CN105670920A - Total bacterial count detection micro-fluidic chip, total bacterial count detection system and total bacterial count detection method - Google Patents
Total bacterial count detection micro-fluidic chip, total bacterial count detection system and total bacterial count detection method Download PDFInfo
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Abstract
The invention discloses a total bacterial count detection micro-fluidic chip.The total bacterial count detection micro-fluidic chip comprises a substrate, a liquid inlet, a detection cavity, a liquid outlet, a liquid inlet passage, a liquid outlet passage and an optical fiber interface.The liquid inlet which forms on the substrate is used for leading detection liquid inside; the detection cavity forms in the substrate; the liquid outlet which forms on the substrate is used for discharging the detection liquid; the liquid inlet passage which forms in the substrate is used for leading the detection liquid from the liquid inlet to the detection cavity; the liquid outlet passage which forms in the substrate is used for leading the detection liquid from the detection cavity to the liquid outlet; the optical fiber interface is used for leading a detection signal out and is arranged above the substrate surrounded by the detection cavity.The whole area of the chip is only several square centimeters, so that the chip is a miniature detection chip.In addition, the micro-fluidic chip has the advantages that human disturbance factors are reduced, attenuation of fluorescence intensity in a detection process is reduced, and detection accuracy is improved.The invention further discloses a total bacterial count detection system and a total bacterial count detection method.
Description
Technical field
The present invention relates to a kind of micro-fluidic chip detection technique, particularly relate to a kind of detection total number of bacterial colony micro-fluidic chip and detection system thereof and detection method.
Background technology
Total number of bacterial colony refers under certain condition (such as aerobic situation, nutritional condition, pH, culture temperature and time etc.) every gram of (every milliliter) sample grown bacterial colony quantity out. In water, the mensuration of total number of bacterial colony is an important indicator of water quality monitoring (WQM), and the microbial contamination of its direct reaction water, artificial swimming-pool water, Drinking Water etc. all need total number of bacterial colony to be detected.
ATP is a kind of high-energy phosphate compound, is made up of with three phosphate groups being connected a molecule VITAMIN B4, a molecule ribose. In chemical reaction, ATP is mainly through release phosphate group thus reaches the object discharging energy. Using ATP bioluminescence detection method, the result of its chemical reaction is that chemical energy is converted to luminous energy, and when having enough fluoresceins and oxygen, the quantity of ATP is directly proportional to the power of optical signal. This kind of optical signal can be detected by particle fluorescence detector, each luminous value can corresponding corresponding total plate count, thus reach the object of instant bacterial detection sum. ATP biloluminescence method has not to be needed to cultivate, easy and simple to handle, complete the advantages such as detection fast.
Having the detection kit for ATP noclilucence in existing market, reagent dosage is big; Detection needs relevant precision instrument to support the use, the purchase of instrument, use and maintenance cost height; On-line checkingi can not be realized in operation, add human factor, reduce accuracy in detection.
Summary of the invention
It is an object of the invention to provide a kind of detection total number of bacterial colony micro-fluidic chip and detection system thereof and detection method, it is possible to the total number of bacterial colony in detection solution fast.
For achieving the above object, the present invention provides following technical scheme: a kind of micro-fluidic chip detecting total number of bacterial colony, comprises substrate; Fluid inlet, for introducing detection liquid and be formed at described substrate; Test chamber, is formed in described substrate; Liquid outlet, for discharging detection liquid, and is formed on described substrate; Feed pathway, for detection liquid is introduced described test chamber from described fluid inlet, is formed in described substrate; Liquid outlet channel; For detection liquid is introduced described liquid outlet from described test chamber, and it is formed in described substrate; And optical fiber interface, draw for signal will be detected, and it is arranged at the top of the substrate that described test chamber is surrounded.
The present invention provides the detection chip of a kind of miniatureization, and detection solution is that micro updating is other, and feed pathway, liquid outlet channel and test chamber are all that milli is meter-sized, and whole chip area is only several square centimeters, is the detection chip of a kind of miniatureization.Simultaneously micro-fluidic chip decreases artificial interference factor, decrease the decay of its fluorescence intensity in testing process, it is to increase accuracy in detection.
Preferably, described test chamber is cylindrical, and the degree of depth is 2mm～5mm, and diameter is 2mm～7mm, and described feed pathway is " Z " shape hybrid channel, and its corner number of degrees are 30 °～60 °, and channel depth is 0.5mm～1mm, and width is 0.2mm～0.8mm.
Preferably, the described substrate described in substrate comprises up and down the interface layer of superposition successively, photic zone, detection layers and reflective immovable bed
Preferably, described fluid inlet and described liquid outlet run through described interface layer and photic zone up and down; Described optical fiber interface runs through described interface layer up and down, and described feed pathway, test chamber and liquid outlet channel are positioned at described detection layers.
Preferably, described euphotic transmittance is 90%～98%.
Preferably, described interface layer and the material of detection layers are independently selected from polymethylmethacrylate or polydimethylsiloxane, and described euphotic material is polyethylene terephthalate, and the material of described reflective immovable bed is acrylonitrile-butadiene-styrene (ABS).
Micro-fluidic chip adopts four Rotating fields, and chip material adopts the material can purchased on market, can be mass, and price is cheap.
Preferably, described fluid inlet comprises the first fluid inlet being connected respectively and the 2nd fluid inlet with described feed pathway one end.
Accordingly, the invention also discloses a kind of detection system detecting total number of bacterial colony, it is characterized in that: comprise micro-fluidic chip and Photoelectric Detection module, described Photoelectric Detection module is used for fluorescence intensity, and described Photoelectric Detection module is connected with the optical fiber interface of described micro-fluidic chip.
This detection system uses Photoelectric Detection module to detect, and avoids large-scale instrument and buys, uses and maintenance cost. It is used in conjunction with light detection module, it is achieved on-line checkingi, decreases human factor, substantially reduce detection time, add accuracy in detection. This micro-fluidic chip design configuration can be connected in Photoelectric Detection module, can be implemented in line detection total number of bacterial colony by related software.
Preferably, described detection system also comprises syringe pump and syringe, and syringe is connected with the fluid inlet of micro-fluidic chip.
Accordingly, a kind of detection method detecting total number of bacterial colony, comprises the steps:
(1) in first fluid inlet and the 2nd fluid inlet of micro-fluidic chip, inject ATP extracting solution and ATP detection solution respectively;
(2), ATP extracting solution and ATP detect solution and fully mix in feed pathway, ATP is luminous with the fluorescein in detection solution and luciferase reaction, enters test chamber;
(3) when detection liquid enters into, through feed pathway, the fluorescence intensity that described test chamber just detects mixing solutions, taking the logarithmic value of ATP concentration as X-coordinate, the logarithmic value of bioluminescence intensity is ordinate zou, draw ATP typical curve, determine its minimum detectability, and then utilize ATP content to be directly proportional to bacterial count this principle, extrapolate total number of bacterial colony;
(4) mixed solution is discharged to the described liquid outlet of inflow through described liquid outlet channel.
This detection method adopts micro-fluidic chip, and detection method is simple, and less reagent consumption, reduces sample cost, decreases environmental pollution.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present application or technical scheme of the prior art, it is briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, the accompanying drawing that the following describes is only some embodiments recorded in the application, for those of ordinary skill in the art, under the prerequisite not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
The structural representation that Fig. 1 show in the specific embodiment of the invention a kind of micro-fluidic chip detecting total number of bacterial colony;
The interface layer structural representation that Fig. 2 show in the specific embodiment of the invention a kind of micro-fluidic chip detecting total number of bacterial colony;
The photic zone structural representation that Fig. 3 show in the specific embodiment of the invention a kind of micro-fluidic chip detecting total number of bacterial colony;
The detection layers structural representation that Fig. 4 show in the specific embodiment of the invention a kind of micro-fluidic chip detecting total number of bacterial colony;
The reflective immovable bed structural representation that Fig. 5 show in the specific embodiment of the invention a kind of micro-fluidic chip detecting total number of bacterial colony;
Fig. 6 is the system schematic of a kind of micro-fluidic chip detecting total number of bacterial colony in invention specific embodiment
Embodiment
Shown in composition graphs 1, the structure of the micro-fluidic chip of detection total number of bacterial colony, the fluid inlet 5 comprising substrate 11 and being formed on the substrate 11. Fluid inlet 5 is connected with one end of feed pathway 8, the other end of feed pathway 8 and one end of liquid outlet channel 9 are formed with test chamber 10, mixing liquid stops in test chamber 10, optical fiber interface 7 it is provided with in test chamber 10, test chamber 10 is connected with one end of liquid outlet channel 9, the other end of liquid outlet channel 9 is connected with liquid outlet 6, and liquid outlet 6 is for discharging mixing solutions.
In use, mixing solutions enters into feed pathway 8 through fluid inlet 5, hybrid reaction in feed pathway 8, enter into test chamber 10, test chamber 10 is collected the fluorescence intensity of mixing solutions, mixing liquid enters into liquid outlet channel 9 from test chamber 10, discharges through the liquid outlet being connected with liquid outlet channel 9.
In the present embodiment, fluid inlet 5 is circular hole, and its diameter is 2mm. Fluid inlet 5 has two, and wherein the first fluid inlet 5 is for ATP extracting solution import, and the 2nd fluid inlet 5 is for detecting solution inlet. Certainly, also it is not limited to two herein. When fluid inlet 5 is 1, directly in fluid inlet 5, inject mixing solutions.
Feed pathway 8 be positioned on substrate circulation passage, the degree of depth of feed pathway 8 be preferably 1mm, width be preferably 0.5mm. Feed pathway 8 is " Z " type hybrid channel, and its corner number of degrees are preferably 40 degree, and channel depth is preferably 1mm, width is preferably 0.5mm, " Z " type hybrid channel is adopted obviously to shorten the mixed solution residence time in the channel, it is to increase its mixing efficiency so that it is enter rapidly detection zone.
Test chamber 10 forms the detection zone of mixing solutions, and its shape is circular, is certainly also not limited to this shape herein, is blended in test chamber 10 and sends fluorescence, and its size to be optimized coupling by light path coupling, and the degree of depth is preferably 3mm, and diameter is preferably 3.5mm.
Being provided with light interface 7 in test chamber 10, the diameter of optical fiber interface 7 is preferably 2.25mm, and optical fiber interface is used for connecing optical fiber.
Liquid outlet 6 is for discharging mixing liquid. The groove of liquid outlet channel 9 for being formed on substrate, the degree of depth is preferably 1mm, and width is preferably 0.5mm. Liquid outlet channel 9 is linear, to discharge mixing liquid with shortest distance. Liquid outlet 6 can be identical with the diameter of liquid outlet, naturally it is also possible to is other sizes.
In the present embodiment, substrate 11 comprises up and down the interface layer of superposition successively, photic zone, detection layers and reflective immovable bed, adopts gluing method to be bonded together between these four layers.
Shown in composition graphs 2-4, fluid inlet and liquid outlet run through interface layer and photic zone up and down; The interface layer that optical fiber interface runs through up and down, feed pathway, test chamber and liquid outlet channel are positioned at detection layers.
The interface layer 1 of the micro-fluidic chip of detection total number of bacterial colony, has offered fluid inlet 5 and liquid outlet 6 on interface layer 1, and the number of fluid inlet 5 is 2, and the number of liquid outlet 6 is 1, is certainly also not limited to this number herein. Fluid inlet 5 enters sample for sample liquid and detection liquid, and liquid outlet 6 goes out sample for what detect mixed solution, and fluid inlet 5 and liquid outlet 6 are the through hole run through up and down. Also offering optical fiber interface 7 on interface layer 1, for connecing optical fiber, this optical fiber interface 7 is also through hole.
Interface layer 1 adopts polymethyl methacrylate materials, it is also possible to adopt the poly-thing material of the higher primary schools such as polydimethylsiloxane. The thickness of this interface layer 1 is preferably 2mm.
Photic zone 2 is for adopting polyethylene terephthalate thin film, and polyethylene terephthalate thin film has good light transmission, can not light detection be had an impact, and avoids fiber end face with solution simultaneously and directly contacts, and pollution fiber end face, affects detected result.
The detection layers 3 of the micro-fluidic chip of detection total number of bacterial colony, feed pathway 8 and liquid outlet channel 9 has been offered on detection layers 3, feed pathway 8 is the groove being positioned at circulation, feed pathway 8 is " Z " type hybrid channel, " Z " type hybrid channel is adopted obviously to shorten the mixed solution residence time in the channel, improve its mixing efficiency so that it is enter rapidly detection zone. The principle of design of feed pathway is under the prerequisite not affecting mixing efficiency, reduces the mixed solution residence time in the channel as far as possible.
The other end of feed pathway 8 and one end of liquid outlet channel 9 are formed with test chamber 10, and mixing liquid stops in test chamber 10, thus forms detection data, and test chamber 10 is circular hole, and its size to be optimized coupling by light path coupling.
One end of the groove of feed pathway 8 is connected with fluid inlet 5 on photic zone 2 with interface layer 1, and the other end of the groove of liquid outlet channel 9 is connected with the liquid outlet 6 on photic zone 2 with interface layer 1.
Test chamber 10 in detection layers 3 coincides with the center of circle of the circular hole of the optical fiber interface 7 of interface layer 1.
Detection layers 3 adopts polymethyl methacrylate materials, and feed pathway 8, test chamber 10 and liquid outlet channel 9 etc. adopt CNC to process, and detection layers 3 can also adopt the high polymer materials such as polydimethylsiloxane.
Reflective immovable bed 4 is used for sealing, and prevents leakage and the spilling of mixed solution at the back side. In the present embodiment, reflective immovable bed 4, it is the acrylonitrile-butadiene-styrene (ABS) of White-opalescent, can scatter light to optical fiber interface 7 by antireflection part, thus improve light detection efficiency.
The present embodiment also discloses a kind of detection system detecting total number of bacterial colony, as shown in Figure 6, comprising micro-fluidic chip 12 and Photoelectric Detection module, described Photoelectric Detection module is used for fluorescence intensity, and described Photoelectric Detection module is connected with the optical fiber interface of described micro-fluidic chip 12. Detecting portion adopts light detection module, and comprising phosphor collection module 13, signal processing module 14 and data display module 15, wherein phosphor collection module 13 comprises photon detector, single photon counter. Single photon counting principle is under utilizing low light condition, photon detector exports the naturally discrete feature of electrical signal, adopting numeral counting technology and pulse screening techniques that extremely weak optical signal is carried out quantization identification, its sensitivity and linear measurement range all substantially exceeds other routine techniquess.
This detection system can also comprise syringe pump and syringe, specifically not shown, and syringe is connected with the fluid inlet of micro-fluidic chip. Syringe pump is that injection liquid provides power.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is described in detail, it is clear that described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, other embodiments all that those of ordinary skill in the art obtain under the prerequisite not making creative work, all belong to the scope of protection of the invention.
In application ATP noclilucence intensity detection water body, the reaction principle of total number of bacterial colony is: ATP noclilucence jointly completes under luciferase, fluorescein and ATP exist. Under the effect of luciferase and Mg2+, fluorescein is provided energy reduction activation by ATP, and the fluorescein after activation combines with luciferase, generates fluorescein (L)-AMP complex body, discharges tetra-sodium. Subsequently under oxygen participates in, the oxidized generation of fluorescein (L)-AMP complex body is electrically excited, and the electronics in fluorescein molecule transits to Polyfluorenes by excited state and goes out photon, produces fluorescence. In theory, luminous intensity is directly proportional to ATP molecular amounts, can determine ATP concentration by fluorescence intensity. At present, existing much research shows and confirms: there is good linear relationship between ATP content and microbe population, it is possible to use ATP content is directly proportional to bacterial count this principle, extrapolates total number of bacterial colony.
Adopt the detecting step of total number of bacterial colony in this micro-fluidic chip detection water body as follows:
(1) putting up the detection system of a kind of micro-fluidic chip, Photoelectric Detection module, wherein Photoelectric Detection is rubbed and is comprised photon detector, single photon counter and corresponding software, tests;
(2) optical fiber interface 7 of the optical fiber end of Photoelectric Detection module and chip is connected. Getting out two syringe pumps, syringe and micro-fluidic chip are connected, syringe is placed on syringe pump.
(3) open syringe pump, under its dynamic action, ATP extracting solution and detection solution are injected in the fluid inlet 5 of micro-fluidic chip simultaneously;
(4) ATP extracting solution and water body enter into described feed pathway 8, ATP extracting solution and detect solution and mix in feed pathway 8, react.
(5) mixing solutions enters into test chamber 10, opens Photoelectric Detection module and test chamber 10 is carried out continuous kinetic measurement, survey its fluorescence intensity.
(6) taking the logarithmic value of ATP concentration as X-coordinate, the logarithmic value of noclilucence intensity is ordinate zou, draws ATP typical curve, it is determined that its minimum detectability, and then utilizing ATP content to be directly proportional to bacterial count this principle, the total number of bacterial colony extrapolating water body is that every mL is no more than 100.
(7) mixed solution is flowed into described liquid outlet 6 from described liquid outlet channel 9 and discharges.
Finally, also it should be noted that, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, so that comprise the process of a series of key element, method, article or equipment not only comprise those key elements, but also comprise other key elements clearly do not listed, or also comprise the key element intrinsic for this kind of process, method, article or equipment.
It should be noted that, herein, the such as relational terms of first and second grades and so on is only used for separating an entity or operation with another entity or operational zone, and not necessarily requires or imply to there is any this kind of actual relation or sequentially between these entities or operation. And, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, so that comprise the process of a series of key element, method, article or equipment not only comprise those key elements, but also comprise other key elements clearly do not listed, or also comprise the key element intrinsic for this kind of process, method, article or equipment. When not more restrictions, the key element limited by statement " comprising ... ", and be not precluded within process, method, article or the equipment comprising described key element and also there is other identical element.
The above is only the embodiment of the application; it is noted that for those skilled in the art, under the prerequisite not departing from the application's principle; can also making some improvements and modifications, these improvements and modifications also should be considered as the protection domain of the application.
Claims (10)
1. one kind is detected the micro-fluidic chip of total number of bacterial colony, it is characterised in that, comprising:
Substrate;
Fluid inlet, for introducing detection liquid and be formed at described substrate;
Test chamber, is formed in described substrate;
Liquid outlet, for discharging detection liquid, and is formed on described substrate;
Feed pathway, for detection liquid is introduced described test chamber from described fluid inlet, is formed in described substrate;
Liquid outlet channel; For detection liquid is introduced described liquid outlet from described test chamber, and it is formed in described substrate;
And optical fiber interface, draw for signal will be detected, and it is arranged at the top of the substrate that described test chamber is surrounded.
2. the micro-fluidic chip of detection total number of bacterial colony according to claim 1, it is characterized in that: described test chamber is cylindrical, the degree of depth is 2mm～5mm, diameter is 2mm～7mm, described feed pathway is " Z " shape hybrid channel, its corner number of degrees are 30 °～60 °, and channel depth is 0.5mm～1mm, and width is 0.2mm～0.8mm.
3. the micro-fluidic chip of detection total number of bacterial colony according to claim 1, it is characterised in that: described substrate comprises up and down the interface layer of superposition successively, photic zone, detection layers and reflective immovable bed.
4. the micro-fluidic chip of detection total number of bacterial colony according to claim 3, it is characterised in that: described fluid inlet and described liquid outlet run through described interface layer and photic zone up and down; Described optical fiber interface runs through described interface layer up and down, and described feed pathway, test chamber and liquid outlet channel are positioned at described detection layers.
5. the micro-fluidic chip of detection total number of bacterial colony according to claim 3, it is characterised in that: described euphotic transmittance is 90%～98%.
6. the micro-fluidic chip of detection total number of bacterial colony according to claim 3, it is characterized in that: described interface layer and the material of detection layers are independently selected from polymethylmethacrylate or polydimethylsiloxane, described euphotic material is polyethylene terephthalate, and the material of described reflective immovable bed is acrylonitrile-butadiene-styrene (ABS).
7. the micro-fluidic chip of detection total number of bacterial colony according to claim 1, it is characterised in that: described fluid inlet comprises the first fluid inlet being connected respectively and the 2nd fluid inlet with described feed pathway one end.
8. one kind is detected the detection system of total number of bacterial colony, it is characterized in that: comprise the micro-fluidic chip described in the arbitrary item of claim 1-7 and Photoelectric Detection module, described Photoelectric Detection module is used for fluorescence intensity, and described Photoelectric Detection module is connected with the optical fiber interface of described micro-fluidic chip.
9. a kind of detection system detecting total number of bacterial colony according to claim 8, it is characterised in that: described detection system also comprises syringe pump and syringe, and syringe is connected with the fluid inlet of micro-fluidic chip, and described syringe is placed on syringe pump.
10. one kind is detected the detection method of total number of bacterial colony, it is characterised in that, comprise the steps:
(1) in first fluid inlet and the 2nd fluid inlet of the micro-fluidic chip described in the arbitrary item of claim 1-7, inject ATP extracting solution and ATP detection solution respectively;
(2), ATP extracting solution and ATP detect solution and fully mix in feed pathway, ATP is luminous with the fluorescein in detection solution and luciferase reaction, enters test chamber;
(3) when detection liquid enters into described test chamber through feed pathway, the fluorescence intensity of detection mixing solutions, taking the logarithmic value of ATP concentration as X-coordinate, the logarithmic value of bioluminescence intensity is ordinate zou, draw ATP typical curve, determine its minimum detectability, and then utilize ATP content to be directly proportional to bacterial count this principle, extrapolate total number of bacterial colony;
(4) mixed solution is discharged to the described liquid outlet of inflow through described liquid outlet channel.
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| CN106591105A (en) * | 2016-12-09 | 2017-04-26 | 东北大学 | Microorganism automatic sample introduction system based on microfluidic chip and preparation method thereof |
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| CN108535241A (en) * | 2018-05-22 | 2018-09-14 | 中国科学院武汉病毒研究所 | A kind of tachysynthesis detection device and its application |
| CN111889150A (en) * | 2020-07-01 | 2020-11-06 | 西安交通大学 | ATP fluorescent microfluidic chip, bioluminescence continuous detection system and detection method |
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