Handheld bacteria detector

Abstract

The utility model discloses a handheld bacterium detector and relates to the technical field of bacterium detection. Comprising a cover plate, a bottom plate and four side plates, the cover plate is arranged at the top of the shell, and a grab handle and a square groove are arranged on one side face of the shell; the fluorescence detection module is parallel to the cover plate and comprises an optical unit, a photoelectric conversion unit and a signal processing unit; the micro-fluidic chip is positioned in the shell, is arranged below the fluorescence detection module in parallel, and is sequentially provided with an enrichment unit, an amplification unit, a cutting unit and a chromatography unit according to a certain interval; the chromatography unit is positioned below the optical unit; the intelligent control module comprises a circular working platform, a servo motor, an intelligent touch display screen, an electromagnet and the like; a mounting through hole is formed in the middle of the circular working platform, a plurality of fan-shaped grooves are formed in the circular working platform in the circumferential direction, a plurality of micro-fluidic chips can be placed in the fan-shaped grooves respectively, and an output shaft of the servo motor and the mounting through hole are detachably and fixedly mounted; the device has the advantages of convenience in handheld operation, high sensitivity and high detection efficiency.

Description

Technical Field

[0001] The utility model relates to the technical field of bacteria detection, in particular to a handheld bacteria detector. Background Art

[0002] Bacteria such as Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli are important pathogens of clinical isolation and hospital infection, which can cause respiratory tract and urogenital infections, trauma, sepsis and diarrhea. In severe cases, they can also cause sepsis and endanger life safety. Therefore, rapid early screening of pathogens is crucial for the prevention and control of related diseases.

[0003] A Chinese patent (grant announcement number CN116356057A, grant announcement date 2023.06.30) discloses a bacterial detection method and system based on digital CRISPR, and the specific steps are as follows: S1, preparing a reaction system;

[0004] S2, the reaction system is formed into several droplet units by the droplet generation method; S3, the reaction is carried out at 37℃ for 30-90min, and then the fluorescence intensity of each droplet unit is obtained by the fluorescence detection equipment, and the fluorescence intensity detection results of all droplet units are counted, so as to calculate the concentration of target bacteria in the bacterial sample to be tested. It can be seen that the droplet operation is cumbersome and demanding, each droplet unit contains at most one cell, and the statistical method and calculation method are traditional.

[0005] A Chinese patent (authorization announcement number CN107084907A, authorization announcement date 2017.08.22) discloses a blood bacteria culture detection method and its application, with the following specific steps: S1, inject blood into a blood culture bottle containing culture medium, mix evenly, and immediately measure the initial gas pressure P0 of the gas space in the blood culture bottle; S2, culture blood bacteria and continuously measure the real-time gas pressure Pt in the blood culture bottle. When Pt>P0, it is determined that bacteria exist in the blood. It can be seen that the technical test cycle is long, the detection results are single, and the level of intelligence is low.

[0006] In summary, the detection technologies for bloodstream infection diseases such as sepsis on the market have the problems of cumbersome operation, long time consumption, and low quality of test result feedback. Therefore, the market urgently needs a convenient, rapid, intelligent and integrated bacterial detection instrument. Utility Model Content

[0007] In view of this, the present invention proposes a handheld bacteria detector, aiming to solve the above technical problems. The handheld bacteria detector proposed in the present invention can achieve the advantages of convenient detection operation, high sensitivity, strong specificity, high efficiency, etc.

[0008] The utility model provides a handheld bacteria detector, comprising:

[0009] A shell, the shell comprising a cover plate, a bottom plate and four side plates, the top of the shell is opened, the cover plate is arranged at the opening; one side plate of the shell is provided with a handle and a square groove; the square groove is located above the handle;

[0010] A fluorescence detection module, the fluorescence detection module is parallel to the cover plate and is arranged at the bottom of the cover plate; the fluorescence detection module includes an optical unit, a photoelectric conversion unit and a signal processing unit; the optical unit includes an ultraviolet excitation light path and a fluorescence collection light path; the photoelectric conversion unit is connected to the fluorescence collection light path; the signal processing unit is connected to the photoelectric conversion unit;

[0011] A microfluidic chip, wherein the microfluidic chip is located in the housing and parallel to the fluorescence detection module; an enrichment unit, an amplification unit, a cutting unit and a chromatography unit are sequentially arranged on the microfluidic chip at certain intervals; the enrichment unit, the amplification unit, the cutting unit and the chromatography unit are all connected through the microfluidic channel; the microfluidic channel is provided with a siphon valve; the chromatography unit is located below the optical unit;

[0012] An intelligent control module, the intelligent control module includes a circular working platform, a servo motor, an intelligent touch display screen and an electromagnet; a mounting through hole is provided in the middle of the circular working platform; a plurality of fan-shaped grooves are provided on the upper surface of the circular working platform along the circumferential direction, and the fan-shaped grooves are used to place the microfluidic chip; the servo motor is located below the circular working platform, and an output shaft is provided on the top of the servo motor; one end of the output shaft is connected to the motor body; the other end of the output shaft is detachably connected to the mounting through hole; the intelligent touch display screen is placed in the square groove and connected to the signal processing unit; the electromagnet is located at the bottom of the circular working platform.

[0013] Compared with the prior art, the utility model has the following beneficial effects: the microfluidic chip can be driven to rotate around a fixed axis by a servo motor to oscillate and mix sample liquids of multiple units; the microfluidic chip is driven to rotate at a variable speed by a servo motor, and the centrifugal force is used to make the sample liquid pass through the enrichment unit, the amplification unit, and the cutting unit in sequence from the internal flow channel, and the sample liquid processed by the first three units enters the chromatography unit for fluorescence color development; the ultraviolet excitation light path of the fluorescence detection technology module emits laser light, which is focused on the area required for detection of the test strip, so that the reaction complex generates fluorescence, and the stimulated fluorescence is focused on the photoelectric conversion unit through the fluorescence collection light path, and the light signal collected by the fluorescence collection light path is converted into an electrical signal, and the signal processing unit analyzes and processes the electrical signal, that is, the fluorescence intensity ratio, and the number of colonies can be obtained by comparing the fluorescence intensity ratio-bacteria concentration standard curve; the utility model performs bacteria detection by fluorescence detection and analysis, and the detection is automated, achieving the effects of high accuracy and high efficiency.

[0014] As a further improvement of the above technical solution, there are multiple fluorescence detection modules; the photoelectric units of the multiple fluorescence detection modules are arranged in a one-to-one correspondence with the chromatography units of the multiple microfluidic chips; and the multiple microfluidic chips work in coordination with the multiple fluorescence detection modules.

[0015] As a further improvement of the above technical solution, the microfluidic chip includes a first chip layer, a second chip layer and a cover layer; the first chip layer and the cover layer are made of transparent materials, and the first chip layer, the second chip layer and the cover layer are sequentially bonded and sealed from bottom to top.

[0016] As a further improvement of the above technical solution, the enrichment unit includes a mixing chamber, a washing chamber, a demagnetization chamber, and a lysis chamber; the mixing chamber, the washing chamber, and the demagnetization chamber are arranged at intervals in a direction away from the mounting through hole and are sequentially connected through the microchannel inside the microfluidic chip;

[0017] The microfluidic flow channel includes a first microfluidic flow channel, a second microfluidic flow channel, a third microfluidic flow channel and a fourth microfluidic flow channel;

[0018] The demagnetization chamber is connected to the lysis chamber through the first microfluidic channel;

[0019] The amplification unit uses RPA amplification technology; the amplification unit is located on the side of the lysis chamber away from the mounting through hole; the amplification unit is connected to the lysis chamber through the second microfluidic channel;

[0020] The cutting unit uses CRISPR / Cas12a cutting technology; the cutting unit is located on the side of the amplification unit away from the mounting through hole; there are multiple cutting units; there are multiple third microfluidic channels; the multiple cutting units are connected to the amplification unit in a one-to-one correspondence through the multiple third microfluidic channels;

[0021] The chromatography unit is a test paper; the chromatography unit is located on the side of the cutting unit away from the mounting through hole; the chromatography unit is connected to multiple cutting units through a fourth microfluidic channel; the fourth microfluidic channel is used to introduce sample liquids from multiple cutting units into the chromatography unit.

[0022] As a further improvement of the above technical solution, the electromagnet includes a first electromagnet and a second electromagnet; when the electromagnet rotates on the circular working platform, the first electromagnet corresponds to the bottom of the mixing chamber, and the top of the first electromagnet is close to the bottom of the mixing chamber; the second electromagnet corresponds to the bottom of the washing chamber, and the top of the second electromagnet is close to the bottom of the washing chamber; when the microfluidic chip rotates, the first electromagnet and the second electromagnet can both move to the bottom of the demagnetization chamber;

[0023] The first electromagnet is used to transfer the mixed sample liquid in the mixing chamber to the washing chamber; the second electromagnet is used to transfer the mixed sample liquid in the washing chamber to the demagnetization chamber; both the first electromagnet and the second electromagnet can be used to demagnetize the sample liquid in the demagnetization chamber.

[0024] The mixing chamber is used to achieve mixing of the sample liquid and concanavalin A-nanomagnetic beads, and concanavalin A is indirectly coupled with streptavidin-coated magnetic nanoparticles to form a concanavalin A-magnetic nanoparticle complex, which is used to capture high-risk pathogens that cause sepsis such as Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus in the sample;

[0025] The sample liquid is further transferred to the washing chamber by the first electromagnet, and washed with the washing liquid to separate the target bacteria from the sample liquid;

[0026] The sample liquid is further transferred to the demagnetization chamber by the second electromagnet; an external magnetic field is applied to the demagnetization chamber by the electromagnet to demagnetize the magnetic beads, thereby separating the target bacteria from the magnetic beads;

[0027] The microfluidic chip is further controlled to rotate at a certain speed to centrifuge the sample in the demagnetization chamber, and the bacterial sample is filtered through the first microfluidic channel and flows into the lysis chamber to mix with the lysis solution;

[0028] Further centrifuging the sample liquid after lysis in the lysis chamber at a certain centrifugal speed, so that the sample liquid after lysis enters the amplification unit after being filtered;

[0029] Further centrifuging the amplified sample liquid in the amplification unit at a certain centrifugal speed, so that the amplified sample liquid enters the cutting unit for cutting;

[0030] The cut sample liquid in the cutting unit is further centrifuged at a certain centrifugal speed, so that the cut sample liquid enters the chromatography unit for chromatography, and finally the chromatography result can be observed;

[0031] The servo motor is further controlled to stop moving, and the fluorescence detection module will detect the chromatography test paper, and finally the number of colonies and the concentration can be obtained.

[0032] As a further improvement of the above technical solution, the intelligent control module also includes a heating device, which includes a heating ring 1 and a heating ring 2; the heating ring 1 and the heating ring 2 are coaxially arranged with the mounting through hole and are both fixed to the bottom of the circular working platform; the heating ring 1 is correspondingly located below the lysis chamber; the heating ring 2 is correspondingly located below the amplification unit;

[0033] After the bacterial sample liquid flows into the lysis chamber, the lysis chamber can be heated by turning on the heating ring 1; after the lysed sample liquid enters the amplification unit, the heating ring 2 can be turned on to perform constant temperature amplification on the amplification unit.

[0034] As a further improvement of the above technical solution, the intelligent control module also includes a controller, and the optical unit, the servo motor, the first electromagnet, the second electromagnet, the heating ring 1, the heating ring 2 and the intelligent touch display screen are all electrically connected to the controller.

[0035] As a further improvement of the above technical solution, the mixing chamber, the washing chamber, the demagnetization chamber, the lysis chamber, the amplification unit, the cutting unit and the chromatography unit are all on the second chip layer; the corresponding positions of the mixing chamber are provided with sample loading holes that can be connected to the corresponding mixing chamber; the top walls of the mixing chamber, the washing chamber, the demagnetization chamber, the lysis chamber, the amplification unit, the cutting unit and the chromatography unit are provided with exhaust holes; the sample loading holes and the exhaust holes are both through holes opened on the cover layer.

[0036] As a further improvement of the above technical solution, the first microfluidic channel, the second microfluidic channel, the third microfluidic channel and the fourth microfluidic channel are all microfluidic grooves located on the top surface of the second chip layer.

[0037] As a further improvement of the above technical solution, a siphon valve 1 is provided on the first microfluidic channel; a siphon valve 2 is provided on the second microfluidic channel; a siphon valve 3 is provided on the third microfluidic channel; and a siphon valve 4 is provided on the fourth microfluidic channel;

[0038] The siphon valve one, the siphon valve, the siphon valve three and the siphon valve four all have a certain centrifugal speed threshold; when the centrifugal speed threshold is exceeded, the siphon valve one, the siphon valve two, the siphon valve three and the siphon valve four can be centrifugally conducted; the centrifugal speed threshold of the siphon valve one can be set to be smaller than the centrifugal speed threshold of the siphon valve two, the centrifugal speed threshold of the siphon valve two can be set to be smaller than the centrifugal speed threshold of the siphon valve three, and the centrifugal speed threshold of the siphon valve three can be set to be smaller than the centrifugal speed threshold of the siphon valve four; by controlling the speed of the microfluidic chip, the conduction state of the sample liquid between the demagnetization chamber and the lysis chamber, between the lysis chamber and the amplification unit, between the amplification unit and the cutting unit, and between the cutting unit and the chromatography unit can be controlled.

[0039] As a further improvement of the above technical solution, different primers are placed in the amplification unit to achieve amplification of different types of bacteria in the same sample liquid; the cutting unit and the third microfluidic channel are multiple and the number is the same; the multiple cutting units are spaced apart and distributed along the direction surrounding the mounting through hole; different primers are placed in the multiple cutting units to achieve cutting of different types of bacteria in the same sample liquid.

[0040] It can be seen from the above technical solution that, compared with the prior art, the utility model discloses a handheld intelligent bacteria detection instrument, which has the following advantages:

[0041] 1. High sensitivity. The functionalized magnetic nanoparticle enrichment technology has high capture sensitivity; the multiplexed RPA nucleic acid amplification technology has high detection sensitivity.

[0042] 2. High specificity and accuracy. RPA amplification technology has high specificity, and its specificity can reach 99.3%. Under the guidance of a guide RNA (gRNA), the WED and PI domains of Cas12a protein can specifically recognize the PAM sequence of the double-stranded DNA (dsDNA) target, thereby promoting the expansion of the dsDNA target.

[0043] 3. High detection efficiency. No need to pre-treat and culture the sample, saving 6-8 hours of bacterial culture time; the whole detection process can be shortened to 60 minutes, achieving rapid diagnosis of bacterial infection.

[0044] 4. High throughput design, multiple bacterial joint tests. Each test can achieve at least 30 throughputs, greatly improving the detection efficiency.

[0045] 5. Low threshold for use. It has low requirements on the operating environment and professional operators, is highly universal, and can be applied in many fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the utility model or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the utility model. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0047] Figure 1 An exploded diagram of the overall structure of a handheld intelligent bacteria detection instrument of the utility model;

[0048] Figure 2 A schematic diagram of the circular working platform structure of a handheld intelligent bacteria detection instrument of the utility model;

[0049] Figure 3 A schematic diagram of the microfluidic chip structure of a handheld intelligent bacteria detection instrument of the utility model;

[0050] Figure 4 A schematic diagram of the cover sheet structure of a handheld intelligent bacteria detection instrument of the utility model;

[0051] Figure 5 A schematic diagram of the working principle of a siphon valve of a handheld intelligent bacteria detection instrument of the utility model;

[0052] Description of main component symbols:

[0053] 1. Shell; 11. Cover; 12. Handle; 13. Square groove; 14. Side plate; 15. Bottom plate; 2. Fluorescence detection module; 21. Optical unit; 211. UV excitation light path; 212. Fluorescence collection light path; 22. Photoelectric conversion unit; 23. Signal processing unit; 3. Microfluidic chip; 31. Enrichment unit; 311. Mixing chamber; 312. Washing chamber; 313. Demagnetization chamber; 314. Lysis chamber; 32. Amplification unit; 33. Cutting unit; 34. Chromatography unit; 35. Microfluidic channel; 351. First microfluidic channel; 352. Second microfluidic channel; 353. Third microfluidic channel Control channel; 354, fourth microfluidic channel; 36, siphon valve; 361, siphon valve 1; 362, siphon valve 2; 363 siphon valve 3; 364 siphon valve 4; 37, sample addition hole; 38, exhaust hole; 4, intelligent control module; 41, circular working platform; 411 installation through hole; 412 fan-shaped groove; 42, servo motor; 421 output shaft; 422 motor body; 43, intelligent touch display screen; 44, electromagnet; 441, first electromagnet; 442, second electromagnet; 45 heating device; 451, heating ring 1; 452, heating ring 2; 46 controller; 47 wireless communication device. The following specific implementation will further illustrate the utility model in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0055] In the description of the present invention, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0056] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present utility model, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0057] In the present invention, unless otherwise clearly specified and limited, the terms "install", "connect", "connect", "fix" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0058] Example:

[0059] like Figures 1 to 5 As shown, the utility model provides a handheld intelligent bacteria detection instrument, comprising:

[0060] The housing 1 comprises a cover plate 11, a bottom plate 15 and four side plates 14; the top of the housing 1 is open, and the cover plate 11 is arranged at the open position; a handle 12 and a square groove 13 are arranged on one side of the housing 1; the square groove 13 is located above the handle 12; the middle of the housing is surrounded and fixed by four side plates 14, and the bottom is provided with a bottom plate 15, and the periphery of the bottom plate 15 is fixed to the bottom of the four side plates 14; one side end of the cover plate 11 is hinged to the top of a side plate 12 (the hinge structure is not shown in the figure), so that the cover plate 11 can be rotated to open toward one side of the housing 1;

[0061] Fluorescence detection module 2, the fluorescence detection module 2 is arranged parallel to the inner side of the cover plate 11; the fluorescence detection module 2 includes an optical unit 21, a photoelectric conversion unit 22 and a signal processing unit 23; the optical unit 21 includes an ultraviolet excitation light path 211 and a fluorescence collection light path 212, the ultraviolet excitation light path 211 is composed of an LED and a reflection path, the fluorescence collection light path 212 is composed of a reflection path, and the ultraviolet excitation light path 211 and the fluorescence collection light path 212 are both hinged to the side plate (the hinge structure is not shown in the figure); the photoelectric conversion unit 22 is connected to the collection terminal of the fluorescence collection light path 212; the signal processing unit 23 is connected to the photoelectric conversion unit 22;

[0062] The microfluidic chip 3 is fan-shaped; the microfluidic chip 3 is located in the housing 1 and is arranged parallel to the bottom of the fluorescence detection module 2; the microfluidic chip 3 is provided with an enrichment unit 31, an amplification unit 32, a cutting unit 33 and a chromatography unit 34 in sequence at a certain interval; the enrichment unit 31, the amplification unit 32, the cutting unit 33 and the chromatography unit 34 are all connected through a microfluidic channel 35; the microfluidic channel 35 is provided with a siphon valve 36; the chromatography unit 34 is located below the optical unit 21;

[0063] Intelligent control module 4, intelligent control module 4 includes a circular working platform 41, a servo motor 42, an intelligent touch display screen 43 and an electromagnet 44, etc.; a mounting through hole 411 is provided in the middle of the circular working platform 41; a plurality of fan-shaped grooves 412 are provided on the upper surface of the circular working platform 41 along the circumferential direction, and a plurality of microfluidic chips 3 can be placed respectively. Along the direction away from the mounting through hole 411, the microfluidic chip 3 is provided with an enrichment unit 31, an amplification unit 32, a cutting unit 33 and a chromatography unit 34; the servo motor 42 is located below the circular working platform 41, and an output shaft 421 is provided on the top of the servo motor 42; one end of the output shaft 421 is connected to the motor body 422; the other end of the output shaft 421 is detachably fixed to the mounting through hole 411; the intelligent touch display screen 43 is placed in the square groove 13 and is connected to the signal processing unit 23; the electromagnet 43 is located at the bottom of the circular working platform 41.

[0064] The utility model discloses a handheld intelligent bacteria detection instrument. A servo motor 42 can drive a microfluidic chip to rotate around a fixed axis to oscillate and mix sample liquids of multiple units. The servo motor 42 drives the microfluidic chip 3 to rotate at a variable speed, and the centrifugal force is used to make the sample liquid pass through the enrichment unit 31, the amplification unit 32, and the cutting unit 33 from the internal flow channel in sequence. The sample liquid processed by the first three units enters the chromatography unit 34 for fluorescence color development. The ultraviolet excitation light path 211 of the fluorescence detection technology module 2 emits laser light, which is focused on the area required for detection of the test strip, so that the reaction combination generates fluorescence. The stimulated fluorescence is focused on the photoelectric conversion unit 22 through the fluorescence collection light path 212, and the light signal collected by the fluorescence collection light path 212 is converted into an electrical signal. The signal processing unit 23 analyzes and processes the electrical signal, that is, the fluorescence intensity ratio, and the number of colonies can be obtained by comparing the fluorescence intensity ratio-bacteria concentration standard curve. The utility model performs bacteria detection through fluorescence detection and analysis, and the detection is automated, achieving the effects of high accuracy and high efficiency.

[0065] Specifically, the cover 11 can be opened to install or remove the microfluidic chip 3; when installing the microfluidic chip 3, align the microfluidic chip 3 with the fan-shaped groove 412 of the circular working platform 41 and place it in the groove; when installing the circular working platform 41, align the installation through hole 411 with the output shaft 421 of the servo motor 42, and the circular working platform 41 can be installed on the output shaft 421, and the installation through hole 411 is adapted to the output shaft 421.

[0066] In some specific embodiments, there are six fluorescence detection modules 2 and six microfluidic chips 3. The photoelectric units 21 of the six fluorescence detection modules 2 and the chromatography units 34 of the six microfluidic chips 3 are arranged one by one and are evenly distributed around the mounting through hole 411 in a fan-shaped shape.

[0067] Specifically, the microfluidic chip 3 includes a first chip layer, a second chip layer and a cover layer; the first chip layer and the cover layer are made of transparent materials, and the first chip layer, the second chip layer and the cover layer are sequentially attached and sealed from bottom to top;

[0068] In some specific embodiments, the enrichment unit 31 includes a mixing chamber 311, a washing chamber 312, a demagnetization chamber 313, and a lysis chamber 314; the mixing chamber 311, the washing chamber 312, and the demagnetization chamber 313 are arranged in a spaced relationship away from the mounting through hole 411 and are sequentially connected through the microchannel 35 inside the microfluidic chip;

[0069] The microfluidic channel 35 includes a first microfluidic channel 351 , a second microfluidic channel 352 , a third microfluidic channel 353 and a fourth microfluidic channel 354 ;

[0070] In some specific embodiments, the waste liquid after washing can enter the waste liquid chamber 315 through the first microfluidic channel 351;

[0071] The demagnetization chamber 313 is connected to the lysis chamber 314 via a first microfluidic channel 351;

[0072] The amplification unit 32 uses RPA amplification technology; the amplification unit 32 is located on the side of the lysis chamber 314 away from the installation through hole 411; the amplification unit 32 and the lysis chamber 314 are connected through the second microfluidic channel 351;

[0073] The cutting unit 33 uses CRISPR / Cas12a cutting technology; the cutting unit 33 is located on the side of the amplification unit 32 away from the mounting through hole 411; there are multiple cutting units 323; there are multiple third microfluidic channels 352; the multiple cutting units 323 are connected to the amplification unit 32 through the multiple third microfluidic channels 353 in a one-to-one correspondence;

[0074] Specifically, CRISPER / Cas12a cutting technology refers to the specific recognition of the PAM sequence of the double-stranded DNA (dsDNA) target by the WED and PI domains of the Cas12a protein under the guidance of a guide RNA (gRNA) after the target nucleic acid appears, thereby promoting the expansion of the dsDNA target. While the crRNA and the target chain are complementary, they also form an R-loop with the non-target chain, causing REC to move away from RuvC, exposing the RuvC domain cutting site, and inducing the Cas12a protein to cis-cut the non-target chain and the target chain;

[0075] Guide RNA (gRNA) is a specific RNA sequence designed to recognize and direct the nuclease to the target DNA region, and is composed of CRISPR RNA (crRNA) and small guide RNA (sgRNA). The crRNA is a 17-20 nucleotide sequence that is complementary to the target DNA, so it varies depending on the target gene. The sgRNA is an RNA sequence that connects the crRNA and the Cas12a protein and recognizes the PAM sequence of the target DNA;

[0076] The chromatography unit 34 is a test paper; the test paper is a rectangular strip; the chromatography unit 34 is located on the side of the cutting unit 33 away from the mounting through hole; the chromatography unit 34 is connected to the multiple cutting units 33 through the fourth microfluidic channel 354; the fourth microfluidic channel 354 collects the sample liquids of the multiple cutting units 33 and flows into the chromatography unit 34;

[0077] The electromagnet 44 includes a first electromagnet 411 and a second electromagnet 442; when the electromagnet 44 rotates on the circular working platform 41, the first electromagnet 441 corresponds to the bottom of the mixing chamber 311, and the top of the first electromagnet 441 is close to the bottom of the mixing chamber 31; the second electromagnet 442 corresponds to the bottom of the washing chamber 312, and the top of the second electromagnet 442 is close to the bottom of the washing chamber 312; during the rotation of the microfluidic chip 3, the first electromagnet 441 and the second electromagnet 442 can be moved to the bottom of the demagnetization chamber 313;

[0078] Specifically, the first electromagnet 441 is used to transfer the mixed sample liquid in the mixing chamber to the washing chamber; the second electromagnet 442 is used to transfer the mixed sample liquid in the washing chamber to the demagnetization chamber 214; the first electromagnet and the second electromagnet can both be used to demagnetize the sample liquid in the demagnetization chamber; the first electromagnet 441 and the second electromagnet 442 are both cylindrical;

[0079] Specifically, the electromagnets 44 are in six groups, and the six groups of electromagnets 44 are evenly distributed around the mounting through hole 411;

[0080] Specifically, the mixing chamber 311 is used to achieve mixing of the sample liquid and concanavalin A-nanomagnetic beads, and concanavalin A is indirectly coupled with streptavidin-coated magnetic nanoparticles to form a concanavalin A-magnetic nanoparticle complex to capture high-risk pathogens that cause sepsis, such as Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus in the sample;

[0081] The first electromagnet 441 further attracts the magnetic nanoparticles to capture the concanavalin A agglutinin-nanomagnetic beads-bacteria in the mixed sample liquid, thereby separating the target bacteria from the remaining substances in the sample liquid;

[0082] The sample liquid in the mixing chamber 311 is further transferred to the washing chamber 312 by the first electromagnet 441; the target bacteria are separated from the sample liquid by washing with washing liquid in the washing chamber 312;

[0083] The sample liquid in the washing chamber 312 is further transferred to the demagnetization chamber 313 by the second electromagnet 442; an external magnetic field is applied to the demagnetization chamber 313 by the electromagnet 44 to demagnetize the magnetic beads;

[0084] The microfluidic chip 3 is further controlled to rotate at a certain speed to centrifuge the sample in the demagnetization chamber 313, and the bacterial sample is filtered through the first microfluidic channel 351 and flows into the lysis chamber 314 to mix with the lysis solution;

[0085] The lysed sample liquid in the lysis chamber 314 is further centrifuged at a certain centrifugal speed, so that the lysed sample liquid enters the amplification unit 22 after being filtered;

[0086] The amplified sample liquid in the amplification unit 22 is further centrifuged at a certain centrifugal speed, so that the amplified sample liquid enters the cutting unit 23 for cutting;

[0087] The cut sample liquid in the cutting unit 23 is further centrifuged at a certain centrifugal speed, so that the amplified sample liquid enters the chromatography unit 24 for chromatography, and finally the chromatography result can be observed;

[0088] The servo motor 42 is further controlled to stop moving, and the fluorescence detection module 2 will detect the chromatography test paper, and finally the number of colonies and the concentration can be obtained.

[0089] In some specific embodiments, the intelligent control module 4 further includes a heating device 45, which includes a heating ring 1 451 and a heating ring 2 452; the heating ring 1 451 and the heating ring 2 452 are coaxially arranged with the mounting through hole 411 and are both fixed to the bottom of the circular working platform 41; the heating ring 1 451 is correspondingly located below the lysis chamber 314; the heating ring 2 452 is correspondingly located below the amplification unit 32;

[0090] Specifically, after the bacterial sample liquid flows into the lysis chamber 214, the lysis chamber 214 can be heated by turning on the heating ring 1 451; after the lysed sample liquid enters the amplification unit 22, the heating ring 2 can be turned on to perform constant temperature amplification on the amplification unit 22.

[0091] In some specific embodiments, the smart touch module 4 further includes a controller 46, and the optical unit 21, the servo motor 42, the first electromagnet 411, the second electromagnet 442, the heating ring 1 451, the heating ring 2 452 and the smart touch display screen 43 are all electrically connected to the controller 46;

[0092] Specifically, the controller 46 is electrically connected to the smart touch screen 43, and the rotation speed of the servo motor 42, the use of the electromagnet 44, the RPA amplification temperature, the use of fluorescence detection, etc. can all be operated through the smart touch screen; the results of fluorescence detection can be displayed through the smart touch screen 43; at the same time, the detection results will also be uploaded to the networked information exchange platform through the wireless communication network 47.

[0093] In some specific embodiments, the mixing chamber 311, the washing chamber 312, the demagnetization chamber 313, the lysis chamber 314, the amplification unit 32, the cutting unit 33 and the chromatography unit 34 are all provided with through holes on the second chip layer; the corresponding positions of the mixing chamber 311 are provided with sample loading holes 37 that can be connected to the corresponding mixing chamber 311; the top walls of the mixing chamber 311, the washing chamber 312, the demagnetization chamber 313, the lysis chamber 314, the amplification unit 315, the cutting unit 33 and the chromatography unit 34 are all provided with exhaust holes 38; the sample loading holes 37 and the exhaust holes 38 are both through holes opened on the cover layer.

[0094] In some specific embodiments, the first microfluidic channel 351, the second microfluidic channel 352, the third microfluidic channel 353 and the fourth microfluidic channel 354 are all microfluidic channels located on the top surface of the second chip layer;

[0095] The first microfluidic channel 351 is provided with a siphon valve 1 361; the second microfluidic channel 352 is provided with a siphon valve 2 362; the third microfluidic channel 353 is provided with a siphon valve 3 363; the fourth microfluidic channel 354 is provided with a siphon valve 4 364;

[0096] Siphon valve one 361, siphon valve two 362, siphon valve three 363 and siphon valve four 364 all have certain centrifugal speed thresholds; they exceed their own centrifugal speed thresholds and can all be centrifugally conducted; the centrifugal speed threshold of siphon valve one 361 can be set to be smaller than the centrifugal speed threshold of siphon valve two 362, the centrifugal speed threshold of siphon valve two 362 is smaller than the centrifugal speed threshold of siphon valve three 363, and the centrifugal speed threshold of siphon valve three 363 is smaller than the centrifugal speed threshold of siphon valve four 364; by controlling the speed of the microfluidic chip, the conduction state of the sample liquid between the washing chamber 213 and the lysis chamber 214, between the lysis chamber 214 and the amplification unit 22, between the amplification unit 22 and the cutting unit 23, and between the cutting unit 23 and the chromatography unit 24 can be controlled;

[0097] Specifically, in order to isolate the reaction chamber, other siphon valves are provided between different units; siphon valve 1 361, siphon valve 2 362, siphon valve 3 363, siphon valve 4 364 and other siphon valves are all microfluidic grooves opened on the top surface of the second chip layer. After the first chip layer, the cover layer and the second chip layer are packaged, a microfluidic channel provided with siphon valve 1 361, siphon valve 2 362, siphon valve 3 363, siphon valve 4 364 and other siphon valves is formed.

[0098] In some specific embodiments, different primers are placed in the amplification unit 22 to achieve amplification of different types of bacteria in the same sample liquid; the cutting units 23 and the third microfluidic channel 353 are multiple and the number is the same; the multiple cutting units 23 are spaced and distributed along the direction surrounding the mounting through hole; different primers are placed in the multiple cutting units 23 to achieve cutting of different types of bacteria in the same sample liquid.

[0099] The working steps of the utility model handheld bacteria detector are:

[0100] Step 1: Add sample to the mixing chamber through the sample injection hole.

[0101] Step 2: Control the microfluidic chip to alternately rotate forward and reverse, and attract the magnetic beads through the first electromagnet to mix the sample liquid and the magnetic beads in the mixing chamber.

[0102] Step 3: The magnetic beads are attracted by the first electromagnet to allow the sample liquid to enter the washing chamber, and the microfluidic chip is rotated back and forth by controlling the rotation speed to wash the sample that has been bound to the magnetic beads in the washing chamber, and other sample liquids that have been washed away from the bacteria-magnetic bead complex.

[0103] Step 4: The magnetic beads are attracted by the second electromagnet and enter the demagnetization chamber, and an external magnetic field is applied by the electromagnet to demagnetize the magnetic beads.

[0104] Step 5: The microfluidic chip is centrifuged at a first centrifugal speed, and the bacteria are filtered through the first microfluidic channel and flow into the lysis chamber to mix with the lysis solution. Once the heating ring is started, the lysis chamber is heated.

[0105] Step 7: Centrifuge the microfluidic chip at a second centrifugal speed so that the lysate enters the amplification unit after being filtered, and start the heating loop 2 to heat the amplification unit at a constant temperature.

[0106] Step 8: Centrifuge the microfluidic chip at a third centrifugal speed so that the amplified sample liquid enters the cutting unit for cutting.

[0107] Step nine: centrifuge the microfluidic chip at the fourth centrifugal speed to allow the amplified sample to enter the chromatography unit, color the chromatography test paper, use the fluorescence detection module to detect the fluorescence intensity ratio, and compare it with the standard curve to obtain the number of colonies.

[0108] Specifically, the first centrifugal speed is greater than the centrifugal speed threshold of siphon valve one, and less than the centrifugal speed threshold of siphon valve two; the second centrifugal speed is greater than the centrifugal speed threshold of siphon valve two, and less than the centrifugal speed threshold of siphon valve three; the third centrifugal speed is greater than the centrifugal speed threshold of siphon valve three, and less than the centrifugal speed threshold of siphon valve four; the fourth centrifugal speed is greater than the centrifugal speed threshold of siphon valve four; the fourth centrifugal speed is greater than the third centrifugal speed; the third centrifugal speed is greater than the second centrifugal speed; the second centrifugal speed is greater than the first centrifugal speed; and their specific values ​​need to be determined according to the structure of the chip.

[0109] The utility model adopts lectin-nanomagnetic bead enrichment technology, real-time multiplex RPA technology, CRISPR / Cas12a cutting technology, colloidal gold immunochromatography technology and high-precision fluorescence detection technology.

[0110] The lectin-nanomagnetic bead enrichment technology specifically recognizes the mannose-type sugars on the surface of bacteria through the sugar recognition domain on concanavalin A, achieving high enrichment efficiency and "no omission" enrichment separation, with a capture rate of 98.7%. At the same time, the magnetic nanoparticle enrichment technology can also quickly separate target bacteria in clinical specimens to achieve bacterial enrichment.

[0111] Based on the bacterial lysis after separation and enrichment by magnetic nanoparticles, the multiple RPA nucleic acid amplification technology designs a pair of primers specific to the target DNA for the characteristic genes, virulence genes, and drug resistance genes of different detected bacteria. The reaction lasts for 15 to 20 minutes under isothermal conditions, and the in vitro amplification of nucleic acids can reach 109-1010 times, with a specificity of 99.3%. In the case of using multiple target gene amplification results as detection indicators in the same reaction chamber, the specificity and accuracy of the experimental results are further improved.

[0112] CRISPR / Cas12a cutting technology selects conserved and specific genes among pathogen species, and the Cas12a protein only needs one crRNA as a guide to recognize DNA, so it can still play a cutting role when the concentration of nucleic acid amplification products is low, and generate high-intensity fluorescence detection signals in a short time, realizing real-time, synchronized and quantitative detection and analysis of RPA reaction products, improving the specificity, convenience and accuracy of detection.

[0113] Colloidal gold immunochromatography technology uses the method of antigen-antibody specific binding, which has strong specificity, high sensitivity and accurate test results. Whether a certain bacteria is infected can be directly judged from the corresponding color-developing area of ​​the chromatography test paper, and the color depth can be used to visually judge the carrying status of the corresponding bacterial species' drug resistance genes and virulence genes. It is intuitive and simple, easy to understand, easy to operate, carry and promote.

[0114] High-precision fluorescence detection technology uses high-precision optical instruments. The ultraviolet excitation light path has high emission efficiency, low power consumption, low cost, and long luminescence life. The fluorescence collection light path has high collection efficiency, high sensitivity, and high spatial resolution, and the detection results are highly accurate.

[0115] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the utility model. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0116] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A handheld bacteria detector, characterized in that: include: A shell, the shell comprising a cover plate, a bottom plate and four side plates, the top of the shell is opened, the cover plate is arranged at the opening; one side plate of the shell is provided with a handle and a square groove; the square groove is located above the handle; A fluorescence detection module, the fluorescence detection module is parallel to the cover plate and is arranged at the bottom of the cover plate; the fluorescence detection module includes an optical unit, a photoelectric conversion unit and a signal processing unit; the optical unit includes an ultraviolet excitation light path and a fluorescence collection light path; the photoelectric conversion unit is connected to the fluorescence collection light path; the signal processing unit is connected to the photoelectric conversion unit; A microfluidic chip, wherein the microfluidic chip is located in the housing and parallel to the fluorescence detection module; an enrichment unit, an amplification unit, a cutting unit and a chromatography unit are sequentially arranged on the microfluidic chip at certain intervals; the enrichment unit, the amplification unit, the cutting unit and the chromatography unit are all connected through a microfluidic channel; the microfluidic channel is provided with a siphon valve; the chromatography unit is located below the optical unit; An intelligent control module, the intelligent control module includes a circular working platform, a servo motor, an intelligent touch display screen and an electromagnet; a mounting through hole is provided in the middle of the circular working platform; a plurality of fan-shaped grooves are provided on the upper surface of the circular working platform along the circumferential direction, and the fan-shaped grooves are used to place the microfluidic chip; the servo motor is located below the circular working platform, and an output shaft is provided on the top of the servo motor; one end of the output shaft is connected to the motor body; the other end of the output shaft is detachably connected to the mounting through hole; the intelligent touch display screen is placed in the square groove and connected to the signal processing unit; the electromagnet is located at the bottom of the circular working platform.

2. The handheld bacteria detector according to claim 1, characterized in that: There are multiple fluorescence detection modules; the photoelectric units of the multiple fluorescence detection modules are arranged in one-to-one correspondence with the chromatography units of the multiple microfluidic chips; and the multiple microfluidic chips work in coordination with the multiple fluorescence detection modules.

3. The handheld bacteria detector according to claim 2, characterized in that: The microfluidic chip comprises a first chip layer, a second chip layer and a cover layer; the first chip layer and the cover layer are made of transparent materials, and the first chip layer, the second chip layer and the cover layer are sequentially attached and sealed from bottom to top.

4. The handheld bacteria detector according to claim 3, characterized in that: The enrichment unit comprises a mixing chamber, a washing chamber, a demagnetization chamber, and a lysis chamber; the mixing chamber, the washing chamber, and the demagnetization chamber are arranged at intervals in a direction away from the mounting through hole and are sequentially connected through the microchannel inside the microfluidic chip; The microfluidic flow channel includes a first microfluidic flow channel, a second microfluidic flow channel, a third microfluidic flow channel and a fourth microfluidic flow channel; The demagnetization chamber is connected to the lysis chamber via a first microfluidic channel; The amplification unit is located on the side of the lysis chamber away from the mounting through hole; the amplification unit is connected to the lysis chamber through the second microfluidic channel; The cutting unit is located on the side of the amplification unit away from the mounting through hole; there are multiple cutting units; there are multiple third microfluidic channels; the multiple cutting units are connected to the amplification unit through the multiple third microfluidic channels in a one-to-one correspondence; The chromatography unit is a test paper; the chromatography unit is located on the side of the cutting unit away from the mounting through hole; the chromatography unit is connected to multiple cutting units through a fourth microfluidic channel; the fourth microfluidic channel is used to introduce sample liquids from multiple cutting units into the chromatography unit.

5. The handheld bacteria detector according to claim 4, characterized in that: The electromagnet includes a first electromagnet and a second electromagnet; when the electromagnet rotates on the circular working platform, the first electromagnet corresponds to directly below the mixing chamber, and the top of the first electromagnet is close to the bottom of the mixing chamber; the second electromagnet corresponds to directly below the washing chamber, and the top of the second electromagnet is close to the bottom of the washing chamber; when the microfluidic chip rotates, the first electromagnet and the second electromagnet can both move to directly below the demagnetization chamber.

6. The handheld bacteria detector according to claim 5, characterized in that: The intelligent control module also includes a heating device, which includes a heating ring 1 and a heating ring 2; the heating ring 1 and the heating ring 2 are coaxially arranged with the mounting through hole and are both fixed at the bottom of the circular working platform; the heating ring 1 is correspondingly located below the cracking chamber; the heating ring 2 is correspondingly located below the amplification unit.

7. The handheld bacteria detector according to claim 6, characterized in that: The intelligent control module also includes a controller, and the optical unit, the servo motor, the first electromagnet, the second electromagnet, the heating ring 1, the heating ring 2 and the intelligent touch display screen are all electrically connected to the controller.

8. The handheld bacteria detector according to claim 7, characterized in that: The mixing chamber, the washing chamber, the demagnetization chamber, the lysis chamber, the amplification unit, the cutting unit and the chromatography unit are all on the second chip layer; the corresponding positions of the mixing chamber are provided with sample loading holes that can be connected to the corresponding mixing chamber; the top walls of the mixing chamber, the washing chamber, the demagnetization chamber, the lysis chamber, the amplification unit, the cutting unit and the chromatography unit are all provided with exhaust holes; the sample loading holes and the exhaust holes are both through holes opened on the cover layer.

9. The handheld bacteria detector according to claim 8, characterized in that: The first microfluidic channel, the second microfluidic channel, the third microfluidic channel and the fourth microfluidic channel are all microfluidic channels located on the top surface of the second chip layer.

10. The handheld bacteria detector according to claim 9, characterized in that: The first microfluidic channel is provided with a siphon valve 1; the second microfluidic channel is provided with a siphon valve 2; the third microfluidic channel is provided with a siphon valve 3; and the fourth microfluidic channel is provided with a siphon valve 4.

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