Quantitative quality control microorganism or particle liquid sample preparation device based on surface acoustic waves

Abstract

The invention relates to a quantitative quality control microorganism or particle liquid sample preparation device based on surface acoustic waves, the quantitative quality control microorganism or particle liquid sample preparation device comprises a sample injection module, a pre-arrangement area, a light scattering counting area, a sorting area and a sample collection and storage module, the sample injection module comprises a sample injection pipeline and two sheath flow pipelines as bypasses; the sample feeding pipeline is sequentially communicated with the main flow channel and the waste liquid pipeline; the sample collection and storage module comprises a waste liquid pipeline and a prepared sample collection pipeline; the sample feeding pipeline, the two sheath flow pipelines, the main flow channel, the waste liquid pipeline and the prepared sample collecting pipeline are all processed on the piezoelectric material substrate and are positioned on the same plane; a group of surface acoustic wave interdigital electrode pairs is arranged on each of the two sides of the main flow channel of the pre-arrangement area and the sorting area, the surface acoustic wave interdigital electrode pairs of the pre-arrangement area are parallel to the main flow channel, and the surface acoustic wave interdigital electrode pairs of the sorting area have an inclined angle relative to the main flow channel; and the inclination direction is consistent with the inclination direction of the prepared sample collecting pipeline relative to the waste liquid pipeline.

Description

Technical Field

[0001] The present invention relates to a precise preparation device for quantitative quality control microorganisms, comprising a sample injection module, a scattering counting module and a sorting module, and can realize precise preparation of quantitative quality control microorganisms or quantitative quality control particle liquid samples. Technical Background

[0002] The precise preparation technology of quantitative quality control microorganisms plays an important role in the field of biosafety metrology. Quantitative quality control microorganisms can be used to verify instrument performance; for food safety and quality assurance; for microbial experiments, whether it is growth promotion experiments, sterility inspection experiments, or antibacterial efficiency tests, quantitative quality control microorganisms are required to participate; for culture medium verification; for biological experiment operation verification, etc. Although quantitative quality control microorganisms are indispensable laboratory standards for many pharmaceutical, food, and microbiological laboratories. However, manual preparation of quantitative microbial suspensions of appropriate concentrations is still commonplace in many microbiological laboratories. At present, quantitative microbial preparation technology can be mainly divided into dilution method and flow cytometry method. The dilution method evenly mixes and dilutes the standard microbial sample, and calculates the number of microorganisms contained in the original solution in proportion. Due to the uncertainty of the standard microbial sample and the unevenness of the mixing, the quantitative quality control microorganisms prepared by this technology have a large uncertainty of up to 400CFU / mL. For example, the specifications of Tailin Bio's Staphylococcus aureus products are 110-1100cfu / pcs. This specification cannot meet applications with higher precision requirements, and therefore also restricts the development of related industries in my country. Flow cytometry refers to a technology that uses microchannels to process or manipulate tiny fluids. Compared with the dilution method, flow cytometry has higher accuracy. However, flow cytometry equipment has the disadvantages of being expensive and the technology is monopolized by foreign countries. From a technical point of view, flow cytometry also has certain technical shortcomings, such as high cell damage rate, low sorting efficiency and insufficient processing of rare cell samples. CHEMUNEX D-Count, a quantitative microbial preparation instrument based on flow cytometry technology independently developed by the French bioMérieux company, has an accuracy of ±3CFU / ml for preparing quantitative microorganisms and a price of no less than 3 million yuan. At present, the dilution method is still the main method used in China to prepare quantitative quality control microbial products, which cannot meet the metrological calibration requirements of high-precision instruments.

[0003] In addition to quantitative quality control of microbial products, other quantitative quality control liquid products also have the above problems. For example, particle size standard substances developed based on polystyrene beads. Currently, only the particle size is considered when calibrating such substances, and their number concentration is not considered. However, in the metrological calibration of the instrument, if the number concentration of the particle size standard substances prepared from polystyrene beads is controllable, the particle size and concentration calibration can be completed simultaneously during the instrument calibration. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies in the above-mentioned background technology, and to provide a device for preparing liquid samples of quantitative quality control microorganisms or quantitative quality control particles based on microfluidics and surface acoustic wave technology. The technical solution is as follows:

[0005] A surface acoustic wave-based quantitative quality control microbial or particle liquid sample preparation device comprises a sample injection module, a pre-arrangement area, a light scattering counting area, a sorting area and a sample collection and storage module, wherein:

[0006] The sample injection module includes a sample injection pipeline 4 and two sheath flow pipelines as bypasses, and the three pipelines are all equipped with peristaltic pumps. The two sheath flow pipelines and the sample injection pipeline 4 converge at the main channel 16; the sample injection pipeline 4 is sequentially connected with the main channel 16 and the waste liquid pipeline 11 and is located on the same axis, and the main channel 16 is located in the pre-arrangement area, the light scattering counting area, and the sorting area; the sample collection and storage module includes the waste liquid pipeline 11, the prepared sample collection pipeline 12 as its bypass, and a collection device 13 for receiving the prepared sample;

[0007] The sample injection pipeline 4, two sheath flow pipelines, the main flow channel 16, the waste liquid pipeline 11 and the prepared sample collection pipeline 12 are all processed on the piezoelectric material substrate and are located in the same plane;

[0008] A group of surface acoustic wave interdigital electrode pairs are respectively arranged on both sides of the main channel 16 in the pre-arrangement area and the sorting area. The surface acoustic wave interdigital electrode pairs 7 in the pre-arrangement area are parallel to the main channel 16. The surface acoustic wave interdigital electrodes in the sorting area have an inclined angle relative to the main channel 16, and the inclined direction thereof is consistent with the inclined direction of the sample preparation collection pipeline 12 relative to the waste liquid pipeline 11.

[0009] The photosensitive area 22 of the light scattering counting area is located at the location of the main channel 16 and is used to count the microorganisms or particles in the main channel 16 .

[0010] Furthermore, the two groups of surface acoustic wave interdigital electrodes are used to load modulation voltage signals with opposite phases, amplitudes and the same frequency.

[0011] Furthermore, the pre-arranged area surface acoustic wave fork-finger electrode pair 7 includes two fork-finger electrodes of the same structure, which are located on both sides of the main channel 16 and symmetrically distributed about the main channel 16. Two columns of coherent waves with the same amplitude are generated by the surface acoustic wave fork-finger electrode pair 7, and a standing wave is generated in the main channel 16, and the node of the generated standing wave is located at the center line of the main channel 16.

[0012] Furthermore, the surface acoustic wave forked electrode pair 10 in the sorting area includes two forked electrodes of the same structure, which are located on both sides of the main channel 16 and symmetrically distributed about the center line of the sample collection pipe 12. Two columns of coherent waves with the same amplitude are generated by the surface acoustic wave forked electrode pair 10, and standing waves are generated in the main channel 16, and the nodes of the generated standing waves are located at the extension line of the center line of the sample collection pipe 12.

[0013] Furthermore, the piezoelectric material substrate is a lithium niobate substrate.

[0014] Furthermore, the two groups of surface acoustic wave interdigital electrode pairs are processed on the same piezoelectric material substrate as the main channel 16 and are located in the same plane as the main channel 16 .

[0015] Furthermore, the light scattering counting area includes a laser 17, a cylindrical mirror 18, an aperture 19, a spherical reflector 20, a light trap 21 and a photodetector 9. The circular spot laser emitted by the laser 17 is converged into a line laser through the cylindrical mirror 18, and is focused on the photosensitive area 22 after passing through the aperture 19. The photosensitive area 22 is located in a plane formed by the laser emission optical axis and the scattered light collection optical axis; the laser beam passing through the photosensitive area 22 enters the light trap 21 and is absorbed by the light trap 21. Each microorganism or particle enters the photosensitive area one by one, and the laser irradiates each microorganism or particle to generate a scattered light pulse signal. The spherical reflector 20 converges the scattered light generated by the microorganism or particle and is collected by the photodetector 9. The number of pulses collected is the number of microorganisms or particles.

[0016] Furthermore, the microorganism or particle liquid sample is pumped in through the sample inlet pipe 4 by a peristaltic pump at a certain flow rate, and two paths of pure water are pumped in through two sheath flow pipes by a peristaltic pump at a certain flow rate as sheath liquid. The flow rate of each peristaltic pump is controlled so that the pumped microorganism or particle liquid sample is located in the middle of the main channel 16.

[0017] The present invention can accurately quantitatively control the preparation of microbial or granular liquid samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a device for preparing liquid samples of microorganisms or particles for quantitative quality control based on surface acoustic waves; Figure 2 It is the light scattering counting device part;

[0019] The figure is marked as follows: 1—peristaltic pump; 2—sheath flow pipeline; 3—peristaltic pump; 4—sample injection pipeline; 5—peristaltic pump; 6—sheath flow pipeline; 7—surface acoustic wave interdigital electrode pair in the pre-arrangement zone; 8—laser; 9—photodetector; 10—surface acoustic wave interdigital electrode pair in the sorting zone; 11—waste liquid pipeline; 12—sample preparation collection pipeline; 13—collection device; 14—line segment where the node of the standing wave of the acoustic field in the pre-arrangement zone is located; 15—line segment where the node of the standing wave of the acoustic field in the sorting zone is located; 16—main flow channel; 17—laser; 18—cylindrical mirror; 19—aperture; 20—spherical reflector; 21—light trap; 22—photosensitive zone DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings:

[0021] The quantitative quality control microorganism or particle liquid sample preparation device based on surface acoustic wave of the present invention comprises a sample injection module, a pre-arrangement area based on surface acoustic wave (SAW) focusing, a counting area based on light scattering technology, a sorting area based on SAW, and a sample collection and storage module based on freeze-drying technology. Sheath flow pipes 2 and 6, sample injection pipe 4, main channel 16, waste liquid pipe 11 and preparation sample collection pipe 12 are all processed on the lithium niobate substrate and are located in the same plane. The material of the flow channel is SU8; lithium niobate is a piezoelectric material, so that the surface acoustic wave fork finger electrode pair 7 in the pre-arrangement area and the surface acoustic wave fork finger electrode pair 10 in the sorting area excite surface acoustic waves, that is, when an electrical signal is loaded on the fork finger electrode pair, the sound wave can be excited through the piezoelectric effect. Since commonly used flow channel materials such as PDMS have bonding problems with lithium niobate, the flow channel on the lithium niobate substrate usually directly uses the photolithography material SU8 to photoetch the flow channel structure on the lithium niobate substrate through a photolithography process, and PDMS is bonded on the top of the flow channel to make a top-closed flow channel system. The pre-arrangement area surface acoustic wave interdigital electrode pair 7 and the sorting area surface acoustic wave interdigital electrode pair 10 are also processed on the same lithium niobate substrate and are located on the same plane. The material of the interdigital electrodes is gold. The light scattering counting area photosensitive area 22 is located at the position where the main channel 16 is located. The plane composed of the laser 17 and the light trap 21, the plane composed of the photodetector 9 and the spherical reflector 20, and the plane where the main channel 16 is located are perpendicular to each other. The intersection of the three planes is the photosensitive area 22.

[0022] The sample injection module is equipped with a peristaltic pump with adjustable flow rate and a microchannel pipeline for sampling. The microchannel pipeline includes two sheath flow pipelines 2 and 6 and one sample sampling pipeline 4. The liquid sampling of each pipeline is controlled by a peristaltic pump (1, 3, 5). The connection between the sampling pipeline and the peristaltic pump is realized by a hose. The arrangement of the three pipelines is shown in the figure, wherein the sample sampling pipeline 4 is located in the middle, and the two sheath flow pipelines 2 and 6 are symmetrically distributed at both ends of the sampling pipeline 4. The standard microbial sample is pumped in by the peristaltic pump 3 through the sample tube at a certain flow rate, and the two pure waters are pumped in by the sheath flow pipelines 2 and 6 as sheath liquids at a certain flow rate. The sheath flow pipelines 2 and 6 and the sample sampling pipeline 4 are collected at the main microchannel. The flow rate of the peristaltic pumps 1, 3, 5 is controlled so that the sample pumped in by the peristaltic pump 3 is basically located in the center of the microchannel.

[0023] The pre-arrangement area is composed of a pre-arrangement area surface acoustic wave interdigital electrode (SAW-IDT) pair 7 and a main channel 16. The surface acoustic wave interdigital electrode (SAW-IDT) pair 7 contains two interdigital electrodes with exactly the same structure, which are arranged on both sides of the main channel 16 and symmetrically distributed about the main channel 16. The surface acoustic wave interdigital electrode pair 7 on both sides of the main channel 16 is loaded with a modulation voltage signal with the same amplitude and frequency and opposite phases, and two coherent waves with the same amplitude are generated by the surface acoustic wave interdigital electrode pair 7, and a standing wave is generated in the main channel 16, and the node of the generated standing wave is located at the line segment 14 where the node of the standing wave of the sound field in the pre-arrangement area is located, that is, the midline of the main channel 16. After the sample pumped in by the peristaltic pump 3 enters the pre-arrangement area, the sample is affected by the standing wave generated by the surface acoustic wave interdigital electrode pair 7, and will be arranged in sequence at the node of the standing wave, and flow to the light scattering counting area.

[0024] The light scattering counting area is composed of a laser 17, a cylindrical mirror 18, an aperture 19, a spherical reflector 20, a light trap 21, and a photodetector 9. The photosensitive area 22 in the light scattering counting device is located in the center of the main channel 16 to count the microorganisms or particles in the main channel. The circular spot laser emitted by the laser 17 is converged into a line laser through the cylindrical mirror 18, and is focused on the photosensitive area 22 after passing through the aperture 19. The photosensitive area 22 is located in the plane formed by the laser emission optical axis and the scattered light collection optical axis, and the position of the photosensitive area 22 is adjusted to the center of the main channel 16. The laser beam passing through the photosensitive area 22 enters the light trap 21 and is absorbed by the light trap 21. Each microorganism enters the photosensitive area one by one, and the laser irradiates each particle to generate a scattered light pulse signal. The spherical reflector 20 converges the scattered light generated by the microorganism and is collected by the photodetector 9. Each microorganism generates a light pulse when passing through the photosensitive area. By counting the collected light pulses, the number of collected pulses is the number of microorganisms.

[0025] The microorganisms passing through the light scattering counting area will flow to the sorting area. The function of the sorting area is to transport the required microorganisms into the sample preparation collection pipe 12 and collect them into the collection device 13, and to transport the remaining microorganism samples to the waste liquid pipe 11. The sorting area is composed of a surface acoustic wave interdigital electrode pair 10 in the sorting area and a main channel 16. The surface acoustic wave interdigital electrode (SAW-IDT) pair 10 in the sorting area contains two interdigital electrodes with completely identical structures, which are arranged on both sides of the main channel 16 and symmetrically distributed about the center line of the sample preparation collection pipe 12. When the surface acoustic wave interdigital electrode pair 10 on both sides of the main channel 16 is loaded with a modulation voltage signal with opposite phase, the same amplitude and frequency, two coherent waves with the same amplitude can be generated by the surface acoustic wave interdigital electrode pair 10, and a standing wave is generated in the main channel 16. Since the surface acoustic wave interdigital electrode (SAW-IDT) pair 10 in the sorting area is symmetrically distributed about the center line of the sample collection pipeline 12, the standing wave node generated is located at the line segment 15 where the standing wave node of the sound field in the sorting area is located, that is, on the extension line of the center line of the sample collection pipeline 12. When microorganisms within the required number and concentration range pass through the sorting area, the surface acoustic wave interdigital electrode pair 10 is loaded with a modulation voltage signal with opposite phases, the same amplitude and frequency, and then, under the action of the sound field, the microorganism will be dragged to the node by the sound field, that is, its movement trajectory is along the line segment 15 where the node of the standing wave of the sound field in the sorting area is located, so the microorganism will move to the entrance of the sample preparation collection pipeline 12, enter the sample preparation collection pipeline 12, and be collected by the collection device 13 (generally a collection device such as an ampoule bottle) under the action of the liquid flow field. The connection between the collection device 13 and the sample preparation collection pipeline 12 is completed by a hose. If the number of signals counted by the light scattering count exceeds the target number of microorganisms, the modulation voltage signal loaded on the surface acoustic wave interdigital electrode pair 10 is turned off, and the main channel 16 in the sorting area is no longer affected by the sound field, and the microorganism will continue to move horizontally forward to the waste liquid pipeline 11 under the action of the liquid flow field.

[0026] The waste liquid pipeline 11 and the sample preparation collection pipeline 12 are both collection branch flow channels. The waste liquid pipeline 11 is connected to the main flow channel 16 and is parallel in direction. The sample collection pipeline 12 is connected to the main flow channel 16 and has a certain angle with the waste liquid pipeline 11. The line segment 15 where the standing wave node of the acoustic field in the sorting area is located is on the same straight line as the center line of the sample preparation collection pipeline 12. The waste liquid pipeline 11, the sample preparation collection pipeline 12 and the collection device 13 together constitute the collection area in the device.

[0027] The following describes the preparation process of quantitative quality control microorganisms through specific examples. Due to the needs of the calibration instrument, a bottle of 100CFU quantitative quality control microbial suspension is required, which is now configured through this device. The existing microbial content is about 10,000CFU and the volume is 1L. It is used as the stock solution and connected to the peristaltic pump 3. The stock solution is pumped into the main channel 16 through the peristaltic pump 3, and two pure waters are pumped in at a certain flow rate through the sheath flow pipes 2 and 6 as sheath liquids. The flow rate of the peristaltic pumps 1, 3, and 5 is controlled so that the sample pumped in by the peristaltic pump 3 is basically located in the center of the microchannel. Calculation shows that the average number concentration of the stock solution is 10CFU / mL. The speed of the peristaltic pump is controlled to be lower than 0.1mL / min, so that the number of microorganisms pumped into the peristaltic pump per minute is maintained at a low level to ensure the preparation accuracy of the prepared samples. Before the peristaltic pumps 1, 3, and 5 work, the light scattering counting module is first turned on to put it in working state, and the modulation voltage signals with opposite phases, same amplitude and frequency loaded on the surface acoustic wave interdigital electrode pair 7 in the pre-arrangement area and the surface acoustic wave interdigital electrode pair 10 in the sorting area are turned on. When the mixed liquid of the stock solution and the pure water is pumped into the main channel 16, in the pre-arrangement area, due to the standing wave field generated by the surface acoustic wave interdigital electrode pair 7 in the pre-arrangement area, the pumped microorganisms are located at the line segment 14 where the standing wave node of the acoustic field in the pre-arrangement area is located, that is, at the center line of the main channel 16, and move to the photosensitive area under the action of the flow field, and the scattered light caused by it is captured by the light scattering counting device to complete the counting of the microorganisms. When the count signal is less than 100, the microorganisms pass through the sorting area in the working state of the surface acoustic wave interdigital electrode pair 10 in the sorting area. Due to the standing wave field generated by the surface acoustic wave interdigital electrode pair 10 in the sorting area in the main flow channel 16, the microorganisms will move along the line segment 15 where the standing wave node of the acoustic field in the sorting area is located. At the same time, due to the effect of the flow field, the microorganisms enter the sample collection pipeline 12 and are collected by the collection device 13. When the count signal reaches 100, the modulation voltage signal loaded on the surface acoustic wave interdigital electrode pair 10 in the sorting area is turned off, and the modulation survey signal loaded on the surface acoustic wave interdigital electrode pair 7 in the pre-arrangement area is maintained, so that the excess microorganisms are collected through the waste liquid pipeline 11. The stock solution is withdrawn from the interface of the peristaltic pump 3, and the interface of the peristaltic pump 3 is replaced with ultrapure water to flush the microorganisms that may remain in the main channel 16. When no light scattering signal is collected after 10 minutes, it is considered that there are no microorganisms remaining in the channel, and the peristaltic pump is turned off, the voltage signal loaded on the surface acoustic wave interdigital electrode pair 7 in the pre-arrangement area is turned off, and the light scattering counting device is turned off. The prepared quantitative quality control microorganisms are used for instrument calibration or freeze-dried storage.

Claims

1. A quantitative quality control microbial or particle liquid sample preparation device based on surface acoustic wave, comprising a sample injection module, a pre-arrangement area, a light scattering counting area, a sorting area and a sample collection and storage module, wherein: The sample injection module comprises a sample injection pipeline (4) and two sheath flow pipelines as bypasses, the three pipelines are all equipped with peristaltic pumps, the two sheath flow pipelines and the sample injection pipeline (4) converge at a main channel (16); the sample injection pipeline (4) is connected with the main channel (16) and the waste liquid pipeline (11) in sequence, the main channel (16) is located in a pre-arrangement area, a light scattering counting area, and a sorting area; the sample collection and storage module comprises the waste liquid pipeline (11), a prepared sample collection pipeline (12) as a bypass thereof, and a collection device (13) for receiving prepared samples; The sample injection pipeline (4), two sheath flow pipelines, the main flow channel (16), the waste liquid pipeline (11) and the prepared sample collection pipeline (12) are all processed on the piezoelectric material substrate and are located in the same plane; A group of surface acoustic wave interdigital electrode pairs are respectively arranged on both sides of the main channel (16) of the pre-arrangement zone and the sorting zone, the surface acoustic wave interdigital electrode pairs (7) of the pre-arrangement zone are parallel to the main channel (16), and the surface acoustic wave interdigital electrodes of the sorting zone have an inclined angle relative to the main channel (16), and the inclined direction thereof is consistent with the inclined direction of the sample preparation collection pipeline (12) relative to the waste liquid pipeline (11); The photosensitive area (22) of the light scattering counting area is located at the location of the main channel (16) and is used to count the microorganisms or particles in the main channel (16).

2. The microbial or particle liquid sample preparation device according to claim 1, characterized in that: Two groups of surface acoustic wave interdigital electrodes are used to load modulation voltage signals with opposite phases, amplitudes and the same frequency.

3. The microbial or particle liquid sample preparation device according to claim 2, characterized in that: The pre-arranged area surface acoustic wave interdigital electrode pair (7) comprises two interdigital electrodes of the same structure, which are arranged on both sides of the main channel (16) and symmetrically distributed about the main channel (16). The surface acoustic wave interdigital electrode pair (7) generates two columns of coherent waves with the same amplitude, generates a standing wave in the main channel (16), and makes the node of the generated standing wave located at the center line of the main channel (16).

4. The microbial or particle liquid sample preparation device according to claim 2, characterized in that: The surface acoustic wave interdigital electrode pair (10) in the sorting area includes two interdigital electrodes of the same structure, which are arranged on both sides of the main channel (16) and symmetrically distributed about the center line of the sample collection pipeline (12). The surface acoustic wave interdigital electrode pair (10) generates two series of coherent waves with the same amplitude, generates standing waves in the main channel (16), and makes the node of the generated standing wave located at the extension line of the center line of the sample collection pipeline (12).

5. The microbial or particle liquid sample preparation device according to claim 1, characterized in that: The piezoelectric material substrate is a lithium niobate substrate.

6. The device for preparing liquid samples of microorganisms or particles according to claim 1, characterized in that: The two groups of surface acoustic wave interdigital electrode pairs are processed on the same piezoelectric material substrate as the main channel (16) and are located in the same plane as the main channel.

7. The device for preparing liquid samples of microorganisms or particles according to claim 1, characterized in that: The sample injection pipeline (4) is connected to the main flow channel (16) and the waste liquid pipeline (11) in sequence and is located on the same axis.

8. The device for preparing liquid samples of microorganisms or particles according to claim 1, characterized in that: The light scattering counting area comprises a laser (17), a cylindrical mirror (18), an aperture (19), a spherical reflector (20), a light trap (21) and a photodetector (9). The circular spot laser emitted by the laser (17) is converged into a linear laser through the cylindrical mirror (18), and is focused on a photosensitive area (22) after passing through the aperture (19); the photosensitive area (22) is located in a plane formed by a laser emission optical axis and a scattered light collection optical axis; the laser beam passing through the photosensitive area (22) enters the light trap (21) and is absorbed by the light trap (21); each microorganism or particle enters the photosensitive area one by one, and a scattered light pulse signal is generated after the laser irradiates each microorganism or particle. The spherical reflector (20) converges the scattered light generated by the microorganism or particle and collects it through the photodetector (9), and the number of pulses collected is the number of the microorganism or particle.

9. The device for preparing liquid samples of microorganisms or particles according to claim 1, characterized in that: The microorganism or particle liquid sample is pumped in through the sample inlet pipe (4) by a peristaltic pump at a certain flow rate, and two pure waters are pumped in through two sheath flow pipes by a peristaltic pump at a certain flow rate as sheath liquid. The flow rate of each peristaltic pump is controlled so that the pumped microorganism or particle liquid sample is located in the middle of the main flow channel (16).

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