CN112697679A - Device for rapidly detecting number of bacteria in beverage - Google Patents
Device for rapidly detecting number of bacteria in beverage Download PDFInfo
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- CN112697679A CN112697679A CN202011411273.7A CN202011411273A CN112697679A CN 112697679 A CN112697679 A CN 112697679A CN 202011411273 A CN202011411273 A CN 202011411273A CN 112697679 A CN112697679 A CN 112697679A
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- 230000001580 bacterial effect Effects 0.000 claims abstract description 8
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- G01N15/1023—
Abstract
The invention relates to the field of bacteria counting, and discloses a device for rapidly detecting the number of bacteria in a beverage, which comprises a microfluidic chip; the micro-fluidic chip comprises a chip substrate and the following flow paths arranged on the chip substrate: the two ends of the sheath flow inflow channel are respectively connected with the sheath flow inlet and the sheath flow fulcrum; both ends of the first sheath flow branch and the second sheath flow branch are respectively connected with a sheath flow fulcrum and a sample focusing point; the sample inlet and the sample channel are arranged between the first sheath flow branch and the second sheath flow branch; two ends of the sample channel are respectively connected with the sample inlet and the sample focusing point; two ends of the focusing fluid channel are respectively connected with the sample focusing point and the waste liquid outlet; an optical detection area is arranged in the middle of the focusing fluid channel. The device has higher accuracy to the bacterial detection to can simplify the flow path, be convenient for wash, thereby prevent that the bacterium from remaining and causing cross contamination in the flow path, can also realize the detection of a small amount of samples in addition, thereby reduce the quantity of fluorescent dye, reduce and detect the cost.
Description
Technical Field
The invention relates to the field of bacteria counting, in particular to a device for rapidly detecting the number of bacteria in a beverage.
Background
The flow cytometer is an analysis instrument taking flow cytometry as a technical core, and can perform multi-parameter and rapid quantitative analysis and sorting on cells or biological particles in a rapid linear flow state, so that the flow cytometer has important application in the fields of clinical medicine, cell biology and the like. The flow cytometer is mainly divided into 4 parts, namely a liquid flow system, an optical system, a detection system and a data acquisition and analysis system, and the flow cytometer with the sorting function also comprises a sorting system.
The liquid flow system comprises a flow chamber and a liquid flow driving system, wherein in the flow chamber, hydrodynamic focusing is realized through a sheath flow technology, a stable liquid flow of single cell arrangement is formed in a channel, and cells in a suspension state pass through a detection area one by one at a high speed at a specific position. For example, chinese patent publication No. CN204903352U discloses a flow cytometer sheath fluid flow chamber, which includes a sheath flow chamber, a sample injection needle, and a sheath flow chamber upper end cap, wherein the center of the sheath flow chamber upper end cap is provided with a sample injection needle hole; the sample injection needle enters from a needle hole of the sample injection needle; the sheath flow chamber upper end cover is provided with a sheath liquid adding groove, a sheath flow channel hole and a sheath liquid drainage plate; the lower part of the sheath flow chamber is provided with a fluid focusing part, and the bottom of the fluid focusing part is provided with a sheath flow outlet. After the sample liquid to be measured and the sheath liquid respectively flow in through the sample injection needle and the sheath flow chamber, the two liquids coaxially flow, the sheath liquid surrounds the sample liquid to be measured and stably flows, then in the focusing part, the liquid flow enters the part with the smaller sectional area from the part with the larger sectional area, the focusing contraction effect is achieved, the diameter of the focused fluid is restricted in a certain range, and cells in the fluid are singly arranged.
After a stable flow of a single cell array is formed using the above-described fluidic system, cell counting can be performed, but when such flow cytometry is used to detect the number of bacteria in a beverage, the following problems arise: (1) the liquid flow system is complicated, the pipeline is long, the cleaning is inconvenient, and the cross contamination of the bacteria-containing sample is difficult to avoid. The practical detection experiment result shows that after the bacteria-containing sample (such as euthanasia) is detected, the sample needs to be cleaned for more than half an hour, so that the bacterial signal of the sample cannot be detected, and the long cleaning time means that the flow cytometer is not suitable for the rapid detection of the quantity of bacteria in the beverage; in addition, the bacteria are different from the animal and plant cells, the latter cannot reproduce after being separated from the environments such as proper culture solution, pH, temperature and the like, the former has strong reproduction capacity, and the fluid system is easy to breed bacteria and cause flow path abandonment as the dead angle is not cleaned. (2) The sample liquid and sheath liquid required by the flow cytometer are too large in volume (because the pipeline is long and the inner diameter is large), so that the consumption of the fluorescent dye is large, and the experiment cost is high. For a single experiment, the flow cytometer requires preparation of a sample fluid volume of typically 1mL, and the cost of staining a single sample is 40-100 yuan, calculated from the cost of Bacterial nucleic acid fluorescent dyes (e.g., LIVE/DEAD. sup. hatch. negative sensitivity Kit). (3) The flow cytometer is designed for cell detection, but not for bacteria detection, and since the sizes of bacteria and cells are not in an order of magnitude, the detection capability of the flow cytometer for bacteria is basically close to the detection lower limit, so that there is a high possibility that a plurality of bacteria are detected together, and it is difficult to ensure the accuracy of the detection result.
Disclosure of Invention
In order to solve the technical problem, the invention provides a device for rapidly detecting the number of bacteria in a beverage. The device has higher accuracy to the bacterial detection to can simplify the flow path, be convenient for wash, thereby prevent that the bacterium from remaining and causing cross contamination in the flow path, can also realize the detection of a small amount of samples in addition, thereby reduce the quantity of fluorescent dye, reduce and detect the cost.
The specific technical scheme of the invention is as follows:
the device for rapidly detecting the number of bacteria in the beverage comprises a fluid module, a detection module and a control module, wherein the fluid module comprises a microfluidic chip; the microfluidic chip comprises a chip substrate, and a sheath flow inlet, a sample inlet, a waste liquid outlet, a sheath flow inlet channel, a first sheath flow branch, a second sheath flow branch, a sample channel, a focusing fluid channel, a sheath flow fulcrum and a sample focusing point which are arranged on the chip substrate; the two ends of the sheath flow inflow channel are respectively connected with the sheath flow inlet and the sheath flow fulcrum; both ends of the first sheath flow branch and the second sheath flow branch are respectively connected with a sheath flow fulcrum and a sample focusing point; the sample inlet and the sample channel are arranged between the first sheath flow branch and the second sheath flow branch; two ends of the sample channel are respectively connected with the sample inlet and the sample focusing point; two ends of the focusing fluid channel are respectively connected with the sample focusing point and the waste liquid outlet; an optical detection area is arranged in the middle of the focusing fluid channel.
The invention realizes hydrodynamic focusing by utilizing the principle of a microfluidic chip laminar flow technology, and the specific mechanism process is as follows: in the microfluidic chip, sheath liquid enters from a sheath inflow port, passes through a sheath flow inflow channel, then branches at a sheath flow branch point, and flows into a first sheath flow branch and a second sheath flow branch; the sample liquid to be detected enters from the sample inlet, and after passing through the sample channel, the sample liquid and the sheath liquid are converged at a sample focusing point, the sheath liquid is annularly wrapped outside the sample liquid to be detected to realize hydrodynamic focusing, and the focusing fluid enters the focusing fluid channel to perform cell number detection in the optical detection area. By controlling the flow rate ratio of the sheath fluid to the sample fluid to be detected, the diameter of the focused fluid can be controlled, and cells can be singly arranged in the fluid.
Compared with the existing flow cytometry, the microfluidic chip is applied to the bacteria quantity detection device for hydrodynamic focusing, has a simple structure and a short flow path, is easy to clean, can prevent bacteria from remaining in the flow path to cause cross contamination, and can realize cell quantity detection by a small amount of samples, so that the consumption of fluorescent dye can be greatly reduced, and the detection cost is reduced. In addition, the flow chamber in the flow cytometer adopts micro-precision numerical control processing, so that the technical difficulty is high, the yield is low, and the cost is very high (one set of flow chamber is sold at a price of tens of thousands yuan); the micro-fluidic chip adopted by the invention has low consumable cost, and the selling price of a single chip is usually between hundreds and thousands of yuan, so that the hardware cost can be obviously reduced.
In addition, in the invention, the sheath liquid flows into the channel through the section of sheath flow before being branched into the two branch flows, which is beneficial to controlling the flow velocity of the two branch flows to be the same, thereby better controlling the fluid width after focusing, preventing cells from overlapping and entering the optical detection area, and improving the detection accuracy.
Preferably, the width of the first sheath flow branch, the width of the second sheath flow branch and the width of the sample channel are 10-500 μm, and the depth of the sample channel is 10-100 μm; the flow rate of the sheath liquid in the first sheath flow branch and the second sheath flow branch and the flow rate of the sample liquid to be detected in the sample channel are 1-100 mu L/min, and the ratio of the total flow rate of the sheath liquid (the sum of the flow rates of the sheath liquid in the first sheath flow branch and the second sheath flow branch) to the flow rate of the sample liquid to be detected is 1-50: 1.
In the microfluidic chip, the width and the flow rate ratio of the sample feeding channel of the sheath fluid and the sample fluid to be detected both affect the hydrodynamic focusing effect and affect the diameter of the focusing fluid. According to the invention, the diameter of the focusing fluid can be controlled to be 10-30 μm by controlling the channel size in the microfluidic chip and the flow rate ratio of the sheath fluid to the sample fluid, so that an ideal hydrodynamic focusing effect is obtained, and bacteria are singly arranged in the focusing fluid (the size of the bacteria is usually 0.5-20 μm).
The existing flow cytometer is designed for cell detection and not for bacteria detection, and because the sizes of bacteria and cells are not in an order of magnitude, the detection capability of the flow cytometer for the bacteria is basically close to the detection lower limit, so that the situation that a plurality of bacteria are detected together is likely to exist, and the accuracy of the detection result is difficult to ensure. The invention sets the channel size in the micro-control flow chip and the flow rate ratio of the sheath fluid and the sample fluid aiming at bacteria, and can ensure that the bacteria are not overlapped in the focusing fluid, thereby improving the detection accuracy.
Preferably, the width of the sheath flow inflow channel and the focusing fluid channel is 10 to 500 μm, and the depth thereof is 10 to 500 μm.
Preferably, in the optical detection area, the cross section of the focusing fluid channel is a rectangle with the diameter of 10-500 mu m multiplied by 10-500 mu m, and the outer wall of the channel is a rectangle with the diameter of 1-5 mm multiplied by 1-5 mm.
Preferably, the chip substrate is made of a material with good light transmittance between 300 nm and 800 nm.
The material of the chip substrate can adopt quartz, glass or sapphire and the like.
Preferably, the fluid module further comprises a sample injection system; the sample introduction system comprises a sample introduction unit and a sheath fluid introduction unit; the sample injection unit comprises a filter and a first precise sample injection pump which are connected in sequence; the sheath liquid sample introduction unit comprises a sheath liquid storage tank and a second precise sample introduction pump which are connected in sequence; and the first precision sample injection pump and the second precision sample injection pump are respectively connected with the sample inlet and the sheath flow inlet.
According to the invention, the first precision sample injection pump and the second precision sample injection pump are arranged, so that the precision sample injection of the sample liquid to be detected and the sheath liquid can be realized, the sample injection volume and the sample injection flow rate of the sample liquid to be detected and the sheath liquid can be precisely adjusted, the flow rate ratio of the sheath liquid to be detected and the sample liquid to be detected reaching the focus point of the sample can be controlled, and the bacteria in the focusing fluid are ensured not to be overlapped; in addition, the precision sample injection pump can ensure that the precision sample injection pump can drive the fluid stably, smoothly and without pulsation, so that the precision sample injection pump can form stable focusing fluid after passing through the micro-fluidic chip, and the detection of the number of bacteria at an optical detection area is facilitated.
The detection object of the existing flow cytometer is usually a cell solution, and it is required that a sample to be detected cannot contain other suspended particulate matters except cells, but a large amount of suspended particulate matters (such as protein, fat, plant powder and the like) exist in a beverage, so that a flow system (particularly a flow chamber in the flow system, the width of a channel is only 100-200 μm) is easily blocked, an optical detection system is interfered, and the flow cytometer cannot normally work. In the invention, the filter is arranged in the sample feeding system to filter the sample liquid to be detected, so that suspended particulate matters (such as protein, fat, plant powder, milk powder and the like) with overlarge sizes in the sample liquid can be removed, and the blockage of the fluid pipeline and the internal channel of the microfluidic chip is prevented.
Preferably, the first precision sample pump and the second precision sample pump are micro plunger pumps, syringe pumps or pneumatic pumps with flow sensors.
Preferably, the first precision sample pump and the sample inlet, and the second precision sample pump and the sheath inflow port are connected through a capillary or a Teflon tube with the inner diameter of 50-500 μm and the length of 5-50 cm.
Capillary, the tubular product that special fluorine dragon pipe has good chemical tolerance can tolerate the washing of bactericidal agent such as 10% sodium hypochlorite solution, is convenient for detect the washing of back pipeline to prevent that remaining bacterium from causing cross contamination.
Preferably, the filter is provided with a filter membrane with the pore diameter of 10-100 mu m.
Preferably, the device also comprises an optical detection module; the optical detection module comprises an incident light unit for forming a laser spot in an optical detection area of the focusing fluid channel, a forward scattered light detection unit for detecting the intensity of forward scattered light, a side scattered light detection unit for detecting the intensity of side scattered light, and a fluorescence detection unit for detecting the intensity of fluorescence.
Preferably, the laser spot has a length of 5 to 20 μm and a width of 0.5 to 5 μm.
Laser forms a light spot on the focusing fluid channel after being focused by the convex lens, the laser light spot adapts to the size of bacteria by controlling the length and the width of the laser light spot, only one bacterium exists in the laser irradiation range every time, and the accuracy of detecting the number of the bacteria is ensured.
In order to meet the requirement of cell detection, the size of a laser spot of the existing flow cytometer is usually 30 micrometers × 70 micrometers, and when the existing flow cytometer is used for detecting bacteria, a plurality of bacteria exist in a laser irradiation range, which causes errors on detection.
Preferably, the incident light unit includes a laser, a collimating aperture and a convex lens, the forward scattering light detection unit includes a first optical filter and a first photoelectric detection sensor, the lateral scattering light detection unit includes a concave lens, a first dichroic mirror, a second optical filter and a second photoelectric detection sensor, and the fluorescence detection unit includes a second dichroic mirror, a third optical filter and a third photoelectric detection sensor; laser emitted by the laser passes through the collimating hole and is focused by the convex lens to form a laser spot in an optical detection area of the focusing fluid channel; the forward scattered light of the laser passing through the optical detection area is emitted into the first photoelectric detection sensor through the first optical filter; the side scattered light of the laser passing through the optical detection area passes through the concave lens and then enters the first dichroic mirror; the transmitted light and the reflected light of the laser passing through the first dichroic mirror are respectively emitted into the second dichroic mirror and the second photoelectric detection sensor after passing through the second optical filter; and the laser passes through the third light filter and then enters the third photoelectric detection sensor after passing through the reflected light of the second dichroic mirror.
The bacteria passing through the optical detection area are irradiated by laser, forward scattered light can be generated along the irradiation direction of the laser, and lateral scattered light can be generated in the direction perpendicular to the incident light; after the side scattered light passes through the first dichroic mirror, the fluorescent light passes through the first dichroic mirror, and the rest of the color light is reflected. The signal intensity of the forward scattered light detected by the first photoelectric sensor can reflect the size of the bacteria, and the larger the signal intensity is, the larger the size of the bacteria is; the signal intensity of the side scattered light detected by the second photoelectric sensor is related to the refractive index of the bacteria and can reflect the internal structure of the bacteria; the signal intensity of the fluorescence detected via the third photosensor reflects the amount of bound fluorescent probe in the bacteria, thereby reflecting information on the relevant target detection object.
Preferably, a baffle is arranged between the optical detection area and the first optical filter.
According to the invention, the baffle is arranged in front of the first optical filter, so that part of laser can be prevented from directly entering the first photoelectric detection sensor without bacterial scattering, and the interference on the detection of forward scattered light is prevented.
Preferably, the fluorescence detection unit further comprises a third dichroic mirror, a fourth optical filter and a fourth photoelectric detection sensor; and the transmitted light of the second dichroic mirror is incident into a third dichroic mirror, and the reflected light of the third dichroic mirror is incident into a fourth photoelectric detection sensor after passing through a fourth optical filter.
When two kinds of fluorescence marks are adopted, the fluorescence filtered by the first dichroic mirror is mixed light and enters the second dichroic mirror, one kind of fluorescence enters the third photoelectric detection sensor after being reflected, and the other kind of fluorescence is received by the fourth photoelectric detection sensor after being reflected by the third dichroic mirror through the second dichroic mirror.
Compared with the prior art, the invention has the following advantages:
(1) the microfluidic chip is adopted for carrying out hydrodynamic focusing, so that a flow path can be simplified, and the cleaning is convenient, thereby preventing bacteria from remaining in the flow path to cause cross contamination; the detection of a small amount of samples can be realized, so that the using amount of the fluorescent dye is reduced, and the detection cost is reduced;
(2) aiming at the bacteria detection, the width of a sample introduction channel, the flow rate ratio of sheath liquid and sample liquid to be detected and the size of a light spot formed by laser in an optical detection area are designed, so that only one bacterium exists in the light spot range, and the detection accuracy is improved;
(3) aiming at the application of bacteria quantity detection, the optical detection module of the flow cytometer is simplified from 2 laser, 6 color and 8 channels to 1 laser, 2 color (or 1 color) and 4 channels (or 3 channels), so that the redundancy of the optical detection module is removed, and the equipment cost can be reduced.
Drawings
FIG. 1 is a schematic diagram of the present invention (sample injection system not shown);
FIG. 2 is a schematic diagram of one configuration of a fluidic module of the present invention;
FIG. 3 is a schematic diagram of a micro flow control chip according to the present invention;
FIG. 4 is an enlarged view at I of FIG. 3;
FIG. 5 is a schematic diagram of the implementation principle of the bacteria counting function of the present invention; wherein, the graph (A) is a fluorescence signal intensity variation curve, the graph (B) is a side scattered light signal intensity variation curve, the graph (C) is a forward scattered light signal intensity variation curve, the graph (a) is a partially enlarged view of the graph (A), the graph (B) is a partially enlarged view of the graph (B), and the graph (C) is a partially enlarged view of the graph (C);
fig. 6 is another schematic structure of the present invention.
The reference signs are: a fluid module 1, a microfluidic chip 1.1, a chip substrate 1.1.1, a sheath flow inlet 1.1.2, a sample inlet 1.1.3, a waste liquid outlet 1.1.4, a sheath flow inlet channel 1.1.5, a first sheath flow branch 1.1.6, a second sheath flow branch 1.1.7, a sample channel 1.1.8, a focusing fluid channel 1.1.9, a sheath flow fulcrum 1.1.10, a sample focusing point 1.1.11, an optical detection area 1.1.12, a sample injection system 1.2, a sample injection unit 1.2.1, a filter 1.2.1.1, a first precise sample injection pump 1.2.1.2, a sheath liquid unit 1.2.2.1, a sheath liquid storage tank 1.2.2.1, a second precise sample injection pump 1.2.2, an optical detection module 2, an incident light unit 2.1, a laser 2.1.1, a collimating aperture 2.1.2, a convex lens 2.1.3, a second light scattering optical filter 2.2, a first light scattering optical filter 2, a second light detection unit 2.2, a second light detection optical detection module 2.2, a first light scattering optical filter 2.1, a second optical filter 2.2, a second optical detection optical filter, a second optical detection unit, a second optical filter, a second optical detection optical filter, a second optical detection module 2.2.2.2., the device comprises a second dichroic mirror 2.4.1, a third optical filter 2.4.2, a third photoelectric detection sensor 2.4.3, a third dichroic mirror 2.4.4, a fourth optical filter 2.4.5, a fourth photoelectric detection sensor 2.4.6, laser 2.5, a data acquisition and processing module 3, a data acquisition system 3.1 and an upper computer 3.2.
Detailed Description
The present invention will be further described with reference to the following examples. The devices, connections, and methods referred to in this disclosure are those known in the art, unless otherwise indicated.
Example 1
As shown in fig. 1, an apparatus for rapidly detecting the number of bacteria in a beverage includes a fluid module 1, an optical detection module 2, and a data acquisition and processing module 3.
As shown in fig. 2, the fluidic module 1 includes a microfluidic chip 1.1 and a sample injection system 1.2.
As shown in fig. 3, the microfluidic chip 1.1 includes a chip substrate 1.1.1, and a sheath flow inlet 1.1.2, a sample inlet 1.1.3, a waste liquid outlet 1.1.4, a sheath flow inflow channel 1.1.5, a first sheath flow branch 1.1.6, a second sheath flow branch 1.1.7, a sample channel 1.1.8, a focusing fluid channel 1.1.9, a sheath flow fulcrum 1.1.10, and a sample focusing point 1.1.11, which are disposed on the chip substrate 1.1.1.1; the two ends of the sheath flow inflow channel 1.1.5 are respectively connected with the sheath flow inlet 1.1.2 and the sheath flow fulcrum 1.1.10; the two ends of the first sheath flow branch 1.1.6 and the second sheath flow branch 1.1.7 are respectively connected with a sheath flow fulcrum 1.1.10 and a sample focusing point 1.1.11; the sample inlet 1.1.3 and the sample channel 1.1.8 are arranged between the first sheath flow branch 1.1.6 and the second sheath flow branch 1.1.7; both ends of the sample channel 1.1.8 are respectively connected with the sample inlet 1.1.3 and the sample focusing point 1.1.11; two ends of the focusing fluid channel 1.1.9 are respectively connected with the sample focusing point 1.1.11 and the waste liquid outlet 1.1.4; an optical detection area 1.1.12 is arranged in the middle of the focusing fluid channel 1.1.9. The width of the first sheath flow branch 1.1.6, the second sheath flow branch 1.1.7, the sample channel 1.1.8, the sheath flow inflow channel 1.1.5 and the focusing fluid channel 1.1.9 is 250 μm, and the depth is 250 μm; the flow rates of the sheath fluids in the first sheath flow branch 1.1.6 and the second sheath flow branch 1.1.7 are both 50 muL/min, the flow rate of the sample fluid to be detected in the sample channel 1.1.8 is 10 muL/min, and the ratio of the total flow rate of the sheath fluids (the sum of the flow rates of the sheath fluids in the first sheath flow branch and the second sheath flow branch) to the flow rate of the sample fluid to be detected is 10: 1. As shown in FIG. 4, the cross-section of the focusing fluid channel 1.1.9 in the optical detection area 1.1.12 is a square of 250 μm × 250 μm, and the outer wall of the channel (which is part of the chip substrate 1.1.1) is a square of 2.5mm × 2.5 mm.
As shown in fig. 2, the sample injection system 1.2 includes a sample injection unit 1.2.1 and a sheath fluid injection unit 1.2.2; the sample injection unit 1.2.1 comprises a filter 1.2.1.1 and a first precision sample injection pump 1.2.1.2 which are connected in sequence; the sheath liquid sample introduction unit 1.2.2 comprises a sheath liquid storage tank 1.2.2.1 and a second precise sample introduction pump 1.2.2.2 which are connected in sequence; the first precision sample injection pump 1.2.1.2 and the second precision sample injection pump 1.2.2.2 are respectively connected with the sample inlet 1.1.3 and the sheath flow inlet 1.1.2. And a filter membrane with the aperture of 20-100 mu m is arranged in the filter 1.2.1.1.
As shown in fig. 1, the optical detection module 2 comprises an incident light unit 2.1 for forming a laser spot within an optical detection zone 1.1.12 of the focused fluid channel 1.1.9, a forward scattered light detection unit 2.2 for detecting the intensity of forward scattered light, a side scattered light detection unit 2.3 for detecting the intensity of side scattered light, and a fluorescence detection unit 2.4 for detecting the intensity of fluorescence. The incident light unit 2.1 comprises a laser 2.1.1, a collimation hole 2.1.2 and a convex lens 2.1.3; the forward scattering light detection unit 2.2 comprises a baffle 2.1.3, a first optical filter 2.2.1 and a first photoelectric detection sensor 2.2.2; the side scattered light detection unit comprises a concave lens 2.3.1, a first dichroic mirror 2.3.2, a second optical filter 2.3.3 and a second photoelectric detection sensor 2.3.4, and the fluorescence detection unit 2.4 comprises a second dichroic mirror 2.4.1, a third optical filter 2.4.2 and a third photoelectric detection sensor 2.4.3. The main optical axis of the convex lens 2.1.3 is perpendicular to the main optical axis of the concave lens 2.3.1. The laser light spot is 5-20 μm in length and 0.5-5 μm in width.
Laser 2.5 emitted by the laser 2.1.1 passes through the collimating hole 2.1.2 and is focused by the convex lens 2.1.3 to form a laser spot in the optical detection area 1.1.12 of the focusing fluid channel 1.1.9; forward scattered light of the laser passing through the optical detection area 1.1.12 is emitted into the first photoelectric detection sensor 2.2.2 through the first optical filter 2.2.1; the side scattered light of the laser passing through the optical detection area 1.1.12 passes through the concave lens 2.3.1 and then is emitted into the first dichroic mirror 2.3.2; the reflected light of the first dichroic mirror 2.3.2 passes through the second optical filter 2.3.3 and then enters the second photoelectric detection sensor 2.3.4, and the transmitted light enters the second dichroic mirror 2.4.1; the reflected light of the second dichroic mirror 2.4.1 is incident on the third photoelectric detection sensor 2.4.3 via the third optical filter 2.4.2.
As shown in fig. 1, the data collecting and processing module 3 includes a data collecting system 3.1 and an upper computer 3.2. The data acquisition system is connected with a first photoelectric detection sensor 2.2.2, a second photoelectric detection sensor 2.3.4 and a third photoelectric detection sensor 2.4.3. The upper computer 3.2 is connected with the data acquisition system 3.1.
The data acquisition system 3.1 adopts a high-speed data acquisition card, simultaneously acquires voltage data of three paths of optical signals (generated by three photoelectric detection sensors respectively), and transmits the data to the upper computer 3.2 for processing. In a single detection experiment, the high-speed acquisition card starts a continuous acquisition mode, and the software of the upper computer 3.2 analyzes acquired voltage data in real time or afterwards to obtain a voltage-time curve. Because the bacteria are dyed, scattered light and fluorescence signals can be simultaneously excited, and the time of the three data of fluorescence, lateral scattered light and forward scattered light is synchronous, when the three data have peaks at the same time, the time point can be judged to be that one bacteria flows through. Based on this principle, the total number of bacteria detected in the experiment can be counted.
After the bacteria are dyed by the green fluorescent dye SYTO9 only dyeing the viable bacteria, the device can detect the number of the viable bacteria: data of 3 channels of green fluorescence, lateral diffused light and forward diffused light are collected simultaneously, as shown in fig. 5, when the peak is detected simultaneously by the fluorescence channel, the lateral diffused light channel and the forward diffused light channel, it is determined that a live bacterium flows through the time point, and the detection of the number of the live bacteria can be realized by the mode.
In addition to performing the function of bacterial enumeration, the device of the present invention can detect the following information: the signal intensity of the forward scattered light can reflect the size of the bacteria, and the larger the signal intensity is, the larger the size of the bacteria is; the signal intensity of the side scattering light is related to the refractive index of the bacteria, and the internal structure of the bacteria can be reflected; the signal intensity of the fluorescence reflects the amount of bound fluorescent probe in the bacteria, and thus the information of the relevant target detection object.
Example 2
As shown in fig. 6, the present embodiment differs from embodiment 1 in that the fluorescence detection unit 2.4 further includes a third dichroic mirror 2.4.4, a fourth optical filter 2.4.5, and a fourth photoelectric detection sensor 2.4.6; the transmitted light of the second dichroic mirror 2.4.1 is emitted into the third dichroic mirror 2.4.4, and the reflected light of the third dichroic mirror 2.4.4 is emitted into the fourth photoelectric detection sensor 2.4.6 after passing through the fourth optical filter 2.4.5; the fourth photoelectric detection sensor 2.4.6 is connected with the data acquisition system 3.1.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.
Claims (10)
1. A device for rapidly detecting the number of bacteria in a beverage, comprising a fluid module (1), wherein the fluid module (1) comprises a microfluidic chip (1.1); the microfluidic chip (1.1) comprises a chip substrate (1.1.1), and a sheath flow inlet (1.1.2), a sample inlet (1.1.3), a waste liquid outlet (1.1.4), a sheath flow inflow channel (1.1.5), a first sheath flow branch (1.1.6), a second sheath flow branch (1.1.7), a sample channel (1.1.8), a focusing fluid channel (1.1.9), a sheath flow fulcrum (1.1.10) and a sample focusing point (1.1.11) which are arranged on the chip substrate (1.1.1.1); the two ends of the sheath flow inflow channel (1.1.5) are respectively connected with the sheath flow inlet (1.1.2) and the sheath flow fulcrum (1.1.10); both ends of the first sheath flow branch (1.1.6) and the second sheath flow branch (1.1.7) are respectively connected with a sheath flow fulcrum (1.1.10) and a sample focusing point (1.1.11); the sample inlet (1.1.3) and the sample channel (1.1.8) are arranged between the first sheath flow branch (1.1.6) and the second sheath flow branch (1.1.7); both ends of the sample channel (1.1.8) are respectively connected with the sample inlet (1.1.3) and the sample focusing point (1.1.11); both ends of the focusing fluid channel (1.1.9) are respectively connected with a sample focusing point (1.1.11) and a waste liquid outlet (1.1.4); an optical detection area (1.1.12) is arranged in the middle of the focusing fluid channel (1.1.9).
2. The device for rapidly detecting the number of bacteria in a beverage according to claim 1, wherein the width of the first sheath flow branch (1.1.6), the width of the second sheath flow branch (1.1.7), the width of the sample channel (1.1.8) are 10-500 μm, and the depth of the sample channel is 10-500 μm; the flow rates of the sheath liquid in the first sheath flow branch (1.1.6) and the second sheath flow branch (1.1.7) and the sample liquid to be detected in the sample channel (1.1.8) are 1-100 mu L/min, and the ratio of the total flow rate of the sheath liquid to the flow rate of the sample liquid to be detected is 1-50: 1.
3. The device for rapidly detecting the number of bacteria in a drink according to claim 1, wherein the cross section of the focusing fluid channel (1.1.9) in the optical detection area (1.1.12) is a rectangle with a diameter of 10-500 μm x 10-500 μm, and the outer wall of the channel is a rectangle with a diameter of 1-5 mm x 1-5 mm.
4. The device for rapidly detecting the bacterial count in a drink according to claim 1, wherein said fluid module (1) further comprises a sample introduction system (1.2); the sample introduction system (1.2) comprises a sample introduction unit (1.2.1) and a sheath fluid introduction unit (1.2.2); the sample injection unit (1.2.1) comprises a filter (1.2.1.1) and a first precision sample injection pump (1.2.1.2) which are connected in sequence; the sheath liquid sample introduction unit (1.2.2) comprises a sheath liquid storage tank (1.2.2.1) and a second precise sample introduction pump (1.2.2.2) which are connected in sequence; the first precision sample injection pump (1.2.1.2) and the second precision sample injection pump (1.2.2.2) are respectively connected with the sample inlet (1.1.3) and the sheath flow inlet (1.1.2).
5. The device for rapidly detecting the number of bacteria in a beverage according to claim 4, wherein the filter (1.2.1.1) is provided with a filter membrane with a pore size of 10-100 μm.
6. The device for rapidly detecting the bacterial count in the beverage according to claim 1, further comprising an optical detection module (2); the optical detection module (2) comprises an incident light unit (2.1) for forming a laser spot in an optical detection zone (1.1.12), a forward scattered light detection unit (2.2) for detecting the intensity of forward scattered light, a side scattered light detection unit (2.3) for detecting the intensity of side scattered light, and a fluorescence detection unit (2.4) for detecting the intensity of fluorescence.
7. The device for rapidly detecting the number of bacteria in a beverage according to claim 6, wherein the laser spot has a length of 5 to 20 μm and a width of 0.5 to 5 μm.
8. The device for rapidly detecting the number of bacteria in a beverage according to claim 6, wherein the incident light unit (2.1) comprises a laser (2.1.1), a collimating hole (2.1.2) and a convex lens (2.1.3), the forward scattering light detection unit (2.2) comprises a first optical filter (2.2.1) and a first photoelectric detection sensor (2.2.2), the lateral scattering light detection unit (2.3) comprises a concave lens (2.3.1), a first dichroic mirror (2.3.2), a second optical filter (2.3.3) and a second photoelectric detection sensor (2.3.4), and the fluorescence detection unit (2.4) comprises a second dichroic mirror (2.4.1), a third optical filter (2.4.2) and a third photoelectric detection sensor (2.4.3); laser (2.5) emitted by the laser (2.1.1) passes through the collimating hole (2.1.2), is focused by the convex lens (2.1.3), and forms a laser spot in an optical detection area (1.1.12) of the focusing fluid channel (1.1.9); the forward scattered light of the laser passing through the optical detection area (1.1.12) is emitted into the first photoelectric detection sensor (2.2.2) after passing through the first optical filter (2.2.1); the laser passes through the side scattering light of the optical detection area (1.1.12) and then enters the first dichroic mirror (2.3.2) after passing through the concave lens (2.3.1); the transmitted light and the reflected light of the laser passing through the first dichroic mirror (2.3.2) are respectively emitted into a second dichroic mirror (2.4.1) and a second photoelectric detection sensor (2.3.4) after passing through a second optical filter (2.3.3); the laser passes through the second dichroic mirror (2.4.1), and then the reflected light passes through the third optical filter (2.4.2) and then enters the third photoelectric detection sensor (2.4.3).
9. The device for rapidly detecting the number of bacteria in a drink according to claim 8, wherein a baffle (2.2.3) is arranged between the optical detection area (1.1.12) and the first optical filter (2.2.1).
10. The device for rapidly detecting the number of bacteria in a drink according to claim 8, wherein the fluorescence detection unit (2.4) further comprises a third dichroic mirror (2.4.4), a fourth optical filter (2.4.5), a fourth photoelectric detection sensor (2.4.6); the transmitted light of the second dichroic mirror (2.4.1) is incident into a third dichroic mirror (2.4.4), and the reflected light of the third dichroic mirror (2.4.4) is incident into a fourth photoelectric detection sensor (2.4.6) after passing through a fourth optical filter (2.4.5).
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