CN110437978A - The integrated detection micro-fluidic chip of bacteria total amount, bacterial spore amount - Google Patents
The integrated detection micro-fluidic chip of bacteria total amount, bacterial spore amount Download PDFInfo
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- CN110437978A CN110437978A CN201910737916.8A CN201910737916A CN110437978A CN 110437978 A CN110437978 A CN 110437978A CN 201910737916 A CN201910737916 A CN 201910737916A CN 110437978 A CN110437978 A CN 110437978A
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Abstract
The invention discloses a kind of bacteria total amount, the integrated detection micro-fluidic chip of bacterial spore amount and its applications, chip includes two independent channels, it is respectively used to ATP biloluminescence method and DPA fluorescence detection, it is divided into two parts after sample introduction passage, a part is by cracking zone, scrubbing section and reaction zone, another part is finally all measured by Systems for optical inspection by cracking zone, scrubbing section, reaction zone and fluorescence lasing region to determine bacteria total amount and spore quantity.The present invention can have many advantages, such as integrated, high sensitivity with operating in a key, detection bacterium total amount and bacterial spores while can be rapid and facilitate.
Description
Technical field
The present invention design micro fluidic chip technical field, and in particular to one kind can be used for spacecraft simultaneously detection bacterium total amount with
The integrated micro-fluidic chip of bacterial spore.
Background technique
Purpose celestial body scene life detection may be will affect from the fly to spaceborne bacterial pollutant of other planetaries of the earth
Experiment and the celestial body celestial body sample returned, the angle protected from planet must select suitable method to carry out bacterium inspection to spacecraft
It surveys, determines its bacteria total amount and endure the bacterial spore amount of the extreme environments such as intense radiation, drying, and then use suitable method
Dissipation is carried out to maintain spacecraft high cleaning environment.Current spacecraft Bacteria Detection is using microbial culture technique.Use this
Kind, which carries out microorganism detection to spacecraft, certain deficiency: precision is insufficient first, because certain micro-organisms can not be cultivated
Property is easy to underestimate the microbial total quantity of spacecraft,.Followed by bacterial spore culture needs the culture with total bacterium to separate, i.e., first
It first passes through 80 DEG C of heating and kills microbial nutrition body cells, then just cultivable bacteria gemma.Final time is too long, cultivation results
Generally require 72 hours or longer time.Therefore needing to develop one kind being capable of quick, easy, sensitive detection spacecraft bacterium
Method.
Biloluminescence method detection ATP has the characteristics that high specific, higher sensitivity, fast and easy and wider measurement range, with
Society development, the mankind continue to develop and improve ATP biloluminescence method, make it in food hygiene, environmental monitoring, drug sieve
The fields such as choosing, medical research have a wide range of applications.The method of the detection bacteriums such as traditional the method for plate culture count is compared, and is used
ATP biloluminescence method carries out total number of bacteria detection, does not need prolonged incubation, has easy to operate, high sensitivity, sound
The advantages that rapid is answered, in food hygiene and environmental monitoring, can quickly have found that it is likely that existing germ contamination, and determine that it contains
Amount.
DPA is distinctive ingredient in bacterial spore, account for about gemma dry weight~10%, can be split by physics, the chemistry of spore
The modes such as solution or rudiment release.So DPA can be used as the important biomolecule marker of bacterial spore for detection bacterium bud
The presence of spore.DPA is also a kind of inorganic chemistry ligand of classics, it combines metal ion with high-affinity.DPA and europium
Ion (or other luminous group of the lanthanides or transition metal ions) combines, and strong luminescence phenomenon is triggered under ultraviolet excitation.According to
DPA fluorescent staining technique, which is carried out, according to this principle measures bacterial spores.
Micro-fluidic chip (Microfluidic chip) is also known as Microfluid based Lab on a chip or chip lab (Lab-on-a-
Chip).Micro-fluidic chip is that actually detected main platform is realized in microflow control technique.Micro-fluidic chip is mainly characterized by it
The channel for accommodating fluid is micron order scale, and the manipulation of fluid is also to realize in short space, to realize in small chip
Upper building chemistry or biology laboratory, can be by the process of a variety of chemistry and biologies under microfluidic system rapidly and automatically
It completes.
In current existing research, there is report of the micro-fluidic chip based on ATP bioluminescence for Bacteria Detection, but
It is up to now, there has been no by ATP bioluminescence, DPA fluorescent light combination, while the person's of can detecte bacteria total amount and bacterium spore
The integrated micro-fluidic chip report of son.
Summary of the invention
The present invention provides a kind of bacteria total amount, the integrated detection micro-fluidic chip of bacterial spore amount and its applications, are used for
Realize sample feeding, sample treatment, the whole process process of example reaction and sample detection.It realizes on micro-fluidic chip simultaneously
In conjunction with two methods of ATP fluorogenic quantitative detection bacteria total amount and DPA fluorogenic quantitative detection bacterial spore, have small in size, integrated
Change, sample and the advantages that reagent consumption is few, high sensitivity.
The purpose of the present invention is achieved through the following technical solutions:
The integrated detection micro-fluidic chip of bacteria total amount, bacterial spore amount, which is characterized in that including chip basal body, fluid
Channel, sample process unit, reaction member, fluorescence detection unit.The fitting of sample process unit is arranged on chip body, including
Cracking zone and scrubbing section, sample enter sample process unit by sample intake passage and are pre-processed；Reaction member fitting setting exists
On chip body, including sample application zone and fluorescence lasing region, reactant and reaction reagent can be transported to reaction zone by fluid channel
In domain, to complete to react；Fluorescence detection unit includes single photon counter, data show and is connected to the end computer PC to show
As a result.
Further, include two accesses in chip basal body, be respectively equipped with the sample process unit and the reaction member, and
It is connected to by the fluid channel with the fluorescence detection unit.
Further, the sample process unit, the reaction member and the fluorescence detection unit are in chip basal body in straight
Line arrangement.
Further, the sample treatment unit and the reaction member include first in the channel 1 of chip basal body side
Cracking zone, the first scrubbing section, the first sample application zone；And it the second cracking zone in the channel 2 of the chip basal body other side, second washes
Wash area, third sample application zone and fluorescence lasing region.
Further, sample respectively enters two passes by Y type fluid channel and is reacted and detected.
Further, the fluid channel between the first cracking zone and the first scrubbing section between the second cracking zone and the second scrubbing section
It is wrapped up by the tube wall equipped with cold water for cooling down.
Micro-fluidic chip provided by the invention has the advantage that
It is fast to detect speed, analysis efficiency is high.Realize that the relevant function of automatically analyzing and detect only needs on micro-fluidic chip
Several seconds or even shorter time, the analysis efficiency of sample is substantially increased, fast and accurately obtains testing result.
Sample and reagent consumption are few.Since channel size is micro-nano rank, required test sample and phase in micro-fluidic chip
Close biological reagent amount be microlitre even nanoliter, for expensive biological reagent, a large amount of testing cost can be saved, and
It can equally be detected under conditions of certain sample extremely pettiness.
Function is integrated.Integrate sample feeding, sample pretreatment, sample washing, example reaction, sample detection, greatly
Operating process is simplified, sample and reagent consumption is reduced, there is huge economic value and social value.
Detailed description of the invention
In order to illustrate more clearly of the technical solution in the present invention, the attached drawing of embodiment will be briefly described below, show and
Easy insight, the accompanying drawings in the following description is only a part of the present embodiment, for those of ordinary skill in the art, not
Under the premise of making the creative labor, it is also possible to obtain other drawings based on these drawings.
Fig. 1 is the schematic diagram of micro-fluidic chip proposed by the present invention.
Wherein, each appended drawing reference in figure:
1- chip basal body；The first cracking zone 2-；The first scrubbing section of 3-；The first reaction zone of 4-；The first detection zone of 5-；6- second is cracked
Area, the second scrubbing section of 7-；The second reaction zone 8-；9- fluorescence lasing region；The second detection zone of 10-；11- sample holes；12- sample introduction is logical
Road；13- sample intake passage；14- excites optical channel；15- sample outlet hole；16- fluid channel.
Specific embodiment
In order to which the purpose of the present invention, structure and function is more clearly understood, below in conjunction with attached drawing, to a kind of miniflow of the invention
Control chip and its application are described in further detail.It is to be appreciated that based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
As shown in Figure 1, micro-fluidic chip proposed by the present invention includes: chip basal body 1, it is equipped with parallel two in chip basal body and leads to
Road a, wherein channel is successively arranged the first cracking zone 2, the first scrubbing section 3, the first reaction zone 4 and the first inspection by arranged in a straight line
Survey area 5；Another channel is equally by the second cracking zone 6, the second scrubbing section 7, second reaction zone 8, glimmering arranged in a straight line of being successively arranged
Light lasing region 9 and the second detection zone.So that the micro-fluidic chip collection sample feeding, sample pretreatment, sample washing, sample
Reaction, sample detection greatly simplifie operating process in one, reduce sample and reagent consumption, and do not have to be equipped with high
Expensive instrument, significantly reduces use cost, makes it possible that scene is detected immediately, has huge economic value and society
Value.
The chip basal body 1 is additionally provided with sample holes 11 and sample outlet hole 15, the sample dissociation area, scrubbing section, reaction zone and detection
Pass through 16 connection of microfluidic channel between area.The sample that detection zone provides after reaction carries out it using Systems for optical inspection glimmering
The space of light detection, sample is completed on the micro-fluidic chip after reacting and detecting, by fluid channel from 15 row of sample outlet hole
Out.
Specifically, a kind of specific embodiment of micro-fluidic chip proposed by the invention is on the micro-fluidic chip
In combination with two methods of ATP fluorescent quantitation and DPA fluorescent quantitation, fast and effeciently to detect Intracellular bacterial total amount and thin
Bacterium spore total amount.
Further, as shown in Figure 1, a kind of specific embodiment as micro-fluidic chip provided by the invention, for ATP
Fluorescent quantitation, sample are entered the first cracking zone 2, are eliminated extracellular ATP using heating means, and by intracellular ATP
It all releases, enters the first reaction zone 4 by the first scrubbing section 3.In addition, fluorescein-luciferase reagent passes through fluid
Channel 12 enters the first reaction zone 4, is reacted with pretreated sample, enters the first detection zone by fluid channel later
5, it is detected and is quantified using Systems for optical inspection.
Further, as shown in Figure 1, a kind of specific embodiment as micro-fluidic chip provided by the invention, for DPA
Fluorescent quantitation, sample enter the second cracking zone 6, using the DPA in heating means release bacterial spore, in the second scrubbing section 7
It is inside centrifuged and collects the supernatant containing DPA；In addition, entering second instead by fluid channel by pretreated sample
Area 8 is answered, is reacted with the Eu-GNPS-TNFB complex solution entered by fluid channel 13；Solution after reaction enters glimmering
Light lasing region 9, imports fluid channel 14 for exciting light, carries out fluorescence excitation to sample, enters the second detection zone 10 later and utilizes
Systems for optical inspection is detected and is quantified to it.
Further, as shown in Figure 1, a kind of specific embodiment as micro-fluidic chip provided by the invention, described first
The volume of reaction tank 4 and the second reaction tank 8 is greater than the volume in sample dissociation area and scrubbing section, can guarantee to accommodate completely in this way
The reagent of required addition.
Further, as shown in Figure 1, a kind of specific embodiment of the micro-fluidic chip provided as invention, above-mentioned micro-fluidic
Chip further includes light shield layer (attached drawing is not made), which can be used for reaction zone, fluorescence lasing region, detection zone and connection institute
The fluid channel for stating reaction chamber covers in, and can guarantee chip interior sample solution and reaction product not by extraneous visible in this way
The interference of light and detection light source.
Structure setting between the reaction chamber and sample holes and sample outlet hole can carry out diversification, the same miniflow according to demand
It can be set side by side with multiple reaction chambers simultaneously on control chip, pass through a plurality of Micro-flow pipe connection each other.
Claims (6)
1. the integrated detection micro-fluidic chip of bacteria total amount, bacterial spore amount, which is characterized in that including chip basal body, stream
Body channel, sample process unit, reaction member, fluorescence detection unit.The fitting of sample process unit is arranged on chip body, packet
Cracking zone and scrubbing section are included, sample enters sample process unit by sample intake passage and pre-processed；Reaction member fitting setting
On chip body, including reaction zone and fluorescence lasing region, reactant and reaction reagent can be transported to reaction by fluid channel
In region, to complete to react；Fluorescence detection unit fitting is arranged on chip body.
2. micro-fluidic chip as described in claim 1, which is characterized in that include two accesses in chip basal body, be respectively equipped with
The sample process unit and the reaction member, and be connected to by the fluid channel with the fluorescence detection unit.
3. micro-fluidic chip as claimed in claim 2, which is characterized in that the sample process unit, the reaction member and
The fluorescence detection unit is arranged in a linear in chip basal body.
4. micro-fluidic chip as claimed in claim 2, which is characterized in that the sample treatment unit and the reaction member packet
Include the first cracking zone, the first scrubbing section, the first sample application zone in the channel 1 of chip basal body side；And it is another in chip basal body
The second cracking zone, the second scrubbing section, third sample application zone and fluorescence lasing region in the channel 2 of side.
5. micro-fluidic chip as described in claim 1, sample respectively enters two passes by Y shape fluid channel and is reacted
With detection.
6. micro-fluidic chip as claimed in claim 4, between the first cracking zone and the first scrubbing section and the second cracking zone and the
Fluid channel between two scrubbing sections is wrapped up by the tube wall equipped with cold water for cooling down.
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Cited By (5)
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| CN111690510A (en) * | 2020-06-11 | 2020-09-22 | 上海前瞻创新研究院有限公司 | Pathogenic bacterium detection reactor and preparation and application methods thereof |
| CN111804356A (en) * | 2020-07-16 | 2020-10-23 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
| CN111920973A (en) * | 2020-08-12 | 2020-11-13 | 北京航空航天大学 | Integrated method, process and device for killing planet protection microorganisms |
| CN113514453A (en) * | 2021-04-21 | 2021-10-19 | 清华大学 | Micro-fluidic chip for detecting bacteria, detector, detection method and application |
| WO2023221023A1 (en) * | 2022-05-19 | 2023-11-23 | 京东方科技集团股份有限公司 | Gene detection substrate, gene detection chip, and preparation method for gene detection sample |
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| CN111690510A (en) * | 2020-06-11 | 2020-09-22 | 上海前瞻创新研究院有限公司 | Pathogenic bacterium detection reactor and preparation and application methods thereof |
| CN111690510B (en) * | 2020-06-11 | 2023-07-21 | 上海前瞻创新研究院有限公司 | Pathogenic bacteria detection reactor and preparation and application methods thereof |
| CN111804356A (en) * | 2020-07-16 | 2020-10-23 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
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| CN111920973A (en) * | 2020-08-12 | 2020-11-13 | 北京航空航天大学 | Integrated method, process and device for killing planet protection microorganisms |
| CN111920973B (en) * | 2020-08-12 | 2021-12-17 | 北京航空航天大学 | Integrated method, process and device for killing planet protection microorganisms |
| CN113514453A (en) * | 2021-04-21 | 2021-10-19 | 清华大学 | Micro-fluidic chip for detecting bacteria, detector, detection method and application |
| CN113514453B (en) * | 2021-04-21 | 2022-07-15 | 清华大学 | Micro-fluidic chip for detecting bacteria, detector, detection method and application |
| WO2023221023A1 (en) * | 2022-05-19 | 2023-11-23 | 京东方科技集团股份有限公司 | Gene detection substrate, gene detection chip, and preparation method for gene detection sample |
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