CN101726591A - Wax-patterned nitrocellulose membrane and preparation method and application thereof - Google Patents
Wax-patterned nitrocellulose membrane and preparation method and application thereof Download PDFInfo
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- CN101726591A CN101726591A CN200910220594A CN200910220594A CN101726591A CN 101726591 A CN101726591 A CN 101726591A CN 200910220594 A CN200910220594 A CN 200910220594A CN 200910220594 A CN200910220594 A CN 200910220594A CN 101726591 A CN101726591 A CN 101726591A
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Abstract
The invention provides a wax-patterned nitrocellulose membrane and a preparation method and application thereof. The nitrocellulose membrane is in a structure that all the front side, the back side and the inner part are provided with wax areas and nitrocellulose areas which alternately appear as well as hydrophilic areas and hydrophobic area which alternately appear. The preparation method comprises the following steps of: designing the required pattern by using a computer, then printing wax on the surface of the untreated nitrocellulose membrane by using a printer; and placing the printed nitrocellulose membrane into a high-temperature vessel for roasting, taking out the roasted nitrocellulose membrane and cooling to obtain the wax-patterned nitrocellulose membrane. The invention is applied to the adsorption and the fixation of protein, the patterning of the protein, immunoassay, sample purification realized by intercepting impurities with micrometer size and other analysis operations; and the method has the advantages of simple process, directness, low cost and high manufacturing cost, is especially suitable for preparing the wax-patterned nitrocellulose membrane on a large scale and has commercial prospect.
Description
Technical field
The present invention relates to biochemical analysis product development and application thereof, particularly a kind of Wax-patterned nitrocellulose membrane, its preparation method and application.
Background technology
Nitrocellulose filter has obtained using widely as a kind of protein adsorption commonly used, base material fixing, reaction, such as common spot immune, and early pregnancy test paper etc.And with respect to traditional nitrocellulose filter, the resulting Wax-patterned nitrocellulose membrane of method for making that the present invention utilizes wax spray to print has the alternately structure between composition, close and distant water of wax/cellulose nitrate.
Micro-fluidic chip (Microfluidics, Lab-on-a-chip) be a frontier interdisciplinary, refer to integrated or be integrated into substantially on the chip of more than square centimeters (even littler) basic operation units such as specimen preparation related in the biochemical field, reaction, separation, detections, form network by the microchannel, run through total system with controlled fluid, in order to replace a kind of technology of the breadboard various functions of conventional biological or chemical.It is the important component part of micro-nano technology, also is one of major technique platform of systems biology research.
The main manufacturing materials of micro-fluidic chip comprises silicon, glass, quartz, polymkeric substance (the most frequently used is dimethyl silicone polymer and organic glass).In recent years, Whitesides group of Harvard University brings into use cheaply filter paper as base material (the Patterned Paper as a Platform forInexpensive of chip manufacturing, Low-Volume, Portable Bioassays Andres W.Martinez, Scott T.Phillips, Manish J.Butte, and George M.Whitesides Angew.Chem.Int.Ed.2007,46,1318-1320).This is that the micro-fluidic chip of base material has following advantage with paper: 1, method for making simple, fast, cost is low; 2, reaction member and driver element are fully integrated on the chip; 3, do not need the control of software, need not extraneous interface; 4, do not need exterior power source, be fit to open-air the analysis; 5, volume is little, cost is low, the operation is simple especially.Because it has the prospect of development portability, low-cost diagnostic analysis platform, thereby the research of this respect is also among launching rapidly.And the base material of the making papery micro-fluidic chip of bibliographical information is mainly the common filter paper that pure cellulose is formed at present, such as a filter paper of Whatman company production.But the common filter paper protein adsorption ability that this pure cellulose is formed, thereby be not suitable as protecpectic carrier, can not on such chip, finish diagnostic analysis operations such as immunoassay.
The present invention adopts this material with very strong protein adsorption ability commonly used of nitrocellulose filter of being made up of nitrocellulose to replace common filter paper and has made the papery micro-fluidic chip, have following advantage: 1, the refill sheet of being made by nitrocellulose filter has very strong protein adsorption, fixing ability, thereby immunoassay can well be finished on this chip; 2, compare with the average pore size (10 to 100 microns) of common filter paper, the average pore size of nitrocellulose filter (0.45 micron) is littler, thereby in the bake process after wax spray is printed, the diffusion of wax on nitrocellulose filter can be controlled better, thereby can obtain the microchannel of minimum 100 micron-scales, the edge of the pattern that obtains simultaneously is also more even; 3, the average pore size of nitrocellulose filter is 0.45 micron, thereby the solid of micron-scale can be trapped, so Wax-patterned nitrocellulose membrane can be used for filtering the impurity of micron level, the sample preparation ability is stronger; 4, it is more even to compare the surface of cellulose nitrate with common filter paper, thereby liquid flowing in the above more is and repeats; 5, it is more fine and close to compare the cellulose of nitrocellulose filter with common filter paper, thereby the physical strength that can bear is bigger.Therefore utilize cellulose nitrate film preparation papery micro-fluidic chip to have crucial meaning.
Summary of the invention
The purpose of this invention is to provide a kind of Wax-patterned nitrocellulose membrane, its preparation method and application.
The invention provides a kind of Wax-patterned nitrocellulose membrane, this nitrocellulose filter is front, the back side and inner wax zone and the regional alternately structure of appearance of appearance, hydrophilic region and hydrophobic region that replaces of cellulose nitrate of being.Fluid sample just can arrive by the restriction of wax and specify conversion zone like this, finishes response analysis.Owing to the restriction of wax, each conversion zone can the phase mutual interference simultaneously.
The present invention also provides the preparation method of nitrocellulose filter, the pattern of wanting with the computer design, with printer wax is printed to the surface of untreated nitrocellulose filter then, printed nitrocellulose filter is put into elevated temperature vessel (as baking oven, hot plate etc.) baking in, baking temperature is between 100 degrees centigrade to 150 degrees centigrade, stoving time is between 4 minutes to 1 hour, make the wax fusing and see through nitrocellulose filter, forming wax/cellulose nitrate alternately forms, nitrocellulose filter between close and distant water, take out cooling then, promptly obtain Wax-patterned nitrocellulose filter.Whole process can be finished within 5-10 minute, and is easy and simple to handle.
The method for making of nitrocellulose filter provided by the invention, described tools adopt business-like wax spray printer and baking oven, and printing is flexible, easy, quick, and the baking operation is easy too fast.Thereby utilize this method can large batch of making Wax-patterned nitrocellulose membrane cheaply, be very suitable for business development.
The preparation method of nitrocellulose filter provided by the invention, described elevated temperature vessel is a kind of in the containers such as baking oven, hot plate; Described printer is the wax spray printer.
Nitrocellulose filter provided by the invention can be applicable to protein adsorption, fixing, and albumen patterning etc. are analyzed operation, and the coffee toroidal effect that usually exists during effectively Profilin is fixed; Can be applicable to immunoassay (as spot immune etc.) etc. and analyze operation; Also can utilize the aperture of the sub-micron of nitrocellulose filter that the impurity of micron-scale is held back analysis operations such as realizing the sample purifying.Also can be applicable to many quota students fractional analysis of various liquid samples such as urine, blood, saliva, water, as be used for urine detection test paper, water quality monitoring test paper etc.
Wax-patterned nitrocellulose membrane of the present invention can be finished the filtration of various liquid samples such as urine, blood, saliva, water, remove the wherein impurity of micron-scale (such as cell etc.), various liquid samples such as urine, blood, saliva, water are directly applied in the biochemical analysis.
Creativeness of the present invention is: 1, this common filter paper of material replacement with very strong protein adsorption ability commonly used of nitrocellulose filter has been made the papery micro-fluidic chip, and has finished the immunoassay operation on this film; 2, make quick Wax-patterned nitrocellulose membrane fast, in enormous quantities with the wax spray printer, easy and simple to handle, manufacturing speed is fast, is highly suitable for commercialization.
The present invention have method for making simple, fast, low cost and other advantages.
Description of drawings
The flow process of Fig. 1 wax spray printing and making Wax-patterned nitrocellulose membrane, wherein: 1 is nitrocellulose filter, and 2 is printing step, and 3 is baking procedure, and 4 is Wax-patterned nitrocellulose membrane, and 5 is outboard profile, and 6 is wax, and 7 is cellulose nitrate.
Fig. 2 prints the variation of nitrocellulose filter front before and after baking of waxing.
Fig. 3 prints the variation of nitrocellulose filter back before and after baking of waxing.
Fig. 4 prints the variation of nitrocellulose filter square section before and after baking of waxing, and wherein: 1 is wax.
Fig. 5 is that wherein: 1 is the front of parallel printing by the variation of Wax-patterned formation cellulose nitrate microchannel before and after making, and 2 is vertical front of printing, 3 is the back side of parallel printing, 4 is the vertical back side of printing, and 5 is 600 microns passages of parallel printing, and 6 is the vertical 600 microns passages of printing.
Fig. 6 is the comparison (scale be 3 micron) of aperture before and after making of nitrocellulose filter.
Fig. 7 is the minimum stamp with the size of the passage of Wax-patterned formation.
Fig. 8 is the minimum dimension that wax can play hydrophobic interval action, and wherein: 1 is 60 microns.
Fig. 9 is the Wax-patterned minimum dimension that forms circular and square pattern at nitrocellulose filter, and wherein: 1 is 200 microns, and 2 is 1000 microns, and 3 is 400 microns.
Figure 10 be Wax-patterned nitrocellulose membrane protein adsorption ability sign and with untreated nitrocellulose filter protein adsorption result's comparison, wherein: 1 is Wax-patterned nitrocellulose membrane, and 2 is nitrocellulose filter.
Figure 11 is the relation (3 millimeters reaction size) of Wax-patterned nitrocellulose membrane protein adsorption result and application of sample volume, has only as can be seen when bag is surpassed 4 microlitres by volume from figure, and bag is even by zone ability result.
Figure 12 be various difform albumen bags by the result, wherein: 1 is 1 millimeter * 1 millimeter, and 2 is 1 millimeter, and 3 is 800 microns, and 4 is 300 microns, and 5 is 1000 microns.
Figure 13 Wax-patterned nitrocellulose membrane is used for the working curve of human immunoglobulin(HIg) fluoroimmunoassay and 50 mcg/ml human immunoglobulin(HIg) immunoassay unit results' analysis of Uniformity, and wherein: 1 is the analysis result of the human immunoglobulin(HIg) of 50 mcg/ml.
Figure 14 Wax-patterned nitrocellulose membrane is used for human immunoglobulin(HIg) colour developing immunoassay result (utilizing collaurum as immune labeled and combination silver dyeing amplifying signal), wherein: 1 is blank, 2 is 1 nanograms/milliliter human immunoglobulin(HIg), 3 is 10 nanograms/milliliter human immunoglobulin(HIg)s, 4 is 100 nanograms/milliliter human immunoglobulin(HIg)s, 5 is 1000 nanograms/milliliter human immunoglobulin(HIg)s, and 6 is 10000 nanograms/milliliter human immunoglobulin(HIg)s.
Figure 15 Wax-patterned nitrocellulose membrane is used for the comparison (the amaranth solution of 1 micron silica gel particle and small-molecular weight is as test solution) of sample filtering and filtration capacity thereof and Wax-patterned common filter paper, wherein: 1 is 1 micron silica gel particle, 2 is amaranth solution, 3 is Wax-patterned common filter paper, and 4 is Wax-patterned nitrocellulose membrane, and 5 is front view (FV), 6 is back view, 7 is 50 mg/ml, and 8 is 5 mg/ml, and 9 is 0.5 mg/ml.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Embodiment 1: the preparation method of Wax-patterned nitrocellulose membrane
Design needed pattern on computers with mapping software, on nitrocellulose filter, print the wax pattern with the wax spray printer then, again printout surface there is the nitrocellulose filter of wax to put into 125 degrees centigrade baking oven baking 5 minutes (this process wax will melt and through nitrocellulose filter), take out cooling then, promptly arrived Wax-patterned nitrocellulose filter.Idiographic flow (comprises mainly and prints and toasted for two steps that whole flow process can be finished) as shown in Figure 1 within 10 minutes.With the outward appearance of the Wax-patterned nitrocellulose membrane of this preparation method preparation, size, surface topography etc. characterize as Fig. 2, Fig. 3, and Fig. 4,5, Fig. 6, Fig. 7, Fig. 8, shown in Figure 9.
This method for making is simple, direct, cost is low, and manufacturing speed is very fast, is very suitable for making on a large scale Wax-patterned nitrocellulose membrane, has Commercial Prospect.
Embodiment 2:
Carry out immunoassay with Wax-patterned nitrocellulose filter, specific implementation process is as follows: whole fluoroimmunoassay process (with human immunoglobulin(HIg) (IgG) as the model analysis thing) is described as follows:
1, in the patterned nitrocellulose membrane reaction member of 3 mm sizes that limit by wax, adds goat anti-human immunoglobulin's solution, because cellulose nitrate has stronger protein adsorption ability, thereby antibody protein can adsorb in its surface, the bag of albumen on Wax-patterned nitrocellulose membrane characterized as Figure 10, Figure 11, shown in Figure 12;
2, the bovine serum albumin solution (BSA) that adds 5 mg/ml, BSA solution seals to reduce non-specific adsorption conversion zone;
3, add cleansing solution PBS+0.05%Tween 20, washing reaction zone;
4, the sample that adds human immunoglobulin(HIg), goat anti-human immunoglobulin's immune response of going up the bag quilt in human immunoglobulin(HIg) in the sample and the conversion zone;
5, add cleansing solution PBS+0.05%Tween 20 again, the washing reaction zone;
6, add goat anti-human immunoglobulin's solution of fluorescein isothiocynate (FITC) mark, goat anti-human immunoglobulin's solution of marked by fluorescein isothiocyanate combines with immunoreactive product in the previous step reactions steps, forms immune complex to be detected;
7, fluoroscopic examination obtains the immunodiagnosis result.
The volume of the solution that per step adds in the above step is 4 microlitres.
The working curve of the human IgG that on this immunoassay system, obtains as the model analysis thing with human IgG, as shown in figure 13.
Wax-patterned nitrocellulose filter carries out the colloid gold label immunoassay and carries out the process that silver dyeing amplifies as follows:
1, with 1-5 step in the fluoroimmunoassay;
2, add goat anti-human igg's solution of colloid gold label, goat anti-human igg's solution of colloid mark combines with immunoreactive product in the previous step reactions steps, forms immune complex to be detected;
3, add cleansing solution PBS+0.05%Tween 20 again, the washing reaction zone;
4, add distilled water washing reaction zone;
5, add the silver-colored staining solution of existing preparation, join conversion zone, carry out silver dyeing.Colloid gold immune obtains analysis result as shown in figure 14 in conjunction with silver dyeing.
Embodiment 3:
Remove the impurity of sample with Wax-patterned nitrocellulose filter.Because the aperture of nitrocellulose filter is very little, has only 0.45 micron, thereby the impurity of micron-scale can be held back, as shown in figure 15.The silica gel particle of 1 micron-scale has obtained holding back on Wax-patterned nitrocellulose filter, and the silica gel particle of 1 micron-scale is not trapped on Wax-patterned common filter paper.And the micromolecule amaranth can be passed through on two kinds of Wax-patterned nitrocellulose membranes and common filter paper, thereby the sample of Wax-patterned nitrocellulose filter can be used for removing the impurity of various biological liquid samples such as blood, urine micron-scale such as cell etc., and carries out micromolecular response analysis operation overleaf.
Claims (5)
1. Wax-patterned nitrocellulose membrane is characterized in that: this nitrocellulose filter is positive, the back side and the inner alternately structure of appearance of wax zone and the alternately appearance of cellulose nitrate zone, hydrophilic region and hydrophobic region that is.
2. the preparation method of the described nitrocellulose filter of claim 1, it is characterized in that: the pattern of wanting with the computer design, with printer wax is printed to the surface of untreated nitrocellulose filter then, printed nitrocellulose filter is put into elevated temperature vessel to be toasted, baking temperature is between 100 degrees centigrade to 150 degrees centigrade, stoving time is between 4 minutes to 1 hour, takes out cooling then, promptly obtains Wax-patterned nitrocellulose filter.
3. according to the preparation method of the described nitrocellulose filter of claim 2, it is characterized in that: described elevated temperature vessel is a kind of in baking oven, some containers of hot plate.
4. according to the preparation method of the described nitrocellulose filter of claim 2, it is characterized in that: described printer is the wax spray printer.
5. the described nitrocellulose filter of claim 1 is applied to protein adsorption, fixing, albumen patterning; Immunoassay; The impurity of micron-scale is held back some analysis operations of realization sample purifying.
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| CN104181153B (en) * | 2013-05-28 | 2017-01-04 | 郑兆珉 | Handwriting paper raw medical detection test piece and manufacturing method thereof |
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| CN105954151A (en) * | 2016-04-27 | 2016-09-21 | 浙江工业大学 | Method for detecting authenticity of Baijiu by using gradient wetting surface |
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Application publication date: 20100609 |