step 2 treated chromatographic paper is placed in cleaned in plasma cleaner after；
Step 4, micro-fluidic detection region be added dropwise monoamine oxidase antibody, the monoamine oxidase be monoamine oxidase A or
Monoamine oxidase B, and cleaned with PBST；
Step 5, bovine serum albumin is added dropwise in micro-fluidic detection region, and is cleaned with PBST and is corresponded to get detection antibody used
Monoamine oxidase content paper base device.
It is that baking temperature is 120 DEG C in step 2,1 min of baking time as improved.
It is that the running parameter of plasma cleaner is 13.56 MHz of working power, oxygen function in step 3 as improved
Rate is 100 W, handles 4 min of time.
It is that the volume of the antibody of monoamine oxidase is 3 μ L in step 4, and concentration is 1 μ g/mL, PBST's as improved
Concentration is 1 mol/L, cleaning 4 times, every time 10 mL.
It is that the bovine serum albumin concentration in step 5 is 50 mg/mL as improved, volume is the concentration of 6 μ L, PBST
It is 1 mol/L, cleans 4 times, 10 mL every time.
The paper base device of above-mentioned detection monoamine oxidase content is preparing glioma or Parkinson disease early diagnosis production
Application in product, i.e., when the antibody of dropwise addition is the antibody of monoamine oxidase A, paper base device can be used for preparing glioma early stage
Application in diagnostic products；When the antibody of dropwise addition is the antibody of monoamine oxidase B, paper base device can be used for preparing Parkinson's disease
Application in disease early diagnosis product
Beneficial effect
Compared with prior art, the paper base device of a kind of detection monoamine oxidase content of the invention, structure is simple and fast, passes through
The mode of wax spray printing forms hydrophobe microfluidic channel, and distinct between each detection zone, can set on same paper base
Multiple detection zones are counted, while realizing high-throughput detection；Its detection for being applied to middle monoamine oxidase is faster, more quasi-.
Detailed description of the invention
Fig. 1 is the paper base device of present invention detection monoamine oxidase A content；
Fig. 2 is the standard curve of monoamine oxidase A content；
Fig. 3 is the standard curve of monoamine oxidase B content；
Fig. 4 is the interference figure of monoamine oxidase A；
Fig. 5 is the interference figure of monoamine oxidase B；
Fig. 6 is to detect monoamine oxidase A paper base device in the application drawing of cell sample；
Fig. 7 is to detect monoamine oxidase B paper base device in the application drawing of cell sample；
Fig. 8 is Western blotting figure.
Specific embodiment
The present invention is further described in detail below by specific embodiment.
Embodiment 1
As shown in Figure 1, a kind of paper base device for detecting monoamine oxidase content, including chromatographic paper and prints on micro- on chromatographic paper
Flow control detection zone, the micro-fluidic detection region are printed by wax spray printer, are contained on the micro-fluidic detection region
There is the antibody of monoamine oxidase, the micro-fluidic detection region is the circle that internal diameter is 8mm, and edge is close and distant aquaporin.
Embodiment 2
Utilize a kind of preparation method of the paper base device of detection monoamine oxidase content described in embodiment 1, including following step
It is rapid:
Step 1, the clean chromatographic paper of surface cleaning is chosen；
Step 2, on chromatographic paper surface, wax prints micro-fluidic detection region, and the chromatographic paper after printing is placed baking so that wax
Back is penetrated into, the microfluidic channel of hydrophobe is formed；
Step 3, will through step 2 treated chromatographic paper is placed in cleaned in plasma cleaner after；
Step 4, the antibody of monoamine oxidase A is added dropwise in micro-fluidic detection region, and is cleaned with PBST；
Step 5, bovine serum albumin is added dropwise in micro-fluidic detection region, and is cleaned with PBST and is contained to get detection monoamine oxidase A
The paper base device of amount.
When the antibody of monoamine oxidase A is changed to the antibody of monoamine oxidase B to get detection monoamine oxidase B content
Paper base device.
Embodiment 3
When choosing the paper base device for detecting monoamine oxidase A content in embodiment 2, the mark of measurement monoamine oxidase A content is formulated
Directrix curve, steps are as follows:
(1) PBST of 10 mL is added dropwise to the different micro-fluidic detection regions of paper base device, activates 5 minutes；
(2) the micro-fluidic detection region that drop has PBST is added dropwise to a series of biological monoamine oxidase A content of various concentration gradients
Sample, after hatching 30 minutes, each micro-fluidic detection region cleans 4 times with PBST, every time 10 μ L；
(3) the antibody MAO-A that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region cleans 4 with PBST after hatching 4 minutes
It is secondary, 10 μ L every time；
(4) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(5) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 2.
Embodiment 4
When choosing the paper base device for detecting monoamine oxidase B content in embodiment 2, the mark of measurement monoamine oxidase B content is formulated
Directrix curve, steps are as follows:
(1) PBST of 10mL is added dropwise in the different micro-fluidic detection regions of the paper base device prepared to embodiment 2, activates 5 minutes；
(2) a series of various concentration ladders are added dropwise in the micro-fluidic detection region that the drop of paper base device prepared by embodiment 2 has PBST
The biological monoamine oxidase B content sample of degree, after hatching 30 minutes, each micro-fluidic detection region cleans 4 times with PBST, every time
10 μL；
(3) the antibody MAO-B(that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region is different from capture antibody kind), it incubates
After changing 4 minutes, 4 times are cleaned with PBST, every time 10 μ L；
(4) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(5) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 3.
Embodiment 5
Monoamine oxidase antibody is detected to homology MAO-A and MAO-B the interfering with each other property on paper base
The paper base units test MAO-A and MAO-B and outer inhibiting prepared with embodiment 2 is anti-on paper base device to it
Interference, step are as follows:
(1) PBST of 10 mL is added dropwise in the different micro-fluidic detection regions of the paper base device prepared to embodiment 2, activates 5 minutes；
(2) MAO-A standard items (standard items (label the sample b), MAO-A of label sample a), MAO-B of isoconcentration is prepared respectively
The inhibitor clorgiline (CL) (label sample a-CL) of MAO-A is added in standard items, the suppression of MAO-B is added in MAO-A standard items
Preparation pargyline (PA) (marks sample a-PA), and inhibitor clorgiline (CL) (the label sample of MAO-A is added in MAO-B standard items
Product b-CL), the inhibitor pargyline (PA) (label sample b-PA) of MAO-B and certain density is added in MAO-B standard items
Pbs(marks sample blank), respectively by sample a, b, a-CL, a-PA, b-CL, b-PA, 3 μ L of blank drop is in micro-fluidic detection
Region is hatched 30 minutes, and each micro-fluidic detection region cleans 4 times with PBST later, every time 10 μ L；
(3) the antibody MAO-A that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region cleans 4 with PBST after hatching 4 minutes
It is secondary, 10 μ L every time；
(4) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(5) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 4.
The immune knot of antibody is captured to MAO-A by the sample of the standard items and its inhibitor that detect MAO-A and MAO-B
Fruit is it is found that the antibody of the device cannot identify the sample of homology MAO-B and only have immune effect to the sample of MAO-A, therefore,
The false positive that the device occurs in use can be reduced, its accuracy is improved.
Embodiment 6
Monoamine oxidase antibody is detected to homology MAO-A and MAO-B the interfering with each other property on paper base
Paper base units test MAO-A and MAO-B prepared with embodiment 3 and outer inhibiting are anti-on paper base device to it
Interference, step are as follows:
(1) PBST of 10 mL is added dropwise in the different micro-fluidic detection regions of the paper base device prepared to embodiment 3, activates 5 minutes；
(2) MAO-A standard items (standard items (label the sample b), MAO-A of label sample a), MAO-B of isoconcentration is prepared respectively
The inhibitor clorgiline (CL) (label sample a-CL) of MAO-A is added in standard items, the suppression of MAO-B is added in MAO-A standard items
Preparation pargyline (PA) (marks sample a-PA), and inhibitor clorgiline (CL) (the label sample of MAO-A is added in MAO-B standard items
Product b-CL), the inhibitor pargyline (PA) (label sample b-PA) of MAO-B and certain density is added in MAO-B standard items
Pbs(marks sample blank), respectively by sample a, b, a-CL, a-PA, b-CL, b-PA, 3 μ L of blank drop is in micro-fluidic detection area
Domain is hatched 30 minutes, and each micro-fluidic detection region cleans 4 times with PBST later, every time 10 μ L；
(3) the antibody MAO-B that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region cleans 4 with PBST after hatching 4 minutes
It is secondary, 10 μ L every time；
(4) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(5) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 5.
It can to the immune result of MAO-B antibody by the sample of the standard items and its inhibitor that detect MAO-A and MAO-B
Know, the antibody of the device cannot identify the sample of homology MAO-A and only have immune effect to the sample of MAO-B, therefore, can drop
The false positive that the low device occurs in use, improves its accuracy.
Embodiment 7
When choosing the paper base device for detecting monoamine oxidase A content in embodiment 2, monoamine oxidase A in cell line sample is detected
Content, step are as follows:
(1) it chooses SHSY5Y cell line lysate (sample is labeled as S) and inhibiting CL(marks sample in lysate sample
This is S-CL), inhibiting PA(marker samples are S-PA), (sample is labeled as H) and adds suppression for HepG-2 cell line lysate
Formulation C L(marker samples are H-CL), inhibiting PA(marker samples are H-PA), (sample is labeled as Hela cell line lysate
HE it is) and in lysate inhibiting CL(marker samples HE-CL), inhibiting PA(marker samples are HE-PA), Yi Jiyi
Determine the pbs(label sample blank of concentration)；
(2) PBST of 10 mL is added dropwise in the different micro-fluidic detection regions for the paper base device for preparing embodiment 2, activates 5 minutes；
(3) sample S, S-CL, S-PA are taken, HH-CL, H-PA, HE, HE-CL, HE-PA, blank drip 3 μ L respectively incubates in detection zone
Change 30 minutes, each micro-fluidic detection region cleans 4 times with PBST later, every time 10 μ L；
(4) the antibody MAO-A that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region cleans 4 with PBST after hatching 4 minutes
It is secondary, 10 μ L every time；
(5) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(6) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 6.
From the cell sample result of Fig. 6 it is found that whether being overexpressed in the device qualitative detection cell sample of the invention
MAO-A and Western blots result (Fig. 8) are consistent.Therefore, which can detect MAO-A, can be used as early diagnosis colloid
The tool of tumor patient.
Embodiment 8
When choosing the paper base device for detecting monoamine oxidase B content in embodiment 2, monoamine oxidase B in cell line sample is detected
Content, step are as follows:
(1) it chooses SHSY5Y cell line lysate (sample is labeled as S) and inhibiting CL(marks sample in lysate sample
This is S-CL), inhibiting PA(marker samples are S-PA), (sample is labeled as H) and adds suppression for HepG-2 cell line lysate
Formulation C L(marker samples are H-CL), inhibiting PA(marker samples are H-PA), (sample is labeled as Hela cell line lysate
HE it is) and in lysate inhibiting CL(marker samples HE-CL), inhibiting PA(marker samples are HE-PA), Yi Jiyi
Determine the pbs(label sample blank of concentration)；
(2) the different micro-fluidic detection regions of paper base device are added dropwise to the PBST of 10 mL, are activated 5 minutes；
(3) sample S, S-CL, S-PA are taken, HH-CL, H-PA, HE, HE-CL, HE-PA, blank drip 3 μ L respectively incubates in detection zone
Change 30 minutes, each micro-fluidic detection region cleans 4 times with PBST later, every time 10 μ L；
(4) the antibody MAO-B(that the label HRP of 3 μ L is added dropwise into micro-fluidic detection region is different from capture antibody kind), it incubates
After changing 4 minutes, 4 times are cleaned with PBST, every time 10 μ L；
(5) peroxide horseradish zymolyte peroxide and luminol are prepared, and is added dropwise 10 μ L's in micro-fluidic detection region
Liquid substrate is exposed, and is immediately placed in chemical detector detection chemiluminescence；
(6) picture is converted into numerical value by ImageJ mode, formulates standard curve, as shown in Figure 7.
From the cell sample result of Fig. 7 it is found that whether crossing table in the paper base device qualitative detection cell sample of the invention
It is consistent up to MAO-B and Western blots result (Fig. 8).Therefore, which can detect MAO-B, can be used as early diagnosis pa
One of the tool of the gloomy disease of gold.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (8)
1. a kind of paper base device for detecting monoamine oxidase content, which is characterized in that including chromatographic paper and print on chromatographic paper
Micro-fluidic detection region, the micro-fluidic detection region printed by wax spray printer, the micro-fluidic detection region
On the antibody containing monoamine oxidase, the monoamine oxidase be monoamine oxidase A or oxidase B.
2. a kind of paper base device for detecting monoamine oxidase content according to claim 1, which is characterized in that the detection
Region shape is the circle that internal diameter is 8 mm, and edge is close and distant aquaporin.
3. based on a kind of preparation method for the paper base device for detecting monoamine oxidase content described in claim 1, feature exists
In, comprising the following steps: step 1, the clean chromatographic paper of surface cleaning is chosen, it is spare；Step 2, on chromatographic paper surface, wax is beaten
Micro-fluidic detection region is printed, and the chromatographic paper after printing is placed into baking so that wax penetrates into back, forms the miniflow of hydrophobe
Control channel；Step 3, will through step 2 treated chromatographic paper is placed in cleaned in plasma cleaner after；Step 4, in micro-fluidic inspection
The antibody that monoamine oxidase is added dropwise in region is surveyed, the monoamine oxidase is monoamine oxidase A or oxidase B and is cleaned with PBST；
Step 5, bovine serum albumin is added dropwise in micro-fluidic detection region, and is cleaned with PBST to get the corresponding list of antibody used is detected
The paper base device of amine oxidase.
4. a kind of preparation method of paper base device for detecting monoamine oxidase content according to claim 3, feature exist
In baking temperature is 120 DEG C in step 2,1 min of baking time.
5. a kind of preparation method of paper base device for detecting monoamine oxidase content according to claim 3, feature exist
In the running parameter of plasma cleaner is 13.56 MHz of working power in step 3, and oxygen power is 100 W, handles the time 4
min。
6. a kind of preparation method of paper base device for detecting monoamine oxidase content according to claim 3, feature exist
In the volume of the antibody of monoamine oxidase is 3 μ L in step 4, and concentration is that the concentration of 1 μ g/mL, PBST is 1 mol/L, cleaning
4 times, 10 mL every time.
7. a kind of preparation method of paper base device for detecting monoamine oxidase content according to claim 3, feature exist
In, the bovine serum albumin concentration in step 5 is 50 mg/mL, and volume is that the concentration of 6 μ L, PBST is 1 mol/L, it cleans 4 times,
10 mL every time.
8. the paper base device based on a kind of detection monoamine oxidase content described in claim 1 or 3 prepare glioma or
Parkinson disease early diagnoses the application in product.
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