CN105954273A - "Relief" type paper-based micro-fluidic detection device and production method thereof - Google Patents
"Relief" type paper-based micro-fluidic detection device and production method thereof Download PDFInfo
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- CN105954273A CN105954273A CN201610288961.6A CN201610288961A CN105954273A CN 105954273 A CN105954273 A CN 105954273A CN 201610288961 A CN201610288961 A CN 201610288961A CN 105954273 A CN105954273 A CN 105954273A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of design and production of rapid detection devices of biological residues, and especially relates to a "relief" type paper-based micro-fluidic detection device. The "relief" type paper-based micro-fluidic detection device comprises a substrate reaction zone and a substrate basement zone, and the substrate reaction zone is arranged on the surface of the substrate basement zone, and is made from a plurality of miniature hydrophilic wood fiber filaments, and a reaction substrate and color development reagent layer which is embedded in the miniature hydrophilic wood fiber filaments in an infiltration mode; and the substrate basement zone is made of a hydrophobic organic material. The production method comprises the following steps: 1, taking a commercial hydrophobic material, carrying out aseptic treatment on the commercial hydrophobic material, and cutting the treated material to form the substrate basement zone; and 2, placing hydrophilic whatman filter paper in the substrate basement zone under an aseptic condition to form the substrate reaction zone, adding a prepared reaction substrate and color development reagent solution to the whatman filter paper in a dropwise manner, infiltrating the filter paper at 25-40DEG C for 0.5-2h, and airing the infiltrated filter paper under an aseptic condition to obtain the "relief" type paper-based micro-fluidic detection device. The detection device can be used to carry out accurate, rapid and roughly quantitative detection on pathogenic organisms in an animal source food.
Description
Technical field
The invention belongs to design and the preparation and application technical field of the quickly detection equipment of biological residue, particularly relate to one " sculptured " paper substrate micro-fluidic detection equipment and preparation method thereof.
Background technology
It is one of subject matter perplexing food safety that food source pathogenic microorganism pollutes, and it quick and precisely detects and monitor the first line of defence being to ensure food safety.The most conventional detection technique mainly has conventional biochemical culture technique, immunological technique, the Protocols in Molecular Biology that liquid chromatography technology and newly-developed get up, conventional biochemical culture technique has special advantage accurately, but program is complicated, take time and effort, the detection cycle is longer, cannot meet and quickly detect needs, the detection technique grown up based on immunology and Protocols in Molecular Biology has good specificity and susceptiveness, the most quickly, but it is higher to the degree of dependence of expensive instrument and equipment and professional and technical personnel, quickly detection and the monitoring needs of food production one line cannot be met.
Paper substrate micro-fluidic detection device (micro fluidic paper based analytical devices, μ PADs) it is that the one grown up in recent years prepares simplicity, with low cost, several free of contamination Fast Detection Technique, its ultimate principle is: by Computer Design figure, wax spray prints filter paper, prepare hydrophobic isolation area, at isolation area endoperidium reactant, the quick detection to checking matter is realized by reaction on paper, the method and conventional biochemical culture technique, immunological technique, the molecular Biological Detection technology of liquid chromatography technology sum has good complementarity, compensate for deficiency and the limitation of aforementioned several method, be especially suitable for food production one line quickly detects needs.
But, owing to wax spray printing technique had exited market in recent years, wax spray printer market is difficult to find, cause the micro-fluidic detection technique of paper substrate to get into a difficult position, and its alternative method such as photolithographic techniques, octadecyl trimethoxysilane (TMOS) etching technique etc. all exist manufactured paper disc preparation complexity, less stable, be difficult to preservation defect.
Summary of the invention
It is an object of the invention to: a kind of " sculptured " paper substrate micro-fluidic detection equipment of design, use this equipment, animal derived food pathogenic microorganism can be carried out accurate fast qualitative detection；Further utilize the mathematical model in known enzyme substrate reactions principle, on the basis of in default detection equipment, substrate is a certain amount of, by known mathematical model relational expression, indirectly calculate the basal ration of tested microorganism, can preferably meet the easy detection requirements such as the Quality Control of food production one line, sampling observation, primary dcreening operation.
Technical solution of the present invention: a kind of " sculptured " paper substrate micro-fluidic detection equipment, this detection equipment is made up of two parts: substrate reactions district and substrate basal area, and described substrate reactions district is vertically arranged in substrate basal area surface；Described substrate reactions district is by possessing some small hydrophilic wood fibre silks and being uniformly embedded in the reaction substrate in wood fibre silk and colour generation oxidant layer is made by pattern of invasion；Described substrate basal area is made up of hydrophobic organic material.
Described substrate reactions district is circular or rectangle or irregularly shaped, or the arbitrary shape required according to detection mesh.Described substrate reactions district is higher than substrate basal area 1-5mm.Described substrate reactions district is the whatman filter paper containing reaction substrate and colour generation oxidant layer.Described substrate basal area is for possessing hydrophobic single or double glue.Described reaction substrate and colour generation oxidant layer are the material group that can produce chromogenic reaction under the specific enzymes catalysis that tested bacteria produces.
A kind of preparation method of " sculptured " paper substrate micro-fluidic detection equipment, after this preparation method comprises the steps: that (1) takes commercially available hydrophobic single or double adhesive tape aseptic process, it is cut into the area more than more than 2 times of substrate reactions district as hydrophobic substrate basal area, standby；(2) aseptically, hydrophilic whatman filter paper is fabricated to the shape required, is placed on the substrate basal area that step (1) prepares, i.e. form substrate reactions district；Then, the reaction substrate prepared in advance and photoghraphic coupler solution are uniformly dripped on whatman filter paper, in wood fibre silk tiny in uniformly reaction substrate and photoghraphic coupler being embedded in whatman filter paper by pattern of invasion, under the conditions of 25-40 DEG C, infiltrate 0.5-2h；Dry under aseptic condition, i.e. can be made into " sculptured " paper substrate micro-fluidic detection equipment.
Beneficial effect: use this detection equipment that animal derived food pathogenic microorganism can carry out accurate Fast Coarse detection by quantitative；Utilize the mathematical model in known enzyme substrate reactions principle, on the basis of substrate is a certain amount of in default detection equipment, indirectly calculate the basic quantity of tested microorganism, it is achieved quickly, the testing requirement of basal ration.Simultaneously, instant invention overcomes uses the substitute technology such as photoetching and chemical etching to prepare complexity and the unstability of paper substrate micro-fluidic detection equipment after wax spray printing technique is quit listing, maintain the paper substrate micro-fluidic detection simple advantage of equipment preparation method, and material is simple, cheap, several pollution-free.
Accompanying drawing explanation
Accompanying drawing 1 is embodiment 1 main TV structure schematic diagram；Accompanying drawing 2 is A-A sectional view；Accompanying drawing 3 is the main TV structure schematic diagram of embodiment 2；Accompanying drawing 4 is the main TV structure schematic diagram of embodiment 3；Accompanying drawing 5 is the main TV structure schematic diagram of embodiment 4；Accompanying drawing 6 is the linear relationship chart of LM bacterial concentration and gray value.
Detailed description of the invention
Embodiment 1, a kind of " sculptured " paper substrate micro-fluidic detection equipment, as shown in Figure 1-2, this detection equipment is made up of two parts: substrate reactions district 1 and substrate basal area 2, and described substrate reactions district 1 is vertically arranged in substrate basal area 2 surface；Described substrate reactions district 1 is by possessing some small hydrophilic wood fibre silks 4 and being uniformly embedded in the reaction substrate in wood fibre silk 4 and colour generation oxidant layer 5 is made by pattern of invasion；Described substrate basal area 2 is made up of hydrophobic organic material 3.
Described substrate reactions district 1 is higher than substrate basal area 21-5mm.Described substrate reactions district 1 is the whatman filter paper containing reaction substrate and colour generation oxidant layer 5.Described substrate basal area 2 is for possessing hydrophobic single or double glue.Described reaction substrate and colour generation oxidant layer 5 are the material group that can produce chromogenic reaction under the specific enzymes catalysis that tested bacteria produces.
Embodiment 2,3 and 4, as shown in Figure 2-5, described substrate reactions district 1 is circular or rectangle or irregularly shaped, or the arbitrary shape required according to detection mesh.
Embodiment 5, the preparation method of a kind of " sculptured " paper substrate micro-fluidic detection equipment, after this preparation method comprises the steps: that (1) takes commercially available hydrophobic single or double adhesive tape aseptic process, it is cut into the area more than more than 2 times of substrate reactions district as hydrophobic substrate basal area, standby；(2) aseptically, hydrophilic whatman filter paper is fabricated to the shape required, is placed on the substrate basal area that step (1) prepares, i.e. form substrate reactions district；Then, the reaction substrate prepared in advance and photoghraphic coupler solution are uniformly dripped on whatman filter paper, in wood fibre silk tiny in uniformly reaction substrate and photoghraphic coupler being embedded in whatman filter paper by pattern of invasion, under the conditions of 25-40 DEG C, infiltrate 0.5-2h；Dry under aseptic condition, i.e. can be made into " sculptured " paper substrate micro-fluidic detection equipment.
Concrete practice: under aseptic condition, whatman filter paper is tailored into circle or rectangle or the hydrophilic reaction zone of arbitrary shape according to detection mesh requirement, commercially available single or double adhesive tape is prepared as the strip more than hydrophilic reaction zone diameter more than 2 times as hydrophobic group base area；The hydrophilic reaction zone whatman filter paper prepared is pasted on it.The substrate of enzyme-specific microorganism to be checked produced and developer are embedded in hydrophilic reaction zone, such as: the bromo-4-of X-InP(5-chloro-3-indoxyl inositol monophosphate ammonium) etc., under the conditions of 25-40 DEG C, infiltrate 0.5-2h；Aseptic valve bag packaging is put in drying, is used as food source microbial rapid detection and is used.
Wherein, optimal hydrophilic reaction zone diameter, hydrophobic effect screening.(1) reference literature data setting 6,7,8,9 and the hydrophilic area of 10mm diameter, dropping color aqueous solution (blue ink) test, calculate through grayvalue transition, it is thus achieved that optimal chromogenic reaction district a diameter of 6-8 mm.(2) the hydrophilic reaction zone of 7mm diameter is used, dropping commercial inks stock solution, dripping quantity is respectively 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 microlitres totally 13 gradients, measure its optimal hydrophobic effect, through comparison analysis, 1 and the 5 hydrophilic reaction zones of microlitre are full not exclusively, though 10 and 15 microlitres are full completely, but not yet form obvious surface tension, it is not enough to reflect the buffer action of hydrophobic region, 20 microlitre sides see have started to be formed the most convex in the shell of tension on surface, hydrophilic area, after 25 microlitres, surface tension layer raises, still complete hydrophobic effect can be shown to 60 microlitres, bottom hydrophobic matrix layer ne-leakage, consider with optimal economic practical standpoint, screening 20-25 microlitre is as optimal hydrophobic dosage.
Embodiment 6, bacterial concentration determine with gray value dose-effect relationship:
(1) bacteria resuscitation: the bacterium solution of the listeria monocytogenes reference culture CICC 21633 being stored in-20 DEG C with glycerol is recovered, carries out plate streaking afterwards, isolated and purified.(2) bacterium solution enrichment: the Listeria monocytogenes bacterium colony grown fine in picking plate after purification, carries out the bacterium solution enrichment of 8h.(3) bacterium solution being enriched with is carried out respectively the dilution of 2,4,8,10,20 times, is added drop-wise to respectively on test-paper after mixing, after 1.5h, terminate reaction, observe colour developing result and be scanned.(4) count plate
The bacterium solution of enrichment 8h is carried out count plate, obtains the bacterial concentration that shown color is corresponding.(5) gradation conversion: with ImageJ1.4u software, colour developing result is carried out gray scale reading, obtain the gray value of color reaction.(6) set up regression equation: the bacterial concentration of gained and gray value relation use Graph Pad Prism 5 statistical software to carry out data process, and set up regression equation and be: y=-0.03850 x+9.615, r2=0.9357；As shown in Figure 6.
Conclusion: can well be proved by the practice of embodiment 6, by the sculptured Test paper of the present invention (equipment), it can be deduced that the semiquantitative analysis result of tested bacteria.The easy detection requirements such as the Quality Control of food production one line, sampling observation, primary dcreening operation can be met.
The technology of the present invention principle: independent research one Special-purpose puncher prepares the difform filter paper of different-diameter as hydrophilic reaction zone (substrate reactions district), using commercially available common single or double adhesive tape as hydrophobic isolation basic unit's carrier (substrate basal area), hydrophilic reaction zone filter paper is pasted onto in hydrophobic isolation basic unit, forms the hydrophilic detection zone of " embossment " sample (substrate reactions district).
The innovative point of the present invention: initiated and hydrophilic reaction zone (substrate reactions district) is highlighted on hydrophobic isolation area basic unit (substrate basal area), the surface tension and the hydrophobic iris action of surrounding that rely on hydrophilic reaction zone filter paper prepare paper substrate micro-fluidic detection device, method, the chemical reaction that detection occurs can be limited in consistent specific region by the method, it is achieved thereby that reacts can be quantitative, and making material availability is strong, it is easy, with low cost to prepare, the Test paper made can make up the scarcity of current rapid and convenient detection technique, has filled up the blank in this field.
Claims (7)
1. " sculptured " paper substrate micro-fluidic detection equipment, it is characterised in that: this detection equipment is made up of two parts: substrate reactions district (1) and substrate basal area (2), and described substrate reactions district (1) is vertically arranged in substrate basal area (2) surface；Described substrate reactions district (1) is by possessing some small hydrophilic wood fibre silk (4) and being uniformly embedded in the reaction substrate in wood fibre silk (4) and colour generation oxidant layer (5) is made by pattern of invasion；Described substrate basal area (2) is made up of hydrophobic organic material (3).
Detection equipment the most according to claim 1, it is characterised in that: described substrate reactions district (1) is circular or rectangle or irregularly shaped.
Detection equipment the most according to claim 1 and 2, it is characterised in that: described substrate reactions district (1) is higher than substrate basal area (2) 1-5mm.
Detection equipment the most according to claim 1, it is characterised in that: described substrate reactions district (1) is containing reaction substrate and the whatman filter paper of colour generation oxidant layer (5).
Detection equipment the most according to claim 1, it is characterised in that: described substrate basal area (2) is for possessing hydrophobic single or double glue.
Detection equipment the most according to claim 1, it is characterised in that: described reaction substrate and colour generation oxidant layer (5) they are the material group that can produce chromogenic reaction under the specific enzymes catalysis that tested bacteria produces.
7. the preparation method of " sculptured " paper substrate micro-fluidic detection equipment, it is characterized in that: after this preparation method comprises the steps: that (1) takes commercially available common two-sided tape aseptic process, it is cut into the area more than more than 2 times of substrate reactions district as hydrophobic substrate basal area, standby；(2) aseptically, hydrophilic whatman filter paper is fabricated to the shape required, is placed on the substrate basal area that step (1) prepares, i.e. form substrate reactions district；Then, the reaction substrate prepared in advance and photoghraphic coupler solution are uniformly dripped on whatman filter paper, in wood fibre silk tiny in uniformly reaction substrate and photoghraphic coupler being embedded in whatman filter paper by pattern of invasion, under the conditions of 25-40 DEG C, infiltrate 0.5-2h；Dry under aseptic condition, i.e. can be made into " sculptured " paper substrate micro-fluidic detection equipment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580489A (en) * | 2017-09-29 | 2019-04-05 | 深圳先进技术研究院 | The chip and method of blood glucose and blood lipid are detected simultaneously |
| CN110709699A (en) * | 2017-05-24 | 2020-01-17 | 格拉斯哥大学校董会 | Metabolite detection device and method of detecting metabolites |
| US11376582B2 (en) | 2019-03-05 | 2022-07-05 | International Business Machines Corporation | Fabrication of paper-based microfluidic devices |
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| US5801061A (en) * | 1997-04-22 | 1998-09-01 | Environmental Test Systems, Inc. | Method for the colorimetric determination of analytes in the presence of interfering particulate materials |
| CN1800341A (en) * | 2005-12-09 | 2006-07-12 | 清华大学 | Method for immobilizing lipase using microstructure in hydrophilic/ hydrophobic composite membrane |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110709699A (en) * | 2017-05-24 | 2020-01-17 | 格拉斯哥大学校董会 | Metabolite detection device and method of detecting metabolites |
| CN110709699B (en) * | 2017-05-24 | 2022-03-04 | 格拉斯哥大学校董会 | Metabolite detection device and method of detecting metabolites |
| CN109580489A (en) * | 2017-09-29 | 2019-04-05 | 深圳先进技术研究院 | The chip and method of blood glucose and blood lipid are detected simultaneously |
| US11376582B2 (en) | 2019-03-05 | 2022-07-05 | International Business Machines Corporation | Fabrication of paper-based microfluidic devices |
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