Microfluidic cryptococcus sorting device and detection method

Abstract

The invention discloses a microfluidic cryptococcus sorting device and a detection method, and the device comprises a focusing section which is used for receiving a fluid solution and focusing particles in the fluid solution; the sinking section is used for receiving the fluid solution with the focused particles and layering the fluid solution; the liquid separation section is used for separating cerebrospinal fluid in the layered fluid solution; the capturing part is used for receiving the fluid solution from which the cerebrospinal fluid is separated and capturing particles in the fluid solution; the outlet section is used for discharging the fluid solution in the capturing part; the focusing section is in a sawtooth-like shape and is composed of a plurality of inverted-V-like assemblies, a focusing channel is arranged in each inverted-V-like assembly, every two adjacent inverted-V-like assemblies are connected in series through a common endpoint, and the internal focusing channels are communicated; by means of the unique structure, accurate focusing, separation and enrichment of cryptococcus can be achieved in a short time, and therefore the purpose of detection is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical equipment, and in particular to a microfluidic cryptococcal sorting device and a detection method. Background Art

[0002] Cryptococcal meningitis (CM) is a serious infectious disease caused by Cryptococcus neoformans and Cryptococcus gattii, which is mainly seen in patients with impaired immune function, especially in those infected with HIV. The early symptoms of CM are usually hidden, and its diagnosis is not only crucial for the management and treatment of the disease, but also can assist in HIV screening to a certain extent. However, due to the high mortality rate of CM and the lack of specificity of symptoms, clinical diagnosis faces great challenges and is prone to misdiagnosis.

[0003] Common symptoms of CM patients include chronic or subacute headaches, positive meningeal irritation signs, and significantly elevated cerebrospinal fluid pressure. However, these symptoms overlap with the manifestations of other central nervous system diseases and lack specificity. In addition, the biological characteristics of Cryptococcus also make diagnosis difficult. For example, the thick polysaccharide capsule of Cryptococcus accounts for about 70% of its total cell mass, and the cell wall structure is tough. These characteristics make the traditional nucleic acid extraction and detection process complicated and inefficient.

[0004] At present, traditional detection methods for CM mainly include direct examination and isolation and culture. Direct examination such as ink smear microscopy requires the collection of samples by centrifugation and precipitation before testing. Although this method is simple and fast, the sample is easily lost, the sensitivity is low, and the false negative rate is high. Although the isolation and culture method is considered the "gold standard" for CM detection, its diagnostic cycle usually takes 2 to 7 days, which cannot meet the needs of clinical rapid diagnosis.

[0005] Microfluidics is an emerging biomedical detection method. By designing precise fluid channels at the microscopic scale, microfluidics can effectively achieve functions such as sample separation, enrichment, and particle focusing. Microfluidics has shown great potential in the fields of cell counting, biomolecule detection, sample sorting, and biosensor optimization. Compared with traditional detection methods, microfluidics has the advantages of low cost, low energy consumption, and efficient operation, and can achieve precise control of droplets, cells, and microorganisms at the microscopic scale.

[0006] In microfluidic technology, particle focusing is an important function. Its core goal is to arrange dispersed particles into a regular single or multi-column sequence to meet the needs of subsequent operations, such as enrichment, separation or manipulation of target particles. According to the operating principle, microfluidic particle focusing methods can be divided into three categories: active focusing, sheath-assisted focusing and passive focusing. Active focusing relies on external forces such as sound waves, magnetic fields or optical forces to manipulate particle movement. Although it has high precision, it usually requires a complex external energy field control system, and can only ensure full focusing under low flow conditions, which limits the flux improvement. Sheath-assisted focusing compresses the target sample into a narrow single sheath flow through additional sheath flow, thereby achieving high-throughput focusing, but it requires additional microchannel structures and fluid input devices, reducing the integration of microfluidic devices. Passive focusing achieves natural particle focusing without external driving force through the inertia of the fluid, the viscoelasticity of non-Newtonian fluids or the micro-vortex effect induced by the channel structure. This method can effectively avoid cell damage during cell manipulation and achieve continuous and high-throughput particle manipulation. In the application of small particle focusing, due to the significant change in cross-sectional width at the channel corners, the Dean drag force (FD) plays a dominant role, accelerating the focusing of particles along the counter-rotating vortex lines toward the center of the channel, where the Dean drag force tends to entrain particles to follow the counter-rotating vortex streamlines.

[0007] From the perspective of cell sorting, sorting technology can be mainly divided into two categories: label-free sorting and labeled sorting. Label-free sorting is based on the physical properties of cells (such as size, density, and shape, etc.), but it is difficult to distinguish cells with similar physical properties but different biological properties. Labeled sorting achieves cell recognition and separation by introducing specific markers (such as fluorescent dyes or magnetic beads). However, this method may have a certain impact on the activity of cells, and since the cells need to be washed multiple times during the operation, the capture efficiency will be reduced. For the focusing of small particles, the cross-sectional width at the corner of the channel is greatly increased, which increases the Dean drag and dominates the effect, and the particles focus faster toward the center of the channel.

[0008] Among the existing CM detection methods, the direct inspection method is complex to operate and the sample is easy to lose, the sensitivity is low, the false negative rate is high, and the isolation and culture method has a long diagnostic cycle. Therefore, a new technology is needed to improve this phenomenon. Summary of the invention

[0009] In view of the deficiencies in the prior art, the present invention provides a microfluidic cryptococcal sorting device and a detection method, which aims to solve the problems in the background technology.

[0010] To achieve the above object, the present invention provides the following technical solution: a microfluidic cryptococcal sorting device, comprising: A focusing section, used for receiving the fluid solution and focusing the particles in the fluid solution; A sinking section, the sinking section is connected to the output end of the sawtooth-like component, and is used to receive the fluid solution after the particles are focused and perform stratification; A liquid separation section, the liquid separation section is connected to the sinking section and communicated with the sinking section, and is used to separate the cerebrospinal fluid in the stratified fluid solution in the sinking section; A transition section, the transition section is connected to the liquid separation section, and is used to receive and transmit the fluid solution after the cerebrospinal fluid is separated by the liquid separation section; A capture element, which is connected to the transition section and is used to receive the fluid solution transmitted by the transition section and capture particles in the fluid solution; An outlet section, the outlet section is connected to the capture member and is used to discharge the fluid solution in the capture member; The focusing section is sawtooth-like and is composed of a plurality of inverted V-shaped components. A focusing channel is arranged in each inverted V-shaped component. Two adjacent inverted V-shaped components are connected in series through a common end point and the internal focusing channels are connected.

[0011] Furthermore, each quasi-inverted V-shaped component is composed of a lower base missing isosceles trapezoid component and four upper base missing isosceles trapezoid components; two obliquely arranged upper base missing isosceles trapezoid components are sequentially connected at both ends of the bottom of the lower base missing isosceles trapezoid component, and the two adjacent and mutually connected upper base missing isosceles trapezoid components are inverted with each other; The two ends of the missing part of the isosceles trapezoidal component with a missing lower base are respectively provided with a first connecting end, and the two ends of the missing part of the isosceles trapezoidal component with a missing upper base are respectively provided with a second connecting end; the specific connection relationship between the isosceles trapezoidal component with a missing lower base and the isosceles trapezoidal component with a missing upper base is as follows: the two first connecting ends of the isosceles trapezoidal component with a missing lower base are respectively connected to a second connecting end of an isosceles trapezoidal component with a missing upper base, and the other second connecting end of the isosceles trapezoidal component with a missing upper base is connected to a second connecting end of an adjacent isosceles trapezoidal component with a missing upper base; Adjacent inverted V-shaped components are connected through an inverted isosceles trapezoidal component with a missing lower base, that is, a second connecting end of the isosceles trapezoidal component with a missing upper base at the bottom of two adjacent inverted V-shaped components that is not connected is respectively connected to the two first connecting ends of the inverted isosceles trapezoidal component with a missing lower base.

[0012] Furthermore, a second connection end of the isosceles trapezoidal component with the upper base missing at the bottom of the first inverted V-shaped component in the focusing section that is not connected is connected to a third connection end of the isosceles trapezoidal component with the lower base missing in half, and another third connection end of the isosceles trapezoidal component with the lower base missing in half is used as the entrance of the focusing section, that is, the entrance of the sorting device; The isosceles trapezoidal component with a missing lower base, the isosceles trapezoidal component with a missing upper base and the isosceles trapezoidal component with a half missing lower base are all provided with focusing channels and are connected.

[0013] Further, the corner angle range of the focusing channel in the isosceles trapezoidal component with the upper base missing is set to 55°~75°; Suppose the width of the focusing channel in the isosceles trapezoidal component with missing lower base, the isosceles trapezoidal component with missing upper base and the isosceles trapezoidal component with half of the lower base is Z, the inner wall length of the focusing channel in the isosceles trapezoidal component with missing lower base is S, and the inner wall length of the focusing channel in the isosceles trapezoidal component with missing upper base is L, satisfying L=S+Z; among which, the width of the focusing channel, the sinking channel and the transition channel are all Z, and the width of the liquid separation channel satisfies 1.5Z.

[0014] Further, a sinking channel is provided in the sinking section, and an inner cavity is provided in the sinking channel. The sinking channel is connected to a second connecting end that is not connected to the isosceles trapezoidal component with the missing upper bottom edge at the bottom of the last inverted V-shaped component in the focusing section, and is communicated with the focusing channel in the isosceles trapezoidal component with the missing upper bottom edge; the first step, the second step, the third step, the fourth step, the fifth step, the sixth step and the seventh step are sequentially provided from the input end to the output end of the inner cavity of the sinking channel, wherein the bottom of the inner cavity of the sinking channel is taken as the 0th step, and it descends layer by layer from the 0th step to the 5th step, and ascends layer by layer from the 5th step to the 7th step; wherein the 1st step, the 2nd step, the 3rd step, the 4th step, the 5th step, the 6th step and the 7th step are provided with equal widths; The 6th step is flush with the 0th step. Let A be the vertical distance from the 5th step to the 0th step, and B be the vertical distance from the 5th step to the 7th step, satisfying B>A. The sinking channel is connected to a second connecting end that is not connected to the isosceles trapezoidal component with the missing upper base edge at the bottom of the last inverted V-shaped component in the focusing section through a plurality of isosceles trapezoidal components with the missing upper base edge that are connected in sequence and inverted to each other, and is connected to the focusing channel in the isosceles trapezoidal component with the missing upper base edge.

[0015] Furthermore, there are two liquid-separating sections, the input ends of the two liquid-separating sections are respectively connected to the two sides of the sinking section, and the output ends of the two liquid-separating sections are connected to the outside world; the two liquid-separating sections are on the same axis; liquid-separating channels are provided in the two liquid-separating sections, and the liquid-separating channels are connected to the sinking channel in the sinking section; the connection point between the liquid-separating section and the sinking section is at the top of the 5th step in the sinking channel.

[0016] Furthermore, a transition channel is provided in the transition section, and the transition channel in the transition section is connected to the sinking channel in the sinking section.

[0017] Furthermore, a capture cavity is provided in the capture member, and the capture cavity is a semicircular rectangular outline, that is, the capture cavity is formed by splicing a rectangular cavity and a semicircular cavity; a vertical angled baffle array composed of a plurality of angled baffles is provided in the capture cavity, and the openings of all angled baffles in the vertical angled baffle array are all toward one end of the capture member entrance; the rectangular cavity in the capture cavity is connected to the transition channel; The connection point between the transition section and the capture member is arranged on the central axis of the capture member; a depression is arranged at the connection between the capture member and the transition section, and the depth of the depression accounts for 1 / 5 to 1 / 4 of the longest distance between the two ends of the capture member.

[0018] Furthermore, an outlet channel is provided in the outlet section, one end of the outlet channel is connected to the semicircular cavity in the capture cavity, and the other end of the outlet channel is connected to the outside.

[0019] A microfluidic cryptococcal detection method is applied to a microfluidic cryptococcal sorting device, comprising the following steps: S1: Mixing the cerebrospinal fluid sample to be tested with acetic acid and ink to obtain a fluid solution; S2: injecting the fluid solution into the inlet of the focusing section, driving the fluid solution through the focusing channels in the lower base missing isosceles trapezoidal components, the upper base missing isosceles trapezoidal components and the half lower base missing isosceles trapezoidal components in the focusing section, so that the particles in the fluid solution are focused; The fluid solution behind the focused particles is driven to pass through the sinking channel, and the fluid solution behind the focused particles is stratified, so that the cerebrospinal fluid in the fluid solution is focused on the upper layer of the sinking channel, discharged through the transition channel in the transition section, and the particles are focused on the lower layer of the sinking channel; The fluid solution after separation of cerebrospinal fluid is driven to flow into the capture chamber through the transition section, and the fluid solution is enriched by the vertical angled baffle array; S3: Observe the particles in the capture cavity by microscopy to see if they contain cryptococci; When all the fluid solution enters the focusing section, the ink is continuously injected into the focusing section using a syringe to drive all the fluid solution into the capture chamber.

[0020] Compared with the existing technology, the present invention has the following beneficial effects: the present invention forms an inverted V-shaped structure by combining an isosceles trapezoidal structure with a missing lower base and an isosceles trapezoidal structure with a missing upper base; the serrated particle focusing structure composed of multiple inverted V-shaped structures can better focus the fluid solution to the middle of the focusing channel; by arranging multiple steps of different heights in the inner cavity of the sinking channel, the cerebrospinal fluid and cryptococcus in the fluid solution can be stratified by gravity stratification; by connecting a liquid separation channel in the sinking channel, the cerebrospinal fluid in the fluid solution can be separated; by arranging a vertical angle baffle array in the capture cavity, the fluid solution can flow into the capture cavity to generate a larger vortex area; at the same time, the particles can also be effectively stored in the capture cavity, which is convenient for subsequent observation through a microscope to see whether there are cryptococci in the capture cavity; no centrifugation is required, the operation is simple, the sample is not easy to lose, the sensitivity is high, the false negative rate is low, and the accurate focusing, separation and enrichment of cryptococcus can be achieved in a short time, thereby achieving the purpose of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the overall structure diagram of the present invention.

[0022] Figure 2 It is a structural diagram of the inverted V-shaped component of the present invention.

[0023] Figure 3 It is a schematic diagram of the connection structure of the inverted V-shaped component of the present invention.

[0024] Figure 4 This is a comparison diagram of the focusing effects of four structures in series, namely, a 5-periodic arc, a rectangle, an inverted V-shape, and an isosceles trapezoid with a missing bottom edge in an embodiment of the present invention.

[0025] Figure 5 This is a velocity cloud diagram of the fluid solution in an inverted V-shaped component.

[0026] Figure 6 It is a cross-sectional view of the sinking channel of the present invention.

[0027] Figure 7 It is a schematic diagram of the connection structure between the sinking channel and the liquid separation channel of the present invention.

[0028] Figure 8 It is a schematic diagram of the internal structure of the capture member of the present invention.

[0029] In the figure, 1. a quasi-inverted V-shaped component; 101. an isosceles trapezoidal component with a missing lower base; 1011. a first connecting terminal; 102. an isosceles trapezoidal component with a missing upper base; 1021. a second connecting terminal; 103. an isosceles trapezoidal component with one-half of the lower base missing; 1031. a third connecting terminal; 4. a sinking section; 401. a sinking channel; 411. the first step; 412. the second step; 413. the third step; 414. the fourth step; 415. the fifth step; 416. the sixth step; 417. the seventh step; 5. a liquid separation section; 6. a transition section; 7. a capture element; 71. a capture chamber; 72. an array of vertical angled baffles; 721. an angled baffle; 8. an outlet section. DETAILED DESCRIPTION

[0030] like Figure 1-Figure 3 As shown, the present invention provides a technical solution: a microfluidic cryptococcal sorting device, comprising: The focusing segment is used to receive the fluid solution and focus the particles (cells, cryptococci, etc.) in the fluid solution; the fluid solution is obtained by mixing a cerebrospinal fluid sample that may contain cryptococci with acetic acid and ink, and the acetic acid is used to rupture the red blood cells that may exist in the cerebrospinal fluid sample to reduce interference.

[0031] The sinking section 4 is connected to the output end of the sawtooth-like component and is used to receive the fluid solution after the focused particles and perform stratification.

[0032] The liquid separation section 5 is connected to the sinking section 4 and communicated with the sinking section 4, and is used for separating the cerebrospinal fluid in the fluid solution after stratification in the sinking section 4.

[0033] The transition section 6 is connected to the liquid separation section 5 and is used to receive and transmit the fluid solution after the cerebrospinal fluid is separated by the liquid separation section 5.

[0034] The capturing member 7 is connected to the transition section 6 and is used for receiving the fluid solution transmitted by the transition section 6 and capturing particles in the fluid solution.

[0035] The outlet section 8 is connected to the capturing member 7 and is used for discharging the fluid solution in the capturing member 7 .

[0036] Among them, the focusing segment is sawtooth-like and is composed of multiple inverted V-shaped components 1. Each inverted V-shaped component 1 is provided with a focusing channel. In this embodiment, three inverted V-shaped components 1 are arranged, and two adjacent inverted V-shaped components 1 are connected in series through a common end point and the internal focusing channels are connected.

[0037] Among them, each inverted V-shaped component 1 is composed of an isosceles trapezoidal component 101 with a missing lower base and four isosceles trapezoidal components 102 with missing upper bases; the two ends of the bottom of the isosceles trapezoidal component 101 with a missing lower base are respectively connected to two obliquely arranged isosceles trapezoidal components 102 with missing upper bases, and the two adjacent and interconnected isosceles trapezoidal components 102 with missing upper bases are inverted with each other.

[0038] Wherein, a first connection terminal 1011 is respectively disposed at both ends of the missing part of the isosceles trapezoidal component 101 with a missing lower base, and a second connection terminal 1021 is respectively disposed at both ends of the missing part of the isosceles trapezoidal component 102 with a missing upper base.

[0039] The specific connection relationship between the isosceles trapezoidal component 101 with a missing lower base and the isosceles trapezoidal component 102 with a missing upper base is as follows: the two first connecting ends 1011 of the isosceles trapezoidal component 101 with a missing lower base are respectively connected to a second connecting end 1021 of an isosceles trapezoidal component 102 with a missing upper base, and the other second connecting end 1021 of the isosceles trapezoidal component 102 with a missing upper base is connected to a second connecting end 1021 of an adjacent isosceles trapezoidal component 102 with a missing upper base.

[0040] Among them, adjacent inverted V-shaped components 1 are connected through an inverted isosceles trapezoidal component 101 with a missing lower base, that is, a second connecting end 1021 at the bottom of two adjacent inverted V-shaped components 1 with a missing upper base and not connected isosceles trapezoidal components 102 are respectively connected to two first connecting ends 1011 of the inverted isosceles trapezoidal components 101 with a missing lower base.

[0041] Among them, a second connecting terminal 1021 that is not connected to the isosceles trapezoidal component 102 with the upper base missing at the bottom of the first inverted V-shaped component 1 in the focusing section is connected to a third connecting terminal 1031 on the isosceles trapezoidal component 103 with one-half of the lower base missing, and the other third connecting terminal 1031 of the isosceles trapezoidal component 103 with one-half of the lower base missing is used as the entrance of the focusing section, that is, the entrance of the sorting device.

[0042] Among them, the isosceles trapezoidal component 101 with missing lower base, the isosceles trapezoidal component 102 with missing upper base and the isosceles trapezoidal component 103 with missing half lower base are all provided with focusing channels and are connected.

[0043] By optimizing the focusing channel angle, it was found that the smaller the channel angle, the better the focusing effect, but the channel pressure drop increased. Therefore, 60° with better focusing effect and moderate pressure drop was selected as the optimal solution.

[0044] Among them, the corner angle range of the focusing channel in the isosceles trapezoidal component 102 with a missing upper base can be set to 55°~75°, and the optimal corner angle of the focusing channel in the isosceles trapezoidal component 102 with a missing upper base is 60°.

[0045] Assume that the width of the focusing channel in the isosceles trapezoidal component 101 with missing lower base, the isosceles trapezoidal component 102 with missing upper base and the isosceles trapezoidal component 103 with half of the lower base missing is Z, the inner wall length of the focusing channel in the isosceles trapezoidal component 101 with missing lower base is S, and the inner wall length of the focusing channel in the isosceles trapezoidal component 102 with missing upper base is L, satisfying L=S+Z; wherein, the width of the focusing channel, the sinking channel 401 and the transition channel are all Z, and the width of the liquid separation channel satisfies 1.5Z, so that the kinetic energy loss of the fluid solution after liquid separation through the liquid separation channel can be reduced, thereby flowing into the capture chamber 71 at a slower speed.

[0046] like Figure 4 As shown in the figure, when the channel inlet velocity is set to 0.1m / s and the cross-sectional area is the same, the focusing effect of the four structures of arc, rectangle, inverted V-shape and isosceles trapezoid with missing bottom edge is compared in series according to 5 periods. The focusing effect of isosceles trapezoid with missing bottom edge is the best, followed by inverted V-shape. Figure 5 As shown in the figure, from the velocity distribution of the fluid solution at the corners of the inverted V-shaped inner channel and the isosceles trapezoidal structure with the bottom missing, it can be seen that due to the existence of wall shear force, the fluid flow velocity close to the side wall of the channel is lower, while the flow velocity in the central area of ​​the channel is relatively high, which causes particles (such as cells or cryptococci, etc.) to gradually concentrate in the central area of ​​the channel during the flow process to form a streamlined arrangement, and a significant vortex area is formed at the corner of the channel with the isosceles trapezoidal structure with the bottom missing. In this area, the fluid produces rotational motion under the action of Dean drag. The rotation and acceleration of the fluid solution enhance the collision and friction effects of the particles. When the fluid solution flows through these local corners, the magnitude and direction of the flow velocity change dramatically, resulting in a continuous change in the velocity distribution law on the cross section and the generation of additional friction. The setting of the isosceles trapezoidal structure with the bottom missing allows the flow velocity direction of the fluid solution to be adjusted multiple times. At the same time, the vortex area at the corner overlaps with the main velocity distribution area, further enhancing the friction effect of the fluid.

[0047] like Figure 6-Figure 7As shown, a sinking channel 401 is provided in the sinking section 4, and an inner cavity is provided in the sinking channel 401. The sinking channel 401 is connected to a second connecting end 1021 that is not connected to the isosceles trapezoidal component 102 with the upper bottom edge missing at the bottom of the last inverted V-shaped component 1 in the focusing section, and is communicated with the focusing channel in the isosceles trapezoidal component 102 with the upper bottom edge missing; the first step 411, the second step 412, the third step 413 are sequentially provided from the input end to the output end of the inner cavity of the sinking channel 401. , the 4th step 414, the 5th step 415, the 6th step 416 and the 7th step 417, wherein the bottom of the inner cavity of the sinking channel 401 is taken as the 0th step, and it descends layer by layer from the 0th step to the 5th step 415, and ascends layer by layer from the 5th step 415 to the 7th step 417; wherein the 1st step 411, the 2nd step 412, the 3rd step 413, the 4th step 414, the 5th step 415, the 6th step 416 and the 7th step 417 are set with the same width.

[0048] The 6th step 416 is flush with the 0th step. Let the vertical distance from the 5th step 415 to the 0th step be A, and the vertical distance from the 5th step 415 to the 7th step 417 be B, satisfying B>A.

[0049] Among them, the sinking channel 401 is connected to a second connecting end 1021 that is not connected to the isosceles trapezoidal component 102 with the missing upper base at the bottom of the last inverted V-shaped component 1 in the focusing section through a plurality of isosceles trapezoidal components 102 with the missing upper base that are connected in sequence and inverted to each other, and is connected to the focusing channel in the isosceles trapezoidal component 102 with the missing upper base.

[0050] Among them, there are two liquid-separating sections 5, the input ends of the two liquid-separating sections 5 are respectively connected to the two sides of the sinking section 4, and the output ends of the two liquid-separating sections 5 are connected to the outside world; the two liquid-separating sections 5 are on the same axis; liquid-separating channels are provided in the two liquid-separating sections 5, and the liquid-separating channels are connected to the sinking channel 401 in the sinking section 4; the connection point between the liquid-separating section 5 and the sinking section 4 is at the top of the 5th step 415 in the sinking channel 401.

[0051] By setting up five consecutive descending steps and two ascending steps, particles (cells, cryptococci, etc.) can be mainly gathered on the lower side of the sinking channel 401 after passing through the sinking channel 401. When separated through the separation channel, the particles (cells, cryptococci, etc.) in the fluid solution maintain linear motion due to inertia, while the cerebrospinal fluid in the fluid solution is separated into the separation channels on both sides, thereby achieving an enrichment effect.

[0052] Among them, a transition channel is arranged in the transition section 6 , and the transition channel in the transition section 6 is connected with the sinking channel 401 in the sinking section 4 .

[0053] like Figure 8 As shown, a capture chamber 71 is arranged in the capture member 7, and the capture chamber 71 is a semi-rectangular outline, that is, the capture chamber 71 is composed of a rectangular cavity and a semi-circular cavity. The capture chamber 71 composed of a rectangular cavity and a semi-circular cavity has the best vortex effect, and particles are easy to enter but difficult to exit; a vertical angled baffle array 72 composed of a plurality of angled baffles 721 is arranged in the capture chamber 71, and the openings of all angled baffles 721 in the vertical angled baffle array 72 are all facing the entrance end of the capture member 7; the rectangular cavity in the capture chamber 71 is connected with the transition channel; through the design of the vertical angled baffle array 72, the fluid solution has a larger vortex area after entering the capture chamber 71, so that particle cells, cryptococci, etc. can be effectively preserved in the capture chamber 71.

[0054] Among them, the connection point between the transition section 6 and the capture member 7 is set on the central axis of the capture member 7; a depression is set at the connection between the capture member 7 and the transition section 6, and the depth of the depression accounts for 1 / 5~1 / 4 of the longest distance between the two ends of the capture member 7.

[0055] Among them, an outlet channel is arranged in the outlet section 8, one end of the outlet channel is connected with the semicircular cavity in the capture cavity 71, and the other end of the outlet channel is connected with the outside.

[0056] A microfluidic cryptococcal detection method comprises the following steps: S1: A cerebrospinal fluid sample that may contain cryptococci is mixed with acetic acid and ink to obtain a fluid solution. Acetic acid is used to rupture red blood cells that may be present in the cerebrospinal fluid sample to reduce interference.

[0057] S2: Inject the fluid solution into the inlet of the focusing section through a micro pump at a speed of 0.1 m / s, and drive the fluid solution through the focusing channels in the isosceles trapezoidal component 101 with missing lower base, the isosceles trapezoidal component 102 with missing upper base, and the isosceles trapezoidal component 103 with half of the lower base missing in the focusing section, so that the particles (cells, cryptococci, etc.) in the fluid solution are focused.

[0058] The fluid solution behind the focused particles is driven to pass through the sinking channel, and the fluid solution behind the focused particles is stratified, so that the cerebrospinal fluid in the fluid solution is focused on the upper layer of the sinking channel, and discharged through the transition channel in the transition section 6, and the particles (cells, cryptococci, etc.) are focused on the lower layer of the sinking channel.

[0059] The fluid solution after separation of cerebrospinal fluid is driven to flow into the capture chamber through the transition section, and the fluid solution is enriched by the vertical angled baffle array.

[0060] S3: Observe the particles in the capture cavity under a microscope to see if they contain Cryptococcus.

[0061] When all the fluid solution enters the focusing section, the ink is continuously injected into the focusing section using a syringe to drive all the fluid solution into the capture chamber.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic cryptococcal sorting device, characterized in that: include: A focusing section, used for receiving the fluid solution and focusing the particles in the fluid solution; A sinking section (4), the sinking section (4) is connected to the output end of the sawtooth-like component and is used to receive the fluid solution after the particles are focused and perform stratification; A liquid separation section (5), the liquid separation section (5) being connected to the sinking section (4) and in communication with the sinking section (4), and being used for separating cerebrospinal fluid from the fluid solution after stratification in the sinking section (4); A transition section (6), the transition section (6) being connected to the liquid separation section (5) and being used for receiving and transmitting the fluid solution after the cerebrospinal fluid is separated by the liquid separation section (5); A capture element (7), the capture element (7) being connected to the transition section (6) and being used to receive the fluid solution transmitted by the transition section (6) and to capture particles in the fluid solution; An outlet section (8), the outlet section (8) being connected to the capture member (7) and being used to discharge the fluid solution in the capture member (7); The focusing section is shaped like a sawtooth and is composed of a plurality of quasi-inverted V-shaped components (1), each quasi-inverted V-shaped component (1) is provided with a focusing channel, and two adjacent quasi-inverted V-shaped components (1) are connected in series via a common end point and the internal focusing channels are connected; Each quasi-inverted V-shaped component (1) is composed of an isosceles trapezoidal component (101) with a missing lower base and four isosceles trapezoidal components (102) with missing upper bases.

2. A microfluidic cryptococcal sorting device according to claim 1, characterized in that: Two ends of the bottom of the lower base missing isosceles trapezoidal component (101) are respectively connected in sequence to two obliquely arranged upper base missing isosceles trapezoidal components (102), and the two adjacent and mutually connected upper base missing isosceles trapezoidal components (102) are inverted with each other; A first connection end (1011) is respectively arranged at both ends of the missing portion of the lower base missing isosceles trapezoid component (101), and a second connection end (1021) is respectively arranged at both ends of the missing portion of the upper base missing isosceles trapezoid component (102); the specific connection relationship between the lower base missing isosceles trapezoid component (101) and the upper base missing isosceles trapezoid component (102) is as follows: the two first connection ends (1011) of the lower base missing isosceles trapezoid component (101) are respectively connected to a second connection end (1021) of an upper base missing isosceles trapezoid component (102), and the other second connection end (1021) of the upper base missing isosceles trapezoid component (102) is connected to a second connection end (1021) of an adjacent upper base missing isosceles trapezoid component (102); Adjacent quasi-inverted V-shaped components (1) are connected via an inverted lower base missing isosceles trapezoid component (101), that is, a second connection end (1021) not connected to the upper base missing isosceles trapezoid component (102) at the bottom of two adjacent quasi-inverted V-shaped components (1) is respectively connected to two first connection ends (1011) of the inverted lower base missing isosceles trapezoid component (101).

3. A microfluidic cryptococcal sorting device according to claim 2, characterized in that: A second connecting end (1021) not connected to the isosceles trapezoidal component (102) with the upper base missing at the bottom of the first quasi-inverted V-shaped component (1) in the focusing section is connected to a third connecting end (1031) on the isosceles trapezoidal component (103) with the lower base missing in half, and another third connecting end (1031) of the isosceles trapezoidal component (103) with the lower base missing in half is used as the entrance of the focusing section, i.e., the entrance of the sorting device; Focusing channels are provided in the isosceles trapezoidal component (101) with a missing lower base, the isosceles trapezoidal component (102) with a missing upper base, and the isosceles trapezoidal component (103) with a half missing lower base, and are connected.

4. A microfluidic cryptococcal sorting device according to claim 3, characterized in that: The corner angle range of the focusing channel in the isosceles trapezoidal component (102) with the upper bottom edge missing is set to 55° to 75°; Assume that the width of the focusing channel in the isosceles trapezoidal component (101) with a missing lower base, the isosceles trapezoidal component (102) with a missing upper base, and the isosceles trapezoidal component (103) with half the missing lower base is Z, the inner wall length of the focusing channel in the isosceles trapezoidal component (101) with a missing lower base is S, and the inner wall length of the focusing channel in the isosceles trapezoidal component (102) with a missing upper base is L, satisfying L=S+Z; wherein the widths of the focusing channel, the sinking channel (401), and the transition channel are all Z, and the width of the liquid separation channel satisfies 1.5Z.

5. A microfluidic cryptococcal sorting device according to claim 4, characterized in that: The sinking section (4) is provided with a sinking channel (401), and an inner cavity is provided in the sinking channel (401). The sinking channel (401) is connected to a second connection end (1021) that is not connected to the upper bottom edge missing isosceles trapezoidal component (102) at the bottom of the last inverted V-shaped component (1) in the focusing section, and is communicated with the focusing channel in the upper bottom edge missing isosceles trapezoidal component (102); the inner cavity of the sinking channel (401) is provided with a first step (411), a second step (412), a third step (413), a fourth step (414) in sequence from the input end to the output end. The first step (411), the second step (412), the third step (413), the fourth step (414), the fifth step (415), the sixth step (416) and the seventh step (417) are provided, wherein the bottom of the inner cavity of the sinking channel (401) is used as the 0th step, and the steps are gradually descended from the 0th step to the 5th step (415), and gradually ascended from the 5th step (415) to the 7th step (417); wherein the first step (411), the second step (412), the third step (413), the fourth step (414), the fifth step (415), the sixth step (416) and the seventh step (417) are arranged with equal width; The sixth step (416) is flush with the zeroth step. Let the vertical distance from the fifth step (415) to the zeroth step be A, and the vertical distance from the fifth step (415) to the seventh step (417) be B, satisfying B>A; The sinking channel (401) is connected to a second connection end (1021) that is not connected to the upper base missing isosceles trapezoidal component (102) at the bottom of the last inverted V-shaped component (1) in the focusing section via a plurality of upper base missing isosceles trapezoidal components (102) that are sequentially connected and inverted to each other, and is in communication with the focusing channel in the upper base missing isosceles trapezoidal component (102).

6. A microfluidic cryptococcal sorting device according to claim 5, characterized in that: The two liquid separation sections (5) are provided, the input ends of the two liquid separation sections (5) are respectively connected to the two sides of the sinking section (4), and the output ends of the two liquid separation sections (5) are connected to the outside world; the two liquid separation sections (5) are located on the same axis; liquid separation channels are provided in the two liquid separation sections (5), and the liquid separation channels are connected to the sinking channel (401) in the sinking section (4); the connection point between the liquid separation section (5) and the sinking section (4) is located at the top of the fifth step (415) in the sinking channel (401).

7. A microfluidic cryptococcal sorting device according to claim 6, characterized in that: A transition channel is provided in the transition section (6), and the transition channel in the transition section (6) is connected to the sinking channel (401) in the sinking section (4).

8. A microfluidic cryptococcal sorting device according to claim 7, characterized in that: The capture member (7) is provided with a capture cavity (71), and the capture cavity (71) is of a semicircular rectangular outline, that is, the capture cavity (71) is formed by splicing a rectangular cavity and a semicircular cavity; a vertical angled baffle array (72) composed of a plurality of angled baffles (721) is provided in the capture cavity (71), and the openings of all the angled baffles (721) in the vertical angled baffle array (72) are all oriented toward the entrance end of the capture member 7; the rectangular cavity in the capture cavity (71) is connected to the transition channel; The connection point between the transition section (6) and the capture member (7) is arranged on the central axis of the capture member (7); a depression is arranged at the connection between the capture member (7) and the transition section (6), and the depth of the depression accounts for 1 / 5 to 1 / 4 of the longest distance between the two ends of the capture member (7).

9. A microfluidic cryptococcal sorting device according to claim 8, characterized in that: An outlet channel is provided in the outlet section (8), one end of the outlet channel is connected to the semicircular cavity in the capture cavity (71), and the other end of the outlet channel is connected to the outside.

10. A microfluidic cryptococcal detection method, applied to a microfluidic cryptococcal sorting device according to any one of claims 1 to 9, comprising the following steps: S1: Mixing the cerebrospinal fluid sample to be tested with acetic acid and ink to obtain a fluid solution; S2: injecting a fluid solution into the inlet of the focusing section, driving the fluid solution through the focusing channels in the lower base missing isosceles trapezoidal components (101), the upper base missing isosceles trapezoidal components (102) and the half lower base missing isosceles trapezoidal components (103) in the focusing section, so that the particles in the fluid solution are focused; Driving the fluid solution behind the focused particles through the sinking channel, stratifying the fluid solution behind the focused particles, so that the cerebrospinal fluid in the fluid solution is focused on the upper layer of the sinking channel, discharged through the transition channel in the transition section (6), and the particles are focused on the lower layer of the sinking channel; The fluid solution after separation of cerebrospinal fluid is driven to flow into the capture chamber through the transition section, and the fluid solution is enriched by the vertical angled baffle array; S3: Observe the particles in the capture cavity by microscopy to see if they contain cryptococci; When all the fluid solution enters the focusing section, the ink is continuously injected into the focusing section using a syringe to drive all the fluid solution into the capture chamber.

Images