A bacterial enrichment device based on microchannel chip and its preparation method and bacterial identification method

Abstract

The invention discloses a bacteria enrichment device based on a microchannel chip, a preparation method thereof and a bacteria identification method. The bacteria enrichment device comprises: a microchannel chip, an elastic membrane covering the microchannel chip, a liquid conveying device connected to the microchannel chip, an injection device and a collection device connected to the liquid conveying device; the microchannel chip comprises: a silicon wafer substrate with an oxide layer, an oxide layer microchannel structure arranged on the silicon wafer substrate with the oxide layer, and a nanostructure arranged inside the oxide layer microchannel structure; the preparation method of the invention constructs a nanostructure in the channel by adding a microchannel, thereby directly and effectively enriching low-concentration bacteria in the chip; a modification method is designed according to the types of microorganisms, thereby realizing simultaneous capture and identification of mixed bacteria; after enrichment, mass spectrometry identification can be directly performed through the bacteria identification method of the invention, thereby shortening the enrichment and detection time, improving efficiency, effectively enhancing mass spectrometry signals, increasing the number of peaks, and improving detection quality.

Description

Technical Field

[0001] The invention relates to the field of microorganism identification, in particular to a bacteria enrichment device based on a microchannel chip and a preparation method thereof and a bacteria identification method. Background Art

[0002] Bacterial infection can lead to diseases such as urinary tract infection, nephritis, meningitis and sepsis, threatening the patient's life. Rapid and accurate identification of pathogenic bacteria is the key to clinical diagnosis and treatment. At present, clinical bacterial identification is still mainly based on traditional phenotypic detection methods, which mainly rely on the morphology, culture and growth characteristics of bacteria supplemented by biochemical tests to achieve bacterial identification. In recent years, molecular biology-based PCR technology, 16S rRNA sequencing and other genetic detection technologies, as well as proteomics-based mass spectrometry analysis technology have begun to be applied to bacterial identification and analysis.

[0003] Although traditional phenotypic analysis methods can meet some clinical needs, they are cumbersome to operate, highly dependent on experience, and often take 2-5 days or even longer to obtain identification results, which is very time-consuming and not suitable for the analysis of difficult-to-culture bacteria. Molecular biology-based PCR and gene sequencing technologies can obtain identification results within 5-12 hours, but require complex pre-treatment, are expensive, are easily affected by sample contamination, and the results are complex to analyze, making them unsuitable for rapid identification of clinical pathogens in primary hospitals.

[0004] Mass spectrometry technology, especially laser-assisted analytical ionization-time of flight mass spectrometry (MALDI-TOF MS), can detect the protein fingerprint of bacteria. Combined with the existing proteome-based bacterial identification system (such as Biotyper), rapid detection and identification of processed bacterial samples can be achieved within a few minutes. However, before mass spectrometry detection of bacteria, it is still necessary to undergo a culture and pretreatment process. The concentration of bacteria in clinical liquid samples such as blood and urine is not enough to be directly used for mass spectrometry analysis. At present, mass spectrometry identification of bacteria in clinical samples is mainly based on the detection of strains isolated from solid culture media, and the culture and separation of bacteria still takes about 24 hours. Mass spectrometry identification is still limited by bacterial culture. Therefore, there is an urgent need for an effective enrichment method for bacteria in clinical samples, which reduces or eliminates the time required for bacterial culture and realizes rapid identification of bacteria in clinical samples. At present, the basic process of microbial mass spectrometry identification is: obtaining biological samples → microbial culture → picking colonies for identification or treating colonies with lysis solution → mass spectrometry identification; the steps are repeated, time-consuming and costly, and the detection efficiency is low. The market needs a simple process, short time, and high efficiency bacterial identification method, and the present invention solves such problems. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a preparation method of a bacteria enrichment device based on a microchannel chip and a bacteria identification method. The device of the present invention can directly enrich bacteria in biological samples, and can directly perform mass spectrometry identification after enrichment without the need for bacterial culture and lysis, thereby shortening the detection time and improving the efficiency, and can effectively enhance the mass spectrometry signal and increase the number of peaks, thereby improving the detection quality.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A bacteria enrichment device based on a microchannel chip comprises: a microchannel chip, an elastic membrane covering the microchannel chip, a liquid conveying device connected to the microchannel chip, an injection device and a collection device connected to the liquid conveying device; the microchannel chip comprises: a silicon wafer substrate with an oxide layer, an oxide layer microchannel structure arranged on the silicon wafer substrate with the oxide layer, and a nanostructure arranged inside the oxide layer microchannel structure.

[0008] In the aforementioned bacteria enrichment device based on a microchannel chip, the elastic membrane comprises: a PDMS membrane and a rubber membrane.

[0009] The aforementioned bacterial enrichment device based on a microchannel chip, the liquid delivery device includes: a liquid delivery channel connected to both ends of the oxide layer microchannel structure; the liquid delivery channel is a quartz capillary, one end of the quartz capillary is connected to the injection device through a Teflon tube as a sample input end, and the other end is connected to the collection device through a Teflon tube as an enriched sample output end.

[0010] In the aforementioned bacterial enrichment device based on a microchannel chip, the depth of the oxide layer microchannel structure is 40 μm±; the nanostructure is a one-dimensional nanoarray, and the length of the nanowire is greater than or equal to 12 μm.

[0011] A method for preparing a bacteria enrichment device based on a microchannel chip comprises the following steps:

[0012] Step 1, preparing a microchannel chip with a nanostructure;

[0013] 1. On the surface of the silicon wafer substrate with an oxide layer, a portion of the oxide layer is first protected, and then the unprotected oxide layer and the substrate silicon wafer portion are etched to obtain a chip with an oxide layer microchannel structure on the surface;

[0014] 2. Prepare nanostructures inside the oxide layer microchannel structure;

[0015] Step 2, modifying the surface of the nanostructure with chemical groups or biological groups;

[0016] Step 3: Building a bacterial enrichment device, including the following steps:

[0017] 1. Cover the surface of the microchannel chip with an elastic film, which fits tightly to the chip surface;

[0018] 2. Two liquid delivery devices are set at both ends of the microchannel chip and pressed tightly with a fixing plate;

[0019] 3. One end of the liquid delivery device is connected to the injection device and the other end is connected to the collection device. The liquid enters the microchannel chip from the injection device and is enriched and then discharged from the collection device.

[0020] The aforementioned method for preparing a bacteria enrichment device based on a microchannel chip comprises the following steps: step 1, preparing a microchannel chip having a nanostructure;

[0021] 1. On the surface of the silicon wafer substrate with an oxide layer, a portion of the oxide layer is first protected, and then the unprotected oxide layer and the substrate silicon wafer portion are etched to obtain a chip with an oxide layer microchannel structure on the surface;

[0022] a. Spin-coating a photoresist on the surface of a silicon wafer substrate with an oxide layer, and then heating and curing the surface to obtain a chip covered with the photoresist;

[0023] b. Expose and develop the chip with the surface covered with photoresist;

[0024] c. After hardening the film on the heating plate, immerse the chip in the buffer solution for etching, take it out, rinse and blow dry;

[0025] d. Wash off the photoresist on the chip surface and blow dry to obtain a silicon wafer with a patterned oxide layer on the surface;

[0026] e. Immersing the silicon wafer with the patterned oxide layer in an alkaline solution for etching to obtain a chip with an oxide layer microchannel structure;

[0027] 2. Fabricate nanostructures inside the oxide layer microchannel structure;

[0028] a. Depositing silver particles on the surface of a chip having an oxide layer microchannel structure, cleaning to remove residual silver ions on the chip surface, and then immersing the chip in a mixture of an oxidant and a weak acid for etching and cleaning;

[0029] b. Immerse the chip in a strong acid solution to remove the silver particles on the surface of the nanowires, then clean and blow dry.

[0030] The aforementioned method for preparing a bacteria enrichment device based on a microchannel chip, wherein the specific method for modifying chemical groups on the surface of the nanostructure comprises the following steps:

[0031] The obtained microchannel silicon nanowire chip is immersed in a solvent solution of 3-aminopropyltriethoxysilane, and after the reaction, the chip is taken out, rinsed, and blown dry;

[0032] The chip is placed in a solvent solution of bis-N,N'-succinimidyl carbonate for reaction, and then rinsed and dried after the reaction is completed;

[0033] The chip was immersed in a buffer solution of concanavalin A, and then rinsed and stored after the reaction.

[0034] The aforementioned method for preparing a bacterial enrichment device based on a microchannel chip comprises molecules with specific affinity for bacterial biological groups; the molecules with specific affinity include: concanavalin A, aptamers, and antibodies.

[0035] In the aforementioned method for preparing a bacteria enrichment device based on a microchannel chip, the flow rate of the bacteria sample flowing into the microchannel silicon nanowire chip is 15 μL / min.

[0036] A method for identifying bacteria based on a microchannel chip comprises the following steps:

[0037] Step 1: using a bacteria enrichment device to capture and enrich bacteria in a liquid sample;

[0038] The sample is passed into the liquid delivery channel, the bacteria in the sample are captured on the chip surface, and sterilized water is passed to clean the captured silicon nanowires;

[0039] The preparation process of the bacteria enrichment device includes:

[0040] Step A, preparing a microchannel chip with a nanostructure;

[0041] 1. On the surface of the silicon wafer substrate with an oxide layer, a portion of the oxide layer is first protected, and then the unprotected oxide layer and the substrate silicon wafer portion are etched to obtain a chip with an oxide layer microchannel structure on the surface;

[0042] 2. Prepare nanostructures inside the oxide layer microchannel structure;

[0043] Step B, modifying the surface of the nanostructure with chemical groups or biological groups;

[0044] Step C, constructing a bacteria enrichment device, comprises the following steps:

[0045] 1. Cover the surface of the microchannel chip with an elastic film, which fits tightly to the chip surface;

[0046] 2. Two liquid delivery devices are set at both ends of the microchannel chip and pressed tightly with a fixing plate;

[0047] 3. One end of the liquid delivery device is connected to the injection device and the other end is connected to the collection device. The liquid enters the microchannel chip from the injection device and is enriched and then discharged from the collection device;

[0048] Step 2: Add matrix solution to the chip surface after capturing bacteria, and collect fingerprints on a MALDI-TOF mass spectrometer after drying;

[0049] Step three, compare the obtained fingerprint map with the standard map in the bacterial identification software database, and obtain the bacterial identification result based on the identification score.

[0050] In the aforementioned microchannel chip-based bacterial identification method, the liquid sample includes: blood, urine, saliva, and cerebrospinal fluid.

[0051] The present invention is beneficial in that:

[0052] The present invention uses an oxide layer silicon wafer as a substrate, improves the structure and size of the chip, adds a microchannel, and constructs a nanostructure in the channel to achieve effective enrichment of low-concentration bacteria in a liquid sample in the chip. Compared with the detection results without enrichment, the mass spectrometry signal intensity and peak number are significantly improved;

[0053] The present invention can achieve low-concentration bacteria (4×10 6 CFU / mL) without culture detection, the enrichment process is simple and fast, and the entire process of enrichment and detection can be completed within 1 hour;

[0054] After 4 hours of pre-incubation, the concentration of urine samples as low as 4×10 3 The enrichment and identification of CFU / mL bacteria shortens the detection time by more than 20 hours compared with traditional methods.

[0055] The present invention constructs a chip array that selectively modifies different bacteria through different surface modifications, and can achieve simultaneous capture and identification of mixed bacteria in a liquid sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A schematic diagram of a process for preparing a microchannel silicon nanowire chip according to an embodiment of the present invention;

[0057] Figure 2 Characterization diagrams of the microchannel silicon nanowire chip structure of the present invention (A: stereoscopic image characterizing the structure of the microchannel silicon nanowire chip, B: SEM image of the cross section of the microchannel silicon nanowire chip, C: 45° inclined SEM image of the silicon nanowires at the bottom of the channel of the microchannel silicon nanowire chip);

[0058] Figure 3 A schematic diagram of an embodiment of a bacteria enrichment device of the present invention;

[0059] Figure 4A is a comparison of mass spectra before capture and after enrichment with microchannel silicon nanowire chip, B is the number of mass spectra peaks with signal-to-noise ratio ≥ 5 in the range of 3-15 kDa between mass spectra before capture and mass spectra after enrichment with microchannel silicon nanowire chip;

[0060] Figure 5 Electron microscope images of the microchannel silicon nanowire chip before and after capturing bacteria in the present invention (A: before capture, B: after capture);

[0061] Figure 6 The number of mass peaks with a signal-to-noise ratio ≥ 5 in the mass spectrum obtained under the conditions of each chip structure (A), each silicon nanowire length (B) and each flow rate (C) in the present invention within the range of 3-15 kDa;

[0062] Figure 7 A is the mass spectrum obtained after the suspension of Escherichia coli with different concentrations in PBS buffer was enriched by the microchannel silicon nanowire chip; B is the mass spectrum obtained (4×10 8 CFU / mL) and the comparison chart of the standard spectrum in the database;

[0063] Figure 8 The mass spectra obtained after the urine samples with various concentrations of Escherichia coli were enriched by the microchannel silicon nanowire chip in the present invention.

[0064] The meaning of the numbers in the figure:

[0065] 1 fixed plate, 2 elastic membrane, 3 microchannel silicon nanowire chip. DETAILED DESCRIPTION

[0066] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] like Figure 3 As shown, a bacteria enrichment device based on a microchannel chip includes: a microchannel chip, an elastic membrane 2 covering the microchannel chip, a liquid delivery device connected to the microchannel chip, an injection device and a collection device connected to the liquid delivery device; the microchannel chip includes: a silicon wafer substrate with an oxide layer, an oxide layer microchannel structure arranged on the silicon wafer substrate with an oxide layer, and a nanostructure arranged inside the oxide layer microchannel structure. As an embodiment, the elastic membrane 2 includes: a PDMS membrane, a rubber membrane; it should be noted that: as long as the membrane layer does not react with the liquid and has elasticity, it can be applied to the present invention, and this is not an exhaustive list.

[0068] The liquid delivery device includes: a liquid delivery channel connected to both ends of the oxide layer microchannel structure. As a preferred embodiment, the liquid delivery channel is a quartz capillary, one end of the quartz capillary is connected to the injection device through a Teflon tube as a sample input end, and the other end is connected to the collection device through a Teflon tube as an enriched sample output end; it should be noted that: as long as it can complete liquid delivery, it can be applied to the present invention, and this is not exhaustive.

[0069] If the depth of the oxide layer microchannel structure is too shallow, the amount of liquid sample that can be accommodated is small; if the depth of the oxide layer microchannel structure is too deep, it will lead to a large deviation in the molecular weight detected by mass spectrometry, so the depth of the oxide layer microchannel structure is preferably 40μm±.

[0070] The nanostructure is a one-dimensional nanoarray, and preferably is a nanowire, and the length of the nanowire is greater than or equal to 12 μm.

[0071] like Figure 1 As shown, a method for preparing a bacteria enrichment device based on a microchannel silicon nanowire chip 3 comprises the following steps:

[0072] Step 1, preparing a microchannel chip with a nanostructure;

[0073] As a preference, the parameters of the silicon wafer substrate with the oxide layer are 1 cm×1 cm, and the oxide layer thickness is 1 μm.

[0074] 1. On the surface of the silicon wafer substrate with an oxide layer, first protect part of the oxide layer, and then etch the unprotected oxide layer and the substrate silicon.

[0075] The chip part obtains a chip with an oxide layer microchannel structure on the surface;

[0076] a. Expose and develop the chip with the surface covered with photoresist;

[0077] As a preferred embodiment, a chip with a surface covered with photoresist is placed on a heating plate, the photoresist is pre-baked and cured, a photomask is covered on the surface of the chip, the chip is exposed under a UV exposure machine, the chip is immersed in a 0.7% NaOH solution for development, and the photoresist in the exposed area is washed away. It should be noted that these options are only an embodiment and are not exhaustive. As long as they can achieve the protection of a partial oxide film, they are applicable to the present invention.

[0078] b. After hardening the film on the heating plate, immerse the chip in the buffer solution for etching, take it out, rinse and blow dry;

[0079] As a preferred embodiment, the buffer is BHF buffer, the rinse is deionized water, and the drying is nitrogen; the purpose is to remove the oxide film of the part without photoresist protection; it should be noted that these options are only an embodiment, not an exhaustive list, and any reagent that can remove the oxide film of the protected part is applicable to the present invention. For example, the oxide layer of the part without photoresist protection can be removed by sputter etching (SE) or reactive ion etching (RIE) using corrosive gases such as CF4.

[0080] c. Wash off the photoresist on the chip surface and blow dry to obtain a silicon wafer with a patterned oxide layer on the surface;

[0081] d, immersing the silicon wafer with the patterned oxide layer in an alkaline solution for etching to obtain a silicon wafer with microchannels;

[0082] As an embodiment, the alkaline solution is a 30% KOH aqueous solution; the purpose of this step is to etch the silicon without oxide layer protection, and the oxide layer still exists on the surface after this etching step is completed; it should be noted that these options are only an embodiment, not an exhaustive list, and any method that can etch the silicon without oxide layer protection is applicable to the present invention. For example, ethylenediamine catechol (EDP) and tetramethylammonium hydroxide (TMAH) are used for wet etching. Or, the silicon without protection is etched by reactive ion etching (RIE) using corrosive gases such as sulfur hexafluoride (SF6) to obtain a microchannel structure.

[0083] The above examples are not exhaustive: any method that can obtain an oxide layer microchannel structure on the surface of a silicon wafer substrate with an oxide layer is applicable to the present invention.

[0084] 2. Prepare nanostructures inside the oxide layer microchannel structure;

[0085] As an embodiment, the following steps are included:

[0086] a. Depositing silver particles on the surface of a chip having an oxide layer microchannel structure, cleaning to remove residual silver ions on the surface of the chip, and then immersing the chip in a mixture of an oxidant and a weak acid for etching and cleaning;

[0087] As a preferred method, the method of depositing silver particles on the surface of a chip having an oxide layer microchannel structure is: immersing the chip having an oxide layer microchannel structure in an aqueous solution of 0.02M AgNO3 and 4.8M HF to deposit silver particles on the surface; it should be noted that these options are only an embodiment and are not exhaustive. As long as the surface deposition of silver particles can be completed, it is applicable to the present invention.

[0088] As a preferred embodiment, the mixed solution of the oxidant and the weak acid is an aqueous solution of 0.4M H2O2 and 4.8M HF. It should be noted that these options are only an embodiment and are not exhaustive. Any method that can accomplish etching is applicable to the present invention.

[0089] b. As an example, the chip is immersed in a strong acid solution to remove the silver particles on the surface of the nanowires, and then cleaned and dried.

[0090] The obtained chip is Figure 2 shown.

[0091] As another example, a chip having an oxide layer microchannel structure was immersed in a mixed solution containing 4.8M HF and 0.02M AgNO3 for etching and cleaning, and then immersed in a diluted nitric acid (v / v=1:1) aqueous solution to remove silver particles on the surface, and then washed with deionized water and dried with nitrogen to obtain silicon nanowires.

[0092] The above examples are not exhaustive: any method that can prepare a nanostructure inside the oxide layer microchannel structure is applicable to the present invention.

[0093] Step 2, modifying the surface of the nanostructure with chemical groups or biological groups;

[0094] Chemically modifying a microchannel silicon nanowire chip includes the following steps:

[0095] The obtained microchannel silicon nanowire chip 3 is immersed in a solvent solution of 3-aminopropyltriethoxysilane. After the reaction, the chip is taken out, rinsed and blown dry.

[0096] The chip is placed in a solvent solution of bis-N,N'-succinimidyl carbonate for reaction, and then rinsed and dried after the reaction is completed;

[0097] The chip is immersed in a buffer solution of concanavalin, and after the reaction, it is rinsed and stored;

[0098] The present invention constructs a chip array that selectively modifies different bacteria through different surface modifications, which can achieve simultaneous capture and identification of mixed bacteria in liquid samples; as a preferred embodiment, a chip modified with concanavalin is selected, which has affinity for a variety of Gram-negative bacteria, expanding the range of tested bacteria. It should be noted that the reagents selected for modification here are only a preferred embodiment, and any reagent modified based on the chip of the present invention is within the protection scope of the present invention.

[0099] Silicon nanowires are modified to improve their affinity to microorganisms.

[0100] If the microorganism is a bacterium, the biological group includes: molecules having specific affinity for bacteria include concanavalin, aptamers and antibodies.

[0101] Step 3: Building a bacterial enrichment device, including the following steps:

[0102] The surface of the microchannel silicon nanowire chip 3 is covered with a polydimethylsiloxane elastic film 2, and liquid delivery channels connected to a syringe and a waste liquid bottle are set at both ends of the patterned channel, and are pressed tightly with a fixing plate 1, such as Figure 3 As a preferred embodiment, the fixing plate 1 is an acrylic plate.

[0103] A method for identifying bacteria based on a microchannel chip 3 comprises the following steps:

[0104] Step 1: using the microchannel silicon nanowire chip 3 in the bacteria enrichment device described above to capture and enrich the bacteria in the liquid sample;

[0105] The sample is passed into the liquid delivery channel, the bacteria in the sample are captured on the chip surface, and sterile water is passed to clean the captured silicon nanowires; the liquid is enriched in the chip microchannel, and the optimal flow rate for the bacterial sample to flow into the microchannel silicon nanowire chip is 15μL / min.

[0106] Step 2: drip a matrix solution onto the chip surface after capturing bacteria, and collect protein fingerprints using MALDI-TOF MS after drying. Preferably, the matrix solution is: drip 1 μL of a saturated solution of α-cyano-4-hydroxycinnamic acid in a mixed solution of acetonitrile, trifluoroacetic acid and deionized water; the volume ratio of acetonitrile, trifluoroacetic acid and deionized water is 50:2.5:47.5.

[0107] The following experiments are used to verify the beneficial effects of the present invention;

[0108] Microchannel silicon nanowire chips were prepared as follows for use in experiments 1, 2, 3, and 4:

[0109] (1) Spin-coat photoresist (4000rpm, 40s) on the surface of a silicon wafer substrate with an oxide layer (1cm×1cm, oxide layer thickness 1μm) using a spin coater. Place the chip with the photoresist on the surface on a heating plate and pre-bake to cure the photoresist. Then cover the chip surface with a photomask with a specific pattern and expose it under a UV exposure machine. Immerse in a 0.7% NaOH solution for about 40s to develop and wash away the photoresist in the exposed area. Place it on a heating plate to harden the film. Then immerse the chip in BHF buffer and etch at room temperature for 10 minutes to remove the silicon dioxide layer in the part not protected by the photoresist. After taking it out, rinse it with deionized water and blow it dry with nitrogen. Use acetone to wash away the photoresist on the chip surface, and blow it dry with nitrogen to obtain a silicon wafer with a patterned oxide layer on the surface.

[0110] (2) Immerse the silicon wafer with the patterned oxide layer in a 30% KOH aqueous solution at 90°C for 20 minutes. Obtain a silicon wafer with microchannels. Immerse the chip with the oxide layer microchannel structure in an aqueous solution of 0.02M AgNO3 and 4.8M HF, and deposit silver particles on the surface (room temperature, 20 seconds). After washing away the residual silver ions on the surface of the chip with deionized water, immerse the chip in an aqueous solution of 0.4M H2O2 and 4.8M HF, and etch for 15 minutes at room temperature. After taking out the chip, wash away the residual etching solution with deionized water. Finally, immerse the chip in a diluted nitric acid solution (1:1, v / v) to remove the silver particles on the surface of the nanowires. Take out the chip after 30 minutes, rinse it with deionized water 3 times, and blow dry it with nitrogen.

[0111] (3) The obtained microchannel silicon nanowire chip 3 was immersed in an ethanol solution of 3-aminopropyltriethoxysilane (4%, v / v), reacted at room temperature for 1 hour, and then the chip was taken out. After rinsing with ethanol, the chip was blown dry with nitrogen. Then the chip was placed in an acetonitrile solution of bis-N,N'-succinimidyl carbonate (10mM) and reacted at room temperature for 15 minutes. After the reaction was completed, the chip was rinsed with acetonitrile and dried with nitrogen. Finally, the chip was immersed in a PBS solution of concanavalin (30μg / mL), reacted at room temperature for 1 hour, rinsed with PBS buffer, and stored at 4°C. .

[0112] The obtained microchannel silicon nanowire chip 3 was used in the following experiments:

[0113] Experiment 1: Verification of the bacterial enrichment performance of microchannel silicon nanowires. The specific experiment is as follows:

[0114] 0.5 mL of E. coli suspension (4 × 10 8 CFU / mL) flows through the microchannel of the silicon nanowire chip at a rate of 15μL / min to enrich the bacteria in the sample. Subsequently, 0.03mL of sterilized water is passed at a rate of 3μL / min to clean the captured silicon nanowires. After removing the chip, 1μL of matrix solution is dripped on the surface. After drying, the MALDI-TOF MS spectrum with a molecular weight range of 2-20kDa is obtained under linear positive ion conditions. In the comparative experiment without enrichment, 1μL of Escherichia coli suspension is directly dripped on the steel target sample point or the surface of the silicon nanowire target plate. After covering the matrix, the mass spectrometer is obtained under the same instrument conditions.

[0115] Experimental results: Figure 4 A is a comparison of the mass spectra of the present invention before enrichment and after enrichment by the microchannel silicon nanowire chip 3, Figure 4 B is the number of mass peaks with a signal-to-noise ratio ≥ 5 in the range of 3-15 kDa in these two mass spectra. Figure 5This is an electron microscope image of the microchannel silicon nanowires enriched with Escherichia coli in the present invention.

[0116] Result analysis: Figure 4 It can be seen that when the bacterial concentration is 4×10 8 CFU / mL, after bacterial enrichment by microchannel silicon nanowire chip 3, the number of mass spectrometry peaks increased significantly. 7 CFU / mL, the sample without enrichment was directly added to the steel target surface, and the mass spectrum had no signal. After enrichment, there were still a large number of characteristic peaks, indicating that the microchannel silicon nanowire chip 3 can effectively enrich bacteria in liquid samples. Figure 4 As shown in the mass spectrum of the sample directly dripped on the surface of the silicon nanowire, the number of mass spectrum peaks obtained by dripping the bacterial suspension on the surface of the silicon nanowire target plate without enrichment is higher than the number of peaks obtained by dripping on the steel target surface. This proves that the presence of silicon nanowires in the microchannel not only has the function of capturing bacteria, but also can promote the ionization of the molecules to be tested and enhance the mass spectrometry signal. Figure 5 E. coli captured on the surface of the silicon nanowires can be directly observed, further verifying the chip's bacteria capture capability.

[0117] Experiment 2, optimization of factors affecting capture efficiency;

[0118] For capturing bacteria in liquid samples, the interaction between bacteria and the capture platform is very important. This experiment optimized the effects of chip structure, nanowire length and flow rate on capture efficiency. In the optimization of the chip structure, 0.5 mL of Escherichia coli suspension in PBS buffer (4×10 8 CFU / mL) was passed through four chips with different structures at a rate of 15μL / min to enrich the bacteria in the sample. Subsequently, 0.03mL of sterile water was passed at a rate of 3μL / min to clean the captured silicon nanowires. After removing the chip, 1μL of matrix solution was dripped on the surface. After drying, MALDI-TOF MS spectra with a molecular weight range of 2-20kDa were obtained under linear positive ion conditions. The bacterial capture effects of four chips, namely, concanavalin-modified microchannel silicon nanowire chip 3 (McSiNWs-Con A), concanavalin-modified channelless silicon nanowire chip (SiNWs-Con A), unmodified microchannel silicon nanowire chip 3 (McSiNWs), and concanavalin-modified microchannel silicon chip (McSi-Con A), were compared.

[0119] For the optimization of nanowire length, 0.5 mL of E. coli suspension (4 × 10 8CFU / mL) was passed through the microchannel of the silicon nanowire chip with different lengths at a rate of 15μL / min to enrich the bacteria in the sample. Then 0.03mL of sterilized water was passed at a rate of 3μL / min to clean the captured silicon nanowires. After removing the chip, 1μL of matrix solution was dripped on the surface. After drying, MALDI-TOF MS spectra with a molecular weight range of 2-20kDa were obtained under linear positive ion conditions. The differences in mass spectrometry signals after bacteria were captured by silicon nanowires of different lengths were compared.

[0120] For flow rate optimization, 0.5 mL of E. coli suspension (4 × 10 8 CFU / mL) were passed through the microchannel of the silicon nanowire chip at a rate of 5μL / min, 10μL / min, 15μL / min and 20μL / min, respectively, to enrich the bacteria in the sample. Subsequently, 0.03mL of sterile water was passed at a rate of 3μL / min to clean the captured silicon nanowires. After removing the chip, 1μL of matrix solution was added to the surface. After drying, MALDI-TOF MS spectra with a molecular weight range of 2-20kDa were obtained under linear positive ion conditions. The effects of different long flow rates on capture efficiency were compared.

[0121] Experimental results: Figure 6 The present invention compares the number of peaks with a signal-to-noise ratio ≥ 5 between 3 and 15 kDa in mass spectra obtained by capturing bacteria on chips with different structures, different silicon nanowire lengths and different flow rates.

[0122] Result analysis: Figure 6 It can be seen that the synergistic effect of the oxide layer microchannel structure, silicon nanowire structure and surface modification of the microchannel silicon nanowire chip 3 improves the capture efficiency of bacteria. The increase in the length of the silicon nanowires in the microchannel can increase the contact area and binding sites with the bacteria, thereby improving the capture efficiency. When the nanowire length is above 12 μm, the capture efficiency increases less with the increase in the nanowire length, and longer nanowires require longer etching time, so 12 μm silicon nanowires are used in the experiments of the present invention. When the sample flow rate is in the range of 5-15 μL / min, the capture efficiency of bacteria increases with the increase in flow rate. This is because as the flow rate increases, the pressure in the capture channel will increase accordingly, causing the sample to immerse deeper into the nanowires, increasing the contact area between the bacteria and the nanowires, thereby improving the capture efficiency. When the flow rate is further increased, the time for bacteria to bind to the sample is reduced, and the capture efficiency is significantly reduced. Therefore, a flow rate of 15 μL / min is used in the present invention.

[0123] Experiment 3: Direct capture and identification of low-concentration bacteria in liquid samples. The specific experiment is as follows:

[0124] 0.5 mL of E. coli suspension in PBS and healthy human urine spiked with E. coli (concentrations of 4 × 10 8 CFU / mL, 4×10 7 CFU / mL, 4×10 6 CFU / mL, 4×10 5 CFU / mL) flows through the microchannel of the silicon nanowire chip at a rate of 15μL / min to enrich the bacteria in the sample. Then 0.03mL of sterile water is passed at a rate of 3μL / min to clean the captured silicon nanowires. 1μL of matrix solution is added dropwise, and the MALDI-TOF MS spectrum is obtained after drying. The obtained spectrum is imported into the Autof Analyzer software, matched with the database, and the identification result is obtained. A score greater than 9 indicates species-level identification, a score between 6-9 indicates genus-level identification, and a score less than 6 indicates an untrustworthy identification result.

[0125] Experimental results:

[0126] As shown in Figure 1 and Figure 7 As shown, Figure 7 A is the mass spectrum obtained after the suspension of Escherichia coli with different concentrations in PBS buffer was enriched by the microchannel silicon nanowire chip; B is the mass spectrum obtained (4×10 8 CFU / mL) and the comparison chart of the standard spectrum in the database;

[0127] Table 1 shows the identification scores of E. coli suspensions enriched at different concentrations in PBS and the identification scores of E. coli enriched at different concentrations in urine samples of healthy persons.

[0128] Table 1

[0129]

[0130] Results analysis: The identification scores in Table 1 show that the enrichment and identification method based on patterned silicon nanowires achieves the culture-free detection of low-concentration bacteria in liquid samples. The detection limit for bacteria in PBS is 4×10 5 CFU / mL. The complex components in urine samples have a certain impact on bacterial identification, but the concentration can still be as low as 4×10 6 Direct identification of bacteria in CFU / mL.

[0131] Experiment 4: enrichment and identification of low-concentration bacteria in liquid samples after short-term culture. The specific experiment is as follows:

[0132] Take 3 mL of healthy human urine sample spiked with Escherichia coli and add an equal volume of LB liquid culture medium (the bacterial concentration after adding the culture medium is 4×10 6 CFU / mL, 4×105 CFU / mL, 4×10 4 CFU / mL, 4×10 3 CFU / mL). Place in a 37°C constant temperature incubator and maintain shaking culture at 200rpm for 4h. Take 0.5mL of the cultured urine sample and flow it through the capture channel of the microchannel silicon nanowire chip 3 at a rate of 15μL / min to enrich the bacteria in the sample. Then, pass 0.03mL of sterile water at a rate of 3μL / min to wash the captured silicon nanowires. Add 1μL of matrix solution and obtain its MALDI-TOF MS spectrum and identification score after drying.

[0133] Experimental results:

[0134] As shown in Table 2 and Figure 8 As shown;

[0135] Figure 8 It is a mass spectrogram obtained after the urine samples spiked with different concentrations of Escherichia coli are enriched by the microchannel silicon nanowire chip in the present invention;

[0136] Table 2 shows the enrichment and identification scores of urine samples spiked with different concentrations of Escherichia coli after 4 h of incubation.

[0137] Table 2

[0138]

[0139] Result analysis: After 4 hours of culture, 4×10 6 The sample enrichment and identification scores of CFU / mL E. coli were significantly higher than those without culture. 3 After culturing and enrichment, samples with CFU / mL can be identified at the genus level. The entire process of culture capture and identification takes less than 5 hours. Compared with traditional methods, the identification time is shortened by more than 20 hours, achieving rapid identification of bacteria in liquid samples.

[0140] The present invention improves the structure and size of the chip, adds a microchannel, and sets the channel as a nanostructure, so as to effectively enrich low-concentration bacteria in liquid samples in the chip, thereby significantly improving the mass spectrometry signal intensity and peak number; shortening the entire process and time of enrichment and detection, and improving detection efficiency.

[0141] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A bacterial identification method based on a microchannel chip, characterized in that: The steps include: Step 1: using a bacteria enrichment device to capture and enrich bacteria in a liquid sample; The sample is passed into the liquid delivery channel, the bacteria in the sample are captured by the chip surface, and sterilized water is passed to clean the captured silicon nanowires; wherein the flow rate of the sample is 15 μL / min; The preparation process of the bacteria enrichment device comprises: Step A, preparing a microchannel chip with a nanostructure; 1. On the surface of a silicon wafer substrate with an oxide layer, a portion of the oxide layer is first protected, and then the unprotected oxide layer and the substrate silicon wafer portion are etched to obtain a chip with an oxide layer microchannel structure on the surface; wherein the oxide layer is a patterned oxide layer, the thickness of the oxide layer is 1 μm; the depth of the oxide layer microchannel structure is 40 μm±; 2. Preparing a nanostructure inside the oxide layer microchannel structure; wherein the nanostructure is a silicon nanowire, and the length of the silicon nanowire is greater than or equal to 12 μm; Step B, modifying the surface of the nanostructure with concanavalin A; Step C, constructing a bacteria enrichment device, comprises the following steps:

1. Cover the surface of the microchannel chip with an elastic film, which fits tightly to the chip surface; 2. Two liquid delivery devices are set at both ends of the microchannel chip and pressed tightly with a fixing plate; 3. One end of the liquid delivery device is connected to the injection device and the other end is connected to the collection device. The sample enters the microchannel chip from the injection device for enrichment and then is discharged from the collection device; Step 2: Add matrix solution to the chip surface after capturing bacteria, and collect fingerprints on a MALDI-TOF mass spectrometer after drying; Step three, compare the obtained fingerprint map with the standard map in the bacterial identification software database, and obtain the bacterial identification result based on the identification score.

2. A method for bacterial identification based on a microchannel chip according to claim 1, characterized in that: The liquid samples include: blood, urine, saliva, and cerebrospinal fluid.

Images