Microfluidic chip, preparation method thereof and method for rapidly quantitatively detecting and identifying bacteria from whole blood

Abstract

This application relates to the field of bacterial detection and identification in blood, specifically disclosing a microfluidic chip, its preparation method, and a method for rapid quantitative detection and identification of bacteria from whole blood. The microfluidic chip comprises a glass slide layer and a microstructure layer bonded together. The microstructure layer has several parallel main channels, and several microchambers are formed on both sides of the main channels in the width direction. Corresponding ends of the main channels are interconnected. The microstructure layer has an inlet channel and an outlet channel, which are respectively connected to the two ends of the main channels. The method for rapid quantitative detection and identification of bacteria from whole blood in this application involves: blood sample entering the chip, lysis buffer entering the chip, culture medium and indicator entering the chip, microchamber-interval culture, microscopic fluorescence detection, and bacterial extraction and identification. The method of this application has the advantages of short detection time, high sensitivity and specificity, and wide applicability.

Description

Technical Field

[0001] This application relates to the field of bacterial detection and identification technology in blood, and more specifically, it relates to a microfluidic chip, its preparation method, and a method for rapid quantitative detection and identification of bacteria from whole blood. Background Technology

[0002] Bloodstream infection (BSI) refers to a serious systemic infectious disease caused by pathogens entering the bloodstream. It is characterized by rapid onset and high mortality. It can lead to serious complications such as multiple organ failure, sepsis, and septicemia, threatening the patient's life. Rapid detection and identification of bacteria in the early stages of infection is crucial to saving patients' lives.

[0003] Blood culture is one of the most commonly used methods for diagnosing bloodstream infections. It involves placing a patient's blood sample into a culture medium to detect and culture pathogens, thus identifying the type of infection. With sufficient sample volume (at least two to three sets of blood culture bottles, each filled with 10 ml), the sensitivity of blood culture can reach over 95%. It is easy to perform and effectively demonstrates the susceptibility of cultured pathogens to antimicrobial agents, making it a routine and reliable method. However, after a positive alarm from the blood culture instrument, the pathogenic microorganism needs to be isolated and purified for identification and drug susceptibility testing, resulting in a longer reporting cycle, typically 24 hours. 28h. PCR is the most commonly used gene amplification method and has broad application prospects in the rapid diagnosis of blood infections. It is a technique for expanding DNA fragments in vitro under the action of polymerases, including three steps: template DNA denaturation, annealing of template DNA with primers, and primer extension. It can shorten the detection time to several hours, but it is usually not sensitive enough and cannot detect low concentrations (1) in blood. The concentration of bacteria in the blood is 100 CFU / ml, so an enrichment step is still required. Moreover, the target bacteria are surrounded by billions of non-target cells (such as red blood cells) in the blood sample, resulting in high background signal and poor specificity during bacterial detection. Summary of the Invention

[0004] To enable rapid detection and identification of bacteria in blood, this application provides a microfluidic chip, its preparation method, and a method for rapid quantitative testing and identification of bacteria from whole blood.

[0005] In a first aspect, this application provides a microfluidic chip, which adopts the following technical solution: A microfluidic chip includes a glass slide layer and a microstructure layer bonded together. The microstructure layer and the glass slide layer together form a microchannel. A plurality of parallel main channels are formed on the microstructure layer. A plurality of microcavities are formed on both sides of the main channels in the width direction. The microcavities are interconnected with the main channels through side channels. The corresponding ends of the main channels are interconnected. The microstructure layer has at least one sample inlet channel and at least one sample outlet channel, which are respectively connected to both ends of the main channel.

[0006] By adopting the above technical solution, using a glass slide layer as the bottom layer and a microstructure layer as the top layer, and improving the structure of the microstructure layer, the blood sample to be tested enters the main channel through the sample inlet channel. The blood sample then enters the microchamber along the side channel, where bacteria in the blood are dispersed. After a short period of incubation, they can be rapidly identified and detected, achieving effective enrichment of low-concentration bacteria from the blood sample within the chip. The microchannels between the glass slide layer and the microstructure layer are used for blood sample storage, lysis, culture, and color development, and also for identifying bacterial species for subsequent medication guidance. Optionally, the micro-chambers on both sides of the main channel are staggered.

[0007] By adopting the above technical solution, the openings of the micro-chambers located on both sides of the main channel are staggered, which can reduce the vertical disturbance of the contents in the micro-chambers caused by the subsequent introduction of auxiliary agents, such as lysis buffer and culture medium, when they flow in the main channel.

[0008] Optionally, the glass slide layer is made of glass, ITO glass, quartz glass, polydimethylsiloxane, or polymethyl methacrylate, and the microstructure layer is made of polydimethylsiloxane.

[0009] Secondly, this application provides a method for fabricating a microfluidic chip, employing the following technical solution: A method for fabricating a microfluidic chip includes the following steps: S1. Use software to design the microstructure of the microfluidic chip and perform film mask printing to obtain the printed film; S2. After cleaning and drying the silicon wafer with concentrated sulfuric acid, drop photoresist onto the silicon wafer and spin-dry it to form a photoresist layer on the silicon wafer. Cover the photoresist layer with the printed film, expose it, and clean the uncured photoresist with a developer to obtain the silicon wafer template. S3. Mix polydimethylsiloxane and curing agent evenly to obtain PDMS prepolymer. Pour the PDMS prepolymer onto a silicon wafer template to form a prepolymer layer. Dry and separate the silicon wafer template to obtain a PDMS layer with microstructure. S4. A sample inlet and outlet channel is opened on the PDMS layer to obtain a microstructure layer; S5. The microstructure layer and the glass slide layer are treated with oxygen plasma. Then, the side of the microstructure layer with the microstructure is bonded to the glass slide layer to form a microfluidic chip with microchannels.

[0010] By employing the above technical solution and through software design, a planar structure of main flow channels, side flow channels, and microcavities is obtained. A planar pattern is formed on the film using a film mask. Photoresist is then adhered to a silicon wafer. A printed film with the obtained planar structure is placed over the photoresist layer. After exposure, the photoresist cures, forming a raised three-dimensional structure. Excess photoresist is removed with a developing solution. Then, polydimethylsiloxane and a curing agent are mixed and poured onto a silicon wafer template. The curing agent, through a cross-linking reaction, breaks the silicon-oxygen bonds in the PDMS molecules, forming new silicon-oxygen bonds, thus creating a three-dimensional network structure. This transforms the liquid polydimethylsiloxane into a solid silicone rubber. After curing, the silicon wafer template is separated, forming a PDMS chip with main flow channels and other structures. Sample inlet and outlet channels are then created on the PDMS chip, which is bonded to a glass slide layer, forming microchannels between them. Plasma treatment of the PDMS chip introduces hydrophilic hydroxyl groups onto its surface, replacing the... CH groups give the PDMS surface its strong hydrophilicity. The glass slide layer is cleaned with concentrated sulfuric acid, and the surface contains a large amount of Si. O bonds, during oxygen plasma treatment, Si The O bonds are broken, resulting in a large number of Si dangling bonds on the surface, which absorb air... OH, forming Si OH bonds are used to bond the treated PDMS chip to the surface of the glass slide layer, and the Si on both surfaces... An interaction occurs between OH groups, forming a robust Si layer between the glass slide and the PDMS chip. O-bonding is used to achieve irreversible bonding between the two. This method not only simplifies the fabrication process of microfluidic chips but also improves the stability and reliability of microfluidic chip operation, opening up new avenues for the modular production and large-scale integration of microfluidic chips.

[0011] Optionally, the thickness of the photoresist layer is 40. 1000μm, the thickness of the prepolymer layer is 3 10mm.

[0012] By adopting the above technical solution, the thickness of the photoresist layer is less than the thickness of the prepolymer layer. The prepolymer layer covers the photoresist layer. After curing, the prepolymer layer separates from the photoresist layer, forming a main channel, side channel and microcavity with a recessed structure in the prepolymer layer.

[0013] Optionally, the mass ratio of polydimethylsiloxane to curing agent in the PMDS prepolymer is 10. 13:1.

[0014] Optionally, the oxygen plasma treatment time is 1 minute. During bonding, the microstructure layer and the glass slide layer are heated to 80°C for 5 minutes. Bake at 90℃ overnight.

[0015] By adopting the above technical solution, a plasma treatment that is too short cannot functionalize the surface of the glass slide layer and the PDMS chip, while a plasma treatment that is too long will excessively and strongly alter the surface of the PDMS chip and the glass slide layer, resulting in increased surface roughness and decreased adhesion performance. Bonding under baking makes it easier for the PDMS chip and the glass slide layer to chemically connect after they come into contact.

[0016] Thirdly, this application provides a method for rapid quantitative detection and identification of bacteria in whole blood, employing the following technical solution: A method for rapid quantitative detection and identification of bacteria in whole blood, comprising the following steps: Blood sample enters the microfluidic chip: Untreated blood sample is driven through the injection channel of the microfluidic chip and enters the micro chamber along the main channel. The injection of blood sample is stopped after the micro chamber is completely filled. Lysis buffer injection into the microfluidic chip: The lysis buffer is driven through the injection channel into the main channel, and the injection time is 10 minutes. 20 minutes; Culture medium and indicator are introduced into the microfluidic chip: The culture medium and indicator are driven through the injection channel into the main channel, and the injection time for the culture medium and indicator is 15 minutes. 20 minutes; Microchamber-separated culture: Gas is introduced into the main flow channel through the inlet channel, and the liquid in the main flow channel is discharged by the gas. Each microchamber is separated by gas, sealing the inlet and outlet channels, and then incubated for 30 days. Cultured at 37℃ for 3 days 5h; Microscopic fluorescence detection: The cultured microfluidic chip is placed under a fluorescence microscope, and the presence and number of strong fluorescence are observed to determine whether there are live target bacteria. Bacterial extraction and identification: The culture medium is driven into the main channel through the injection channel, and the bacteria in the microchamber are extracted by laser induction. The bacteria are collected on blood agar plates in the sample outlet channel for amplification culture. The colonies obtained from the culture are identified by mass spectrometry to obtain bacterial information.

[0017] By employing the above technical solution, blood samples, after being driven, enter the microchamber along the main flow channel and side flow channel. Once the microchamber is completely filled with blood, the entry of the blood sample stops. Because the microchamber is completely filled with blood, the flow velocity within the microchamber is almost zero, and the lysis buffer does not enter the microchamber; it only flows within the main flow channel, squeezing out any blood that did not enter the microchamber to the sample outlet channel. However, during the flow of the lysis buffer, the lysis buffer in the main flow channel also diffuses into the microchamber along the side flow channel, causing the blood cells to lyse. Simultaneously, the lysed blood cells also diffuse into the main flow channel and are discharged through the sample outlet channel. After injecting the lysis buffer for a period of time, the blood cells in the microchamber are completely lysed and diffused out. The lysis buffer used to lyse blood cells is based on osmotic pressure-induced cell lysis. That is, blood cells in a hypotonic solution, lacking the protection of cell walls, easily absorb water and rupture, while bacteria can survive for a long time in a hypotonic environment. Therefore, the lysis buffer only lyses blood cells and does not lyse bacteria.

[0018] After lysis with lysis buffer, only lysis buffer and bacteria remain in the microchamber. In order to allow the bacteria in the microchamber to proliferate and be detected, culture medium and reaction growth indicator are introduced through the injection channel and diffused into the microchamber along the main channel.

[0019] After the culture medium and indicator fill the microchambers, the microchambers are connected by liquid in the main channel. To make the microchambers independent of each other, gas is injected into the main channel through the sample inlet channel. The gas will displace the liquid in the main channel, so that there is no longer a liquid connection between the microchambers. Therefore, the introduction of the gas phase makes each microchamber in the microchamber array an independent culture bottle. This ensures that each microchamber with indicator changes is produced by the reproduction of the original single bacteria. By counting the number of microchambers that have changed, the number of bacteria contained in the original sample entering the microchamber can be quantitatively reflected. In addition, the gas phase separates the microchambers, which can ensure that the bacteria in each microchamber have the required oxygen, carbon dioxide and other gases. For anaerobic bacteria culture media, it can be carried out in an anaerobic incubator. Then, the isolated microchambers are placed in an incubator for short-term incubation.

[0020] Under a microscope, the microfluidic chip that has been cultured will produce a color change in the indicator due to the proliferation of microcavities. In order to improve the detection speed, rapid detection can be performed under low magnification. The combination of bright field and fluorescence field can quickly detect the number of target microcavities.

[0021] The culture medium is injected into the microchamber through the injection channel, thereby exporting the liquid inside the microchamber and simultaneously removing the bacteria from the microchamber. The bacteria are collected onto blood agar plates for amplification culture. Due to the large bacterial count, colonies can be formed on the blood agar plates after a short incubation period. The bacterial strain information can then be obtained through mass spectrometry identification. Bacteria in the sample can be detected in a very short time without the need for pretreatment of the blood sample, thus avoiding bacterial loss caused by pretreatment.

[0022] Optionally, the injection flow rate of the lysis buffer is 20. 40 μL / min.

[0023] By adopting the above technical solution, the lysis buffer is injected into the main channel at the above speed, which can prevent the lysis buffer flow rate from being too fast, causing disturbance of the blood sample in the microchamber and diffusion to the outside of the microchamber, and can also prevent the lysis buffer flow rate from being too slow, affecting the cell lysis rate and detection time.

[0024] Optionally, the mass ratio of the culture medium to the indicator is 9:1. The ratio of culture medium to indicator was 10:1, and the injection flow rate was 20. 40 μL / min.

[0025] By adopting the above technical solution, the ratio of the above indicator to culture medium can fully enable the bacteria to develop color, making it easy to observe the number of bacteria. Moreover, the injection speed of the culture medium and indicator can allow the culture medium and indicator to diffuse into the micro chamber and fill the micro chamber, thus completing the bacterial proliferation.

[0026] Optionally, during the cultivation of the microfluidic chip, the microfluidic chip is placed in a culture dish, and purified water is added so that its height is level with that of the microfluidic chip.

[0027] By adopting the above technical solution, the culture medium can be saturated with humidity during the cultivation process, preventing the evaporation of droplets in the chip and affecting the proliferation and cultivation of bacteria.

[0028] Optionally, the indicator is selected from one of Alamar Blue, naphthol peptide, bromocresol purple, phenol red, and luciferase.

[0029] In summary, this application has the following beneficial effects: 1. The microfluidic chip in this application has a simple structure, is easy to manufacture, has low liquid flow loss, can effectively enrich low concentrations of bacteria in blood samples, shorten detection time, improve detection sensitivity and specificity, is convenient and quick to use, has a wide range of applications, and consumes little reagent.

[0030] 2. The method in this application can remove infection from blood cells while preserving bacteria in the blood. More importantly, this method retains the advantages of culture-based detection methods, and the micro-culture environment can greatly shorten the time for detecting bacterial growth, thus enabling detection in a short time (3... Bacteria were detected in the sample after 5 hours.

[0031] 3. The method of this application allows for the quantitative detection of bacteria directly from whole blood without the need for blood pretreatment, thus avoiding the loss of bacteria caused by complex blood pretreatment.

[0032] 4. After rapid quantitative detection of bacteria based on this method, the bacteria in the chip can be exported for downstream mass spectrometry identification, which can realize rapid detection and identification of bacteria in blood and provide guidance for clinical medication. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of a microfluidic chip.

[0034] Figure 2 This is a schematic diagram of the microstructure layer.

[0035] Figure 3 This is a flowchart illustrating the changes of a microfluidic chip during blood testing.

[0036] Figure 4 This is a linear fit graph of plate counts and microfluidic chip counts of Escherichia coli detected by the method in Example 3.

[0037] Figure 5 The mass spectrum obtained for detecting Escherichia coli as the target bacteria according to the method in Example 3.

[0038] Figure 6 This is a linear fit graph of plate counts and microfluidic chip counts of Staphylococcus aureus detected according to the method in Example 4.

[0039] Figure 7 The mass spectrum is obtained for Staphylococcus aureus as the target bacteria, as detected according to the method in Example 4.

[0040] Figure 8 The graph shows the linear fit between plate counts and microfluidic chip counts of Acinetobacter baumannii detected according to the method in Example 5.

[0041] Figure 9 The mass spectrum obtained for detecting Acinetobacter baumannii as the target bacteria according to the method in Example 5.

[0042] Figure 10 This is a linear fit graph of plate counts and microfluidic chip counts for detecting Klebsiella pneumoniae according to the method in Example 6.

[0043] Figure 11 The mass spectrum obtained for detecting Klebsiella pneumoniae as the target bacteria according to the method in Example 6.

[0044] In the figure: 1. Slide layer; 2. Microstructure layer; 21. Main channel; 22. Microchamber; 23. Side channel; 24. Sample inlet channel; 25. Sample outlet channel; 26. Connecting channel; 27. Continuous channel; 28. Interconnected channel. Detailed Implementation

[0045] Example Example 1: A microfluidic chip, such as Figure 1 As shown, it includes a glass slide layer 1 and a microstructure layer 2 bonded together, and the microstructure layer 2 and the glass slide layer 1 together constitute a microchannel.

[0046] See Figure 1 and Figure 2 The microstructure layer 2 has several parallel main channels 21, and the number of main channels 21 is even. Several micro chambers 22 are formed on both sides of the width direction of the main channel 21. The number and volume of the micro chambers 22 are designed according to the required sample loading. The cross-section of the micro chambers can be rectangular, square, circular, triangular, etc. In this embodiment, the number of micro chambers 22 on one side of the main channel 21 is 12. The cross-sectional shape of the micro chambers is rectangular in this embodiment. The micro chambers 22 and the main channel 21 are interconnected through side channels 23. The opening width of the side channels 23 is smaller than the opening width of the micro chambers 22. The micro chambers 22 on one side of the same main channel 21 are staggered with the micro chambers 22 on the other side.

[0047] Multiple sample inlet channels 24 and multiple sample outlet channels 25 are provided on the microstructure layer 2. In this embodiment, two sample inlet channels 24 and three sample outlet channels 25 are used as examples. The sample inlet channels 24 and the sample outlet channels 25 are located at both ends of the main channel 21 and are connected to the main channel 21. The sample inlet channels 24 and the sample outlet channels 25 penetrate through the microstructure layer 2.

[0048] In this embodiment, eight main channels 21 are used as an example. Two main channels 21 are grouped together. The corresponding ends of the two main channels 21 are connected to each other through connecting channels 26. The connecting channels 26 at one end of the two groups of main channels 21 are connected through continuous channels 27. The two continuous channels 27 are connected to each other through interconnecting channels 28.

[0049] The glass slide layer 1 is made of glass, and the microstructure layer 2 is made of polydimethylsiloxane.

[0050] Example 2: A method for fabricating a microfluidic chip, comprising the following steps; S1. Using CAD software, design the microstructure of the microfluidic chip in Example 1, and perform film mask printing to obtain the printed film; S2. After cleaning and drying the silicon wafer with concentrated sulfuric acid, drop SU onto the silicon wafer. 8. Photoresist is applied and spin-coated to form a 40μm thick photoresist layer on the silicon wafer. The printed film is then placed over the photoresist layer and exposed for 5 minutes. GS is then used for further processing. The uncured photoresist was cleaned with Type 1 developer to obtain a silicon wafer template. S3. Mix 30 mL of polydimethylsiloxane and curing agent at a volume ratio of 10:1, and stir on a magnetic stirrer for 20 min to prepare PDMS prepolymer. Pour the PDMS prepolymer onto a silicon wafer template, place it in a vacuum drying oven, and evacuate at a negative pressure of 0.1 MPa. Repeat the vacuuming process 5 times until all air bubbles in the PDMS prepolymer disappear, forming a prepolymer layer with a thickness of 10 mm. Place it in an oven and heat to cure for 3 h. After removal, separate the silicon wafer template to obtain a PDMS layer with a microstructure. The PDMS prepolymer is selected from Dow Corning DC... 184; S4. An injection channel 24 and an outlet channel 25 are respectively opened at both ends of the PDMS chip. The injection channel 24 and the outlet channel 25 are interconnected with the microstructure on the PDMS chip to obtain the microstructure layer 2. S5. Both the microstructure layer 2 and the glass slide layer 1 are treated with oxygen plasma for 3 minutes. Then, the side of the microstructure layer 2 with the microstructure is bonded to the glass slide layer 1. During bonding, baking the microstructure layer 2 and the glass slide layer 1 at 80°C overnight is beneficial to improve the bonding performance, and together they form a microfluidic chip with microchannels.

[0051] Example 3: A method for rapid quantitative detection and identification of bacteria in whole blood, comprising the following steps: S1. Sample Preparation: Target bacteria *Escherichia coli* (E. coli ATCC35218) were added to pig blood to prepare blood samples with gradient concentrations (1 cfu / ml, 10 cfu / ml, 100 cfu / ml, and 1000 cfu / ml). Three parallel experiments were set up for each concentration, and the original bacterial suspension was plated on three plates to determine the number of bacteria added to the blood. MH broth medium with 10 wt% Alamar Blue indicator cationic adjustment was prepared. Lysis buffer: sterile water. The microfluidic chip prepared in Example 2 was placed in a vacuum pump and vacuum-treated for 5 min. The cationic-adjusted MH broth medium was selected from Shanghai Guandao Biotechnology Co., Ltd. S2. Blood sample enters the microfluidic chip: Using a 1mL medical syringe, a pig blood sample mixed with the target bacteria is drawn up. Driven by the injection pump, the blood sample enters the interconnecting channel 28 through the injection channel 24, then enters the connecting channel 26 along the continuous channel 27, enters the main channel 21 from the connecting channel 26, and then enters the micro chamber 22 through the side channel. The injection of blood sample is stopped after the micro chamber 22 is completely filled with blood. S3. Lysis buffer enters the microfluidic chip: Sterile water is used as the lysis buffer. Sterile water is injected into the main channel 21 along the injection channel 24 at an injection rate of 40 μL / min. The injection time of sterile water is 20 min. S4. Culture medium and indicator enter the microfluidic chip: MH broth culture medium with cationic adjustment containing 10wt% Alamar Blue indicator is introduced into the main channel 21 at a flow rate of 40μL / min (the flow rate of the main channel 21 is 1.04mm / s) along the injection channel 24. The injection time is 15min. Under the diffusion effect, the culture medium fills the micro chamber 22. S5. Microchamber 22 Interval Culture: Air is injected into the main channel 21 through the injection channel 24 at a flow rate of 0.8 L / min using a syringe for 3 min to drain the liquid from the main channel 21. Each microchamber 22 is separated by gas. The injection channel 24 and the outlet channel 25 are sealed with tape. The microfluidic chip is placed in a culture dish, and purified water is added to the culture dish until the height of the purified water is level with the height of the microfluidic chip. Then, it is cultured in an incubator at 37°C for 5 h. S6. Microscopic Fluorescence Detection: The cultured microfluidic chip is observed under a fluorescence microscope. The microcommunities 22 produced by bacterial proliferation cause the Alma blue indicator to change from its original pale blue to pale pink. The presence and number of fluorescent chambers are observed to determine the presence of live target bacteria. The linear fitting graph of plate counting and chip technology is shown in the figure. Figure 4 As shown, the number of bacteria incorporated is highly consistent with the number detected on the chip.

[0052] S7. Bacterial Extraction and Identification: MH broth medium was injected into the main channel 21 through the injection channel 24 at a rate of 40 μl / min for 20 min. Simultaneously, bacteria were extracted from the microchamber 22 using laser induction. The bacteria were collected onto blood agar plates at the exit channel 25 and then cultured at 37°C for 3 h. Colonies formed on the blood agar plates, which were then spread onto MALDI plates. The spectrum acquired by the TOF mass spectrometer, and the mass spectrometry identification image are as follows. Figure 5 As shown, the obtained spectrum is compared with the standard spectrum in the bacterial identification software database, and the bacterial identification result is obtained based on the identification analysis.

[0053] Example 4: A method for rapid quantitative detection and identification of bacteria in whole blood, differing from Example 3 in that the target bacterium is Staphylococcus aureus (S. aureus ATCC 29213). The linear fitting graph of plate counts and counts on the microfluidic chip is shown below. Figure 6 As shown, the obtained mass spectrum is as follows: Figure 7 As shown.

[0054] Example 5: A method for rapid quantitative detection and identification of bacteria in whole blood, differing from Example 3 in that the target bacterium is *Acinetobacter baumannii*. The linear fitting graph of plate counts and counts on the microfluidic chip is shown below. Figure 8 As shown, the obtained mass spectrum is as follows: Figure 9 As shown.

[0055] Example 6: A method for rapid quantitative detection and identification of bacteria in whole blood, differing from Example 3 in that the target bacteria is Klebsiella pneumoniae. The linear fitting graph of plate counts and counts on the microfluidic chip is shown below. Figure 10 As shown, the obtained mass spectrum is as follows: Figure 11 As shown.

[0056] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for rapid quantitative detection and identification of bacteria in whole blood using a microfluidic chip, characterized in that, The microfluidic chip includes a glass slide layer (1) and a microstructure layer (2) bonded together. The microstructure layer (2) and the glass slide layer (1) together form a microchannel. The microstructure layer (2) has several parallel main channels (21). Several microcavities (22) are formed on both sides of the width direction of the main channel (21). The microcavities (22) and the main channel (21) are interconnected through side channels (23). The corresponding ends of the main channel (21) are interconnected. The microstructure layer (2) is provided with at least one sample inlet channel (24) and at least one sample outlet channel (25), and the sample inlet channel (24) and the sample outlet channel (25) are respectively connected to both ends of the main channel (21); The method includes the following steps: Blood sample enters the microfluidic chip: Untreated blood sample is driven through the injection channel (24) of the microfluidic chip and enters the micro chamber (22) along the main channel (21) until the blood sample completely fills the micro chamber (22) and then the injection of blood sample is stopped; Lysis solution enters the microfluidic chip: The lysis solution is driven through the injection channel (24) into the main channel (21), and the injection time of the lysis solution is 10 minutes. 20 minutes; Culture medium and indicator are introduced into the microfluidic chip: The culture medium and indicator are driven through the injection channel (24) into the main channel (21), and the injection time of the culture medium and indicator is 15 minutes. 20 minutes; Microchamber (22) spaced culture: Gas is introduced into the main channel (21) through the sample inlet channel (24), and the liquid in the main channel (21) is discharged by the gas. Each microchamber (22) is separated by gas, and the sample inlet channel (24) and the sample outlet channel (25) are sealed. Then, at 30°C, the liquid is discharged into the main channel (21). Cultured at 37℃ for 3 days 5h; Microscopic fluorescence detection: The cultured microfluidic chip is placed under a fluorescence microscope, and the presence and number of strong fluorescence are observed to determine whether there are live target bacteria. Bacterial extraction and identification: The culture medium is driven into the main channel (21) through the injection channel (24), and the bacteria in the microchamber (22) are taken out by laser induction. The bacteria are collected on the blood plate in the sample outlet channel (25) for amplification culture. The colonies obtained by culture are identified by mass spectrometry to obtain bacterial information.

2. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 1, characterized in that, The injection flow rate of the lysis fluid is 20. 40 μL / min.

3. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 1, characterized in that, The mass ratio of the culture medium to the indicator is 9:

1. The ratio of culture medium to indicator was 10:1, and the injection flow rate was 20. 40 μL / min.

4. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 1, characterized in that: The micro-cavities (22) on both sides of the main channel (21) are staggered.

5. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 1, characterized in that, The glass slide layer (1) is made of ITO glass, quartz glass, polydimethylsiloxane, or polymethyl methacrylate, and the microstructure layer (2) is made of polydimethylsiloxane.

6. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 1, characterized in that, The fabrication method of the microfluidic chip includes the following steps: S1. Use software to design the microstructure of the microfluidic chip and perform film mask printing to obtain the printed film; S2. After cleaning and drying the silicon wafer with concentrated sulfuric acid, drop photoresist onto the silicon wafer and spin-dry it to form a photoresist layer on the silicon wafer. Cover the photoresist layer with the printed film, expose it, and clean the uncured photoresist with a developer to obtain the silicon wafer template. S3. Mix polydimethylsiloxane and curing agent evenly to obtain PDMS prepolymer. Pour the PDMS prepolymer onto a silicon wafer template to form a prepolymer layer. Dry and separate the silicon wafer template to obtain a PDMS layer with microstructure. S4. An injection channel (24) and an outlet channel (25) are opened on the PDMS layer to obtain a microstructure layer (2). S5. The microstructure layer (2) and the glass slide layer (1) are treated with oxygen plasma, and then the side of the microstructure layer (2) with microstructure is bonded to the glass slide layer (1) to form a microfluidic chip with microchannels.

7. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 6, characterized in that, The thickness of the photoresist layer is 40. 1000μm, the thickness of the prepolymer layer is 3 10mm.

8. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 6, characterized in that, The mass ratio of polydimethylsiloxane to curing agent in the PDMS prepolymer is 10:

1. 13:

1.

9. The method for rapid quantitative detection and identification of bacteria from whole blood according to claim 6, characterized in that, The oxygen plasma treatment time is 1 minute. During bonding, the microstructure layer (2) and the glass slide layer (1) are bonded at 80°C for 5 minutes. Bake at 90℃ overnight.

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