A chip for simultaneous rapid detection of multiple target molds
By designing a multi-target rapid mold detection chip, and using PDMS sponge film and microfluidic technology to achieve simultaneous enrichment and detection of mold, the problems of long time consumption and low sensitivity of traditional methods are solved, and rapid and accurate mold detection is achieved.
Patent Information
- Application Number
- CN202522334263.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-11-06
- Estimated Expiration
- 2035-11-04
AI Technical Summary
Traditional mold detection methods are time-consuming and have low sensitivity, making them prone to missed or false detections and unable to achieve real-time monitoring of mold contamination in the environment.
A multi-target rapid detection chip for molds was designed, employing an upper and lower chamber structure and a PDMS sponge film to trap mold spores. Microfluidic technology was combined to achieve integrated enrichment, lysis, and detection of molds. The LAMP method was used to expand the target genes, and the results were analyzed by HNB dye colorimetry.
It enables simultaneous enrichment, lysis, and detection of molds, shortening detection time, improving detection efficiency and accuracy, and reducing operational complexity and cost, making it suitable for use by non-professionals.
Smart Images
Figure CN224942614U_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a chip for the simultaneous and rapid detection of multiple target molds. Background Technology
[0002] Traditional methods for mold detection generally employ culture methods. For example, potato dextrose agar is used to culture the fungi for 5-7 days, followed by counting the total number of colonies to analyze mold contamination. While culture methods offer high accuracy, they are time-consuming and difficult to use for real-time monitoring of environmental mold contamination. Furthermore, approximately 70% of fungi cannot be cultured, leading to significant false negatives. Alternatively, direct microscopic examination can be used. Although this method is faster, it has low sensitivity and heavily relies on the operator's subjective experience, with an error margin of up to 35%, making it prone to false positives and false negatives. Utility Model Content
[0003] The main objective of this invention is to provide a chip for the simultaneous and rapid detection of multiple target molds.
[0004] The technical solution adopted by this utility model to solve its technical problem is:
[0005] A chip for simultaneous rapid detection of multiple target molds includes an upper layer and a lower layer, and a PDMS sponge located between the upper and lower layers; the upper layer has an upper inlet, and the number of outlets is progressively increased by dividing the upper inlet into two and then into four, with each outlet connected to an upper chamber; the lower layer has a lower outlet, and the number of inlets is progressively increased by dividing the lower outlet into two and then into four, with each inlet connected to a lower chamber; each upper chamber corresponds to a lower chamber, and a membrane for trapping mold spores is provided between the upper and lower chambers.
[0006] Furthermore, in the chip, the upper layer has an upper-layer inlet, which is connected to one end of the upper-layer primary flow channel. The other end of the upper-layer primary flow channel is connected to the middle portion of the upper-layer secondary flow channel. The two ends of the upper-layer secondary flow channel are respectively connected to the middle portions of two upper-layer tertiary flow channels. The two ends of one upper-layer tertiary flow channel are respectively connected to the upper-layer first chamber and the upper-layer second chamber, and the two ends of the other upper-layer tertiary flow channel are respectively connected to the upper-layer third chamber and the upper-layer fourth chamber. The two upper-layer tertiary flow channels are arranged in the same row, and the upper-layer tertiary flow channel is arranged parallel to the upper-layer primary flow channel and the upper-layer secondary flow channel.
[0007] The lower layer has a lower layer outlet, which is connected to one end of the lower layer primary flow channel. The other end of the lower layer primary flow channel is connected to the middle section of the lower layer secondary flow channel. The two ends of the lower layer secondary flow channel are respectively connected to the middle sections of two lower layer tertiary flow channels. The two ends of one lower layer tertiary flow channel are respectively connected to the lower layer first chamber and the lower layer second chamber, and the two ends of the other lower layer tertiary flow channel are respectively connected to the lower layer third chamber and the lower layer fourth chamber. The two lower layer tertiary flow channels are arranged in parallel, and the lower layer tertiary flow channel, the lower layer primary flow channel, and the upper layer secondary flow channel are arranged in parallel.
[0008] Among them, after the upper and lower layers are stacked, the upper first chamber and the lower first chamber are vertically aligned and have a first membrane between them; the upper second chamber and the lower second chamber are vertically aligned and have a second membrane between them; the upper third chamber and the lower third chamber are vertically aligned and have a third membrane between them; the upper fourth chamber and the lower fourth chamber are vertically aligned and have a fourth membrane between them.
[0009] Furthermore, the cross-section of each chamber is circular, square, triangular, or elliptical.
[0010] Furthermore, each upper chamber is pre-embedded with primers targeting the mold;
[0011] Furthermore, the film is a commercially available film.
[0012] Furthermore, the film is a PDMS sponge film.
[0013] Furthermore, the PDMS filter membrane is square and has four PDMS sponge slices on it, each slice corresponding to a chamber; the shape of the PDMS sponge slices corresponds to the shape of the chambers.
[0014] Furthermore, the thickness of the film is 0.05-1 mm.
[0015] Furthermore, the pore size of the film is smaller than the diameter of mold spores.
[0016] Compared with the prior art, this technical solution has the following advantages:
[0017] 1. The design of the upper and lower chamber structure of the chip in this utility model enables the use of PDMS sponge and other films to trap fungal spores in a closed environment, which is safer. On the other hand, the flow channel of the multi-target collaborative detection inlet is divided into two and then into four to ensure that the sample can be evenly distributed in the four reaction chambers.
[0018] 2. This invention employs an integrated microfluidic chip for enrichment and detection, enabling the simultaneous enrichment, lysis, and detection of at least four types of molds. The chip features a four-chamber parallel design, allowing for the simultaneous detection of four molds with a single sample injection. This improves detection efficiency, reduces batch-to-batch variability, and enhances the reliability of the results. The enriched spores can be lysed using a chitinase-assisted 40°C heat treatment process, releasing complete nucleic acids within 15 minutes without subsequent purification, simplifying the process and laying the foundation for automation. Subsequently, the LAMP method is used to expand the specific target genes of the four molds. Each chamber of the chip is relatively independent, with each chamber pre-embedded with a specific LAMP primer for one mold, ensuring high specificity of the detection targets. Simultaneously, the HNB dye colorimetric method is used to indicate the LAMP reaction results. Analysis of the RGB grayscale values of the spots in each reaction chamber after photographing enables simultaneous, rapid, and quantitative detection of multiple molds. Attached Figure Description
[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0020] Figure 1 This is a schematic diagram of the upper structure of the chip for simultaneous rapid detection of multiple target molds according to this utility model.
[0021] Figure 2 This is a schematic diagram of the lower layer structure of the chip for simultaneous rapid detection of multiple target molds according to this utility model.
[0022] Figure 3 This is a schematic diagram of the structure of a PDMS thin film.
[0023] Figure 4 This is a schematic diagram of the overall structure of the chip for rapid detection of multiple mold targets using a commercial filter membrane, as described in this utility model.
[0024] In the picture:
[0025] 100 - Upper layer 110 - Upper layer inlet 120 - Upper layer primary flow channel 130 - Upper layer secondary flow channel 140 - Upper layer tertiary flow channel
[0026] 150 - Upper three-stage flow channel; 160 - Upper first chamber; 170 - Upper second chamber; 180 - Upper third chamber; 190 - Upper fourth chamber
[0027] 200 - Lower Level 201 - Indicator Marker 210 - Lower Level Outlet 220 - Lower Level Primary Flow Channel 230 - Lower Level Secondary Flow Channel 240 - Lower Level Tertiary Flow Channel 250 - Lower Level Tertiary Flow Channel 260 - Lower Level First Chamber 270 - Lower Level Second Chamber 280 - Lower Level Third Chamber 290 - Lower Level Fourth Chamber
[0028] 300-PDMS film, 310-PDMS sponge chips
[0029] 400-Upper mold 410-Inlet 420-Outlet 430-Cavity
[0030] 500-lower mold 530-round cavity
[0031] 600-Commercial Filter Membrane
[0032] Figure 5 This is a schematic diagram of the structure of a PDMS sponge slice.
[0033] Figure 6 This is a standard curve for detecting Aspergillus terreus within the chip. Detailed Implementation
[0034] Example 1
[0035] See Figures 1 to 5 This invention provides a chip for the simultaneous rapid detection of multiple target molds, comprising an upper layer 100, a lower layer 200, and a filter membrane 300 located between the upper and lower layers.
[0036] The upper layer 100 has an upper inlet 110, which is connected to one end of the upper primary flow channel 120. The other end of the upper primary flow channel 120 is connected to the middle part of the upper secondary flow channel 130. The two ends of the upper secondary flow channel 130 are respectively connected to the middle parts of the upper tertiary flow channels 140 and 150. The two ends of the upper tertiary flow channel 140 are respectively connected to the upper first chamber 160 and the upper second chamber 170, and the two ends of the upper tertiary flow channel 150 are respectively connected to the upper third chamber 180 and the upper fourth chamber 190. The upper tertiary flow channels 140 and 150 are arranged in the same row, and the upper tertiary flow channels (140 and 150) are arranged parallel to the upper primary flow channel 120 and the upper secondary flow channel 130.
[0037] The lower layer 200 has a lower layer outlet 210, which is connected to one end of the lower primary flow channel 220. The other end of the lower primary flow channel 220 is connected to the middle portion of the lower secondary flow channel 230. The two ends of the lower secondary flow channel 230 are respectively connected to the middle portions of the lower tertiary flow channel 240 and the lower tertiary flow channel 250. The two ends of the lower tertiary flow channel 240 are respectively connected to the lower first chamber 260 and the lower second chamber 270, and the two ends of the lower tertiary flow channel 250 are respectively connected to the lower third chamber 280 and the lower fourth chamber 290. The lower tertiary flow channels 240 and 250 are arranged in the same row, and the lower tertiary flow channels (240 and 250) are arranged parallel to the lower primary flow channel 220 and the upper secondary flow channel 230.
[0038] Among them, after the upper layer 100 and the lower layer 200 are stacked, the upper first chamber 160 and the lower first chamber 260 are vertically aligned and have a first filter membrane between them; the upper second chamber 170 and the lower second chamber 270 are vertically aligned and have a second filter membrane between them; the upper third chamber 180 and the lower third chamber 280 are vertically aligned and have a third filter membrane between them; the upper fourth chamber 190 and the lower fourth chamber 290 are vertically aligned and have a fourth filter membrane between them.
[0039] Each chamber can have a cross-section that is circular, square, triangular, elliptical, or other shapes.
[0040] The "filter membrane" described in this utility model includes PDMS sponge membranes or commercial filter membranes. For example... Figure 3 As shown, the PDMS filter membrane 300 is square, with four PDMS sponge slices 310 on it. That is, the four PDMS sponge slices 310 are connected together by the PDMS membrane. The structure of the PDMS sponge slice 310 is as follows: Figure 5 As shown, it is cylindrical in shape with multiple holes on its surface. Each PDMS sponge slice 310 corresponds to one chamber.
[0041] The upper first chamber 160, upper second chamber 170, upper third chamber 180, and upper fourth chamber 190 are pre-embedded with primers targeting the target molds (e.g., primers targeting the aflR gene of Aspergillus flavus, the anxC4 gene of Aspergillus niger, the GOD gene of Aspergillus terreus, and the benA gene of Aspergillus terreus), with each chamber targeting one target mold.
[0042] The method of using this invention is as follows: Air samples are collected using a vacuum pump. The air sample is input into the chip through the upper inlet 110, and mold spores are trapped on the filter membrane as they pass through it. After gas collection, a spore lysis buffer containing chitinase is injected into the chamber through the upper inlet 110, and chemical-thermal lysis is performed at a constant temperature of 40°C for 15 minutes. After lysis, LAMP reaction reagent is introduced. Subsequently, the target genes of four molds are amplified at a constant temperature of 65°C. After amplification, qualitative and semi-quantitative detection of the four molds is performed based on HNB colorimetric analysis combined with RGB grayscale value analysis, realizing an integrated operation of collecting, lysing, amplifying, and detecting four molds on a single chip.
[0043] See Figure 4 The PDMS sponge membrane 300 can also be replaced by a commercial filter membrane 600. Furthermore, the commercial filter membrane 600 is a track-etched filter membrane with a thickness of 100 μm and a pore diameter of 0.4 μm.
[0044] This invention uses *Tetrandrobium* as an example, employing a PDMS sponge membrane 300. A quantitative detection standard curve was plotted. A 20 μL LAMP reaction system with different concentrations of spores was constructed, with the specific component configuration as follows:
[0045] Element 2×LAMP Master Mix 10× primer mixture Bst DNA polymerase Nuclease-free water Different concentrations of oxytetracycline samples to be tested Volume (μL) 10 2 1 5 2
[0046] After the system is prepared, gently mix it by pipetting and briefly centrifuge at room temperature to ensure the reaction solution collects at the bottom of the tube. Then place the reaction tube in a PCR instrument and run the following program: amplify at 65 ℃ for 30 minutes; maintain 4 ℃ continuously; set the lid temperature to 105 ℃ to prevent condensation. After 30 minutes, remove the reaction tube and let it stand at room temperature for 2 minutes. Observe the color change against a pure white background. A positive reaction will appear sky blue, while a negative reaction will remain violet.
[0047] Quantitative detection standard curve as follows Figure 6 As shown, the chip of this invention has a detection limit or sensitivity of 10 CFU / mL.
[0048] As can be seen from the examples above:
[0049] 1. Traditional culture methods require 3-7 days or even longer to obtain results. While molecular biology methods such as PCR shorten the identification time, they still require lengthy sample pretreatment (enrichment, DNA extraction, and purification). This invention seamlessly integrates four key steps—air sampling, spore enrichment, cell disruption, and nucleic acid amplification and detection—onto a single microchip, achieving a "sample in, result out" detection model. Time costs are significantly reduced: the entire process, from collecting air samples to obtaining qualitative and semi-quantitative results for four common pathogenic molds, can be completed within tens of minutes. This speed is unattainable with traditional methods, enabling real-time environmental quality assessment and risk warning.
[0050] 2. High Detection Sensitivity and Accuracy: Precise Control from "Million-Level" to "Micro-Level". The core advantage of microfluidic technology lies in the precise control of fluids and reactions at the microscale; low sample consumption and high enrichment efficiency: the chip-integrated thin film has high retention efficiency for 1-5μm particles (corresponding to the size of mold spores), ensuring that target spores in limited air samples are captured and enriched to the maximum extent in the reaction chamber without significant loss. This provides a more robust sensitivity foundation compared to traditional impaction or centrifugation methods, which may result in spore bounce, death, or dispersion.
[0051] 3. Avoid cross-contamination and ensure specificity: Each reaction chamber is physically isolated and can be pre-embedded with different primers. This allows for simultaneous parallel amplification of nucleic acids targeting different targets in four independent "micro-factories" after a sample enters. This design fundamentally eliminates problems such as primer interference and uneven amplification efficiency that may exist in multiplex PCR, ensuring the specificity and accuracy of multi-target detection. The detection results are clear and unambiguous, eliminating the need for complex melting curves or sequencing to distinguish different molds, thus simplifying the interpretation process.
[0052] 4. This invention compresses and solidifies a complex process requiring expensive equipment, multiple specialized laboratories, and professional operators onto a chip the size of a business card. Users only need to perform three simple steps: connect the chip to the vacuum pump for sampling -> sequentially inject lysis buffer and reaction premix through the inlet -> incubate at a constant temperature and observe / photograph the color change. The entire process eliminates the need for complex nucleic acid extraction and purification, cumbersome reagent preparation, expensive real-time fluorescence PCR instruments, and opening the chip for electrophoresis detection. This significantly reduces the technical background and professional training requirements for operators, enabling community health workers, factory quality inspectors, and even home users with minimal training to reliably complete the tests, greatly improving the accessibility and widespread adoption of the technology.
[0053] 5. Extremely low cost per test
[0054] Low chip material cost: PDMS is a common and inexpensive polymer material, ideally suited for large-scale mass production using processes such as soft lithography and molding, thus keeping the cost of a single chip extremely low. Minimal reagent consumption: The micro-scale reaction system means that the amount of expensive reagents such as primers, enzymes, and dNTPs used is reduced by several times or even an order of magnitude compared to traditional methods. The reagent cost per detection is significantly reduced. Low equipment investment and maintenance costs: As mentioned earlier, the absence of expensive large instruments also means no corresponding maintenance, calibration costs, or huge power consumption.
[0055] 6. High-throughput parallel detection capability
[0056] This chip integrates four parallel reaction chambers, enabling simultaneous processing of four different target molds in a single air sample. This not only improves detection efficiency but also further reduces the detection cost per target. The architecture offers excellent scalability; future designs can incorporate more chambers or utilize multi-layered chip structures to achieve simultaneous detection of more microorganisms or environmental indicators (such as endotoxins and allergens). Furthermore, the chip can be divided into eight sections to detect even more types of molds, depending on the specific requirements.
[0057] The above description is only a preferred embodiment of the present utility model, and therefore cannot be used to limit the scope of the present utility model. All equivalent changes and modifications made in accordance with the scope of the present utility model patent and the contents of the specification should still fall within the scope of the present utility model.
Claims
1. A chip for simultaneous rapid detection of multiple targets of molds, characterized in that, It includes an upper layer and a lower layer, as well as a thin film located between the upper and lower layers; the upper layer has an upper inlet, and the number of outlets is gradually increased by dividing the upper inlet into two and then into four, with each outlet connected to an upper chamber; the lower layer has a lower outlet, and the number of inlets is gradually increased by dividing the lower outlet into two and then into four, with each inlet connected to a lower chamber; each upper chamber corresponds to a lower chamber, and a thin film for trapping mold spores is provided between the upper and lower chambers.
2. The chip for simultaneous rapid detection of multiple target molds according to claim 1, characterized in that, The upper layer has an upper inlet, which is connected to one end of the upper primary flow channel; the other end of the upper primary flow channel is connected to the middle part of the upper secondary flow channel; the two ends of the upper secondary flow channel are respectively connected to the middle parts of two upper tertiary flow channels; one upper tertiary flow channel is respectively connected to the upper first chamber and the upper second chamber, and the other upper tertiary flow channel is respectively connected to the upper third chamber and the upper fourth chamber; the two upper tertiary flow channels are arranged in parallel, and the upper tertiary flow channel is arranged in parallel with the upper primary flow channel and the upper secondary flow channel; The lower layer has a lower layer outlet, which is connected to one end of the lower layer primary flow channel; the other end of the lower layer primary flow channel is connected to the middle part of the lower layer secondary flow channel; the two ends of the lower layer secondary flow channel are respectively connected to the middle parts of two lower layer tertiary flow channels; the two ends of one lower layer tertiary flow channel are respectively connected to the lower layer first chamber and the lower layer second chamber, and the two ends of the other lower layer tertiary flow channel are respectively connected to the lower layer third chamber and the lower layer fourth chamber; the two lower layer tertiary flow channels are arranged in the same row, and the lower layer tertiary flow channel is arranged in parallel with the lower layer primary flow channel and the lower layer secondary flow channel; Among them, after the upper and lower layers are stacked, the upper first chamber and the lower first chamber are vertically aligned and have a first membrane between them; the upper second chamber and the lower second chamber are vertically aligned and have a second membrane between them; the upper third chamber and the lower third chamber are vertically aligned and have a third membrane between them; the upper fourth chamber and the lower fourth chamber are vertically aligned and have a fourth membrane between them.
3. The chip for simultaneous rapid detection of multiple target molds according to claim 1, characterized in that, Each upper chamber is pre-embedded with primers targeting the specific mold.
4. A chip for simultaneous rapid detection of multiple target molds according to claim 1, characterized in that, Each chamber has a cross-section that is circular, square, triangular, or elliptical.
5. A chip for simultaneous rapid detection of multiple target molds according to claim 1 or 2, characterized in that, The film is a commercial film.
6. A chip for simultaneous rapid detection of multiple target molds according to claim 1 or 2, characterized in that, The membrane is a PDMS filter membrane.
7. A chip for simultaneous rapid detection of multiple target molds according to claim 6, characterized in that, The PDMS filter membrane is square and has four PDMS sponge slices on it, each slice corresponding to a chamber; the shape of the PDMS sponge slices corresponds to the shape of the chambers.
8. A chip for simultaneous rapid detection of multiple target molds according to claim 1 or 2, characterized in that, The thickness of the film is 0.3-1 mm.
9. A chip for simultaneous rapid detection of multiple target molds according to claim 1 or 2, characterized in that, The pore size of the film is smaller than the diameter of mold spores.