A whole-process micro-fluidic chip for rapid, accurate and high-sensitivity detection of water quality samples and application thereof

Abstract

The application discloses a whole-process micro-fluidic chip for rapid, accurate and high-sensitivity detection of water quality samples and application thereof, and relates to the technical field of micro-fluidic chips. The chip comprises a front cover plate, a rear cover plate, a first aluminum block, a second aluminum block, a conduit, a heating block, a Peltier device and a magnet. When the front cover plate and the rear cover plate are buckled together, the front cover plate and the rear cover plate form an enrichment chamber, a thermal cracking chamber, a first washing chamber, a second washing chamber and a nucleic acid amplification reaction chamber respectively, all the chambers are open at the top and are communicated with each other, and the chambers are used for storing an oil phase. The first aluminum block and the second aluminum block are respectively pasted on the back of the rear cover plate and are respectively aligned with the thermal cracking chamber and the nucleic acid amplification reaction chamber. The heating block and the Peltier device are respectively arranged on the back of the first aluminum block and the second aluminum block. The magnet is used for attracting magnetic beads to realize the transfer of bacteria and / or nucleic acid molecules between the chambers. According to the application, a whole-process micro-fluidic chip capable of realizing rapid, accurate and high-sensitivity detection of VBNC Escherichia coli in water is constructed.

Description

Technical Field

[0001] This invention relates to the field of water quality testing, and more specifically to a full-process microfluidic chip for rapid, accurate, and highly sensitive detection of water quality samples and its applications. Background Technology

[0002] Drinking water is an indispensable part of daily life, and its quality significantly impacts people's well-being, thus requiring professional water quality testing and analysis. Microbial contamination is a major hazard in drinking water, with coliform bacteria being a primary cause of water pollution. Excessive coliform levels render drinking water unsafe, especially for children and the elderly, whose weakened immune systems can pose serious health risks. Currently, drinking water is commonly disinfected using chlorination and ultraviolet light. Under prolonged stress from chlorination and ultraviolet light, E. coli enters a viable butnonculturable (VBNC) state to increase its survival rate. Studies have found that VBNC E. coli retains metabolic activity and toxicity; furthermore, the VBNC state can be reversed under suitable conditions. The gold standard for E. coli detection is the plate assay, which can miss VBNC E. coli, thus underestimating the number of E. coli in drinking water. Once a large number of VBNC E. coli enter the human food chain, they can reactivate in the body, exerting their virulence and posing a threat to human health. Therefore, effective detection of VBNC E. coli in drinking water is a hot research topic in this field.

[0003] Methods for detecting VBNC bacteria include respiration assays, live / dead cell staining, and molecular biology techniques. While these methods have been validated for qualitative and quantitative detection of VBNC E. coli, they still suffer from drawbacks such as numerous laboratory steps, complex procedures, time consumption, and susceptibility to environmental factors. The development of molecular biology has made molecular methods for bacterial detection a major trend. By detecting specific genes in VBNC bacteria, their presence can be accurately determined. These methods offer high sensitivity and specificity, but require specific experimental equipment and techniques, and the procedures are cumbersome and susceptible to external contamination.

[0004] While a range of microfluidic chips have been developed for nucleic acid detection, such as droplet PCR or digital PCR, enabling absolute quantification and thus finding wide application in bacterial detection, these methods typically lack pretreatment steps for real samples, preventing direct detection of actual samples. Furthermore, the low VBNC content in water samples leads to low detection sensitivity, limiting their widespread use. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic chip for rapid, accurate, and highly sensitive detection of water quality samples, thereby solving the problems of cumbersome steps, complex operation, long time consumption, and susceptibility to environmental factors in existing technologies.

[0006] According to a first aspect of the present invention, a full-process microfluidic chip for rapid, accurate, and highly sensitive detection of water samples is provided, comprising: a front cover plate, a rear cover plate, a first aluminum block, a second aluminum block, a conduit, a heating block, a Peltier device, and a magnet; wherein, the surface of the front cover plate facing the rear cover plate has five rectangular grooves arranged sequentially along its length, which, when the front cover plate and the rear cover plate are fastened together, respectively form an enrichment chamber, a pyrolysis chamber, a first washing chamber, a second washing chamber, and a nucleic acid amplification reaction chamber, all of which are open at the top and interconnected for storing an oil phase; the first aluminum block and the second aluminum block are respectively attached to the back of the rear cover plate and aligned with the pyrolysis chamber and the nucleic acid amplification reaction chamber, respectively; the conduit is inserted into the enrichment chamber from above the front cover plate and the rear cover plate; the heating block and the Peltier device are respectively disposed on the back of the first aluminum block and the second aluminum block; the magnet is used to attract magnetic beads to realize the transfer of bacteria and / or nucleic acid molecules between the chambers.

[0007] Preferably, the front cover and the rear cover are made of transparent polycarbonate material. The reasons for choosing polycarbonate material are as follows: 1) PC material has low cost and simple processing, which can reduce costs; 2) It has low hygroscopicity and can be reused; 3) It has good light transmittance, which is convenient for experimental observation and optical detection; 4) It has a high glass transition temperature (about 145°C), which is suitable for PCR experiments.

[0008] Preferably, the front cover and the rear cover are bonded together by an adhesive.

[0009] Preferably, the first aluminum block and the second aluminum block are slightly larger than the thermal pyrolysis chamber and the nucleic acid amplification reaction chamber, respectively.

[0010] Preferably, after the heating block is powered on, it rapidly transfers heat through the first aluminum block to achieve effective lysis of bacteria in the pyrolysis chamber.

[0011] Preferably, the Peltier device can rapidly increase and decrease temperature to facilitate in-situ PCR amplification of nucleic acid molecules.

[0012] Preferably, the catheter includes an inlet catheter and an outlet catheter, used for the entry and exit of samples, respectively.

[0013] According to a second aspect of the present invention, an application of the end-to-end microfluidic chip as described above in the detection of VBNC (Vitamin B coli) in water samples is provided. However, it should be understood that the end-to-end microfluidic chip provided by the present invention is not limited to the detection of VBNC in water samples, but can also be used to detect other bacteria.

[0014] Unlike existing PCR chips that are generally horizontally extended, the microfluidic chip provided by this invention has a vertical structure. Utilizing oil-sealing and magnetic control technology, nucleic acid extraction and amplification reagents can be pre-embedded in the chip. After sampling, specifically binding magnetic beads are added, and enrichment is achieved under the action of a magnetic sheet. Subsequently, an external magnet facilitates the transfer and reaction of nucleic acids in different reagents, completing nucleic acid extraction and amplification. Finally, quantitative detection is performed by observing fluorescence intensity. The entire microfluidic chip design is simple, the manufacturing process is straightforward, pollution is minimal, and the results are reliable, demonstrating excellent prospects for industrial transformation.

[0015] Compared with existing methods for detecting VBNC (Vitamin B cerevisiae), the end-to-end microfluidic chip provided by this invention has the following advantages: 1. The enrichment chamber can effectively pretreat bacteria in the sample, saving reaction time; 2. The microfluidic chip adopts a vertical structure and oil-sealing strategy, which can reduce the use of pumps and the influence of external environmental factors, resulting in more reliable results; 3. The manufacturing process is simple and low-cost; 4. The chip structure can be combined with different types of magnetic beads, and the magnetic beads are modified with different nucleic acid aptamers, which can then be used to enrich other bacteria, thus having versatility and realizing the end-to-end detection of other bacteria; 5. This microfluidic chip can complete the entire process of sample enrichment, thermal lysis, two-step washing, and elution amplification in about 1 hour, realizing a rapid, accurate, and highly sensitive end-to-end detection of VBNC in water with "sample in, result out".

[0016] In summary, according to the present invention, a complete microfluidic chip capable of rapid, accurate, and highly sensitive detection of VBNC Escherichia coli in water has been constructed. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of a full-process microfluidic chip according to a preferred embodiment of the present invention;

[0018] Figure 2 This is the front view of the front cover.

[0019] Figure 3 This is the front view of the rear cover.

[0020] Figure 4 This is a 3D view of the heating block;

[0021] Figure 5A three-dimensional view of the Peltier device;

[0022] The meanings of the reference numerals in the attached figures are as follows:

[0023] 1. 100 Front cover plate; 2. 200 Rear cover plate; 3. 300 Heating block; 4. 400 Peltier device; 5 Sample inlet / outlet conduit; 6 Magnet; 101 Enrichment chamber; 102 Pyrolysis chamber; 103 First washing chamber; 104 Second washing chamber; 105 Amplification chamber; 106 Mineral oil; 107 Encapsulation reagent (aqueous phase); 201 First aluminum block; 202 Second aluminum block; 301 K-type thermocouple; 302 Resistance heating rod; 401 Positive electrode; 402 Negative electrode; 403 P / N type semiconductor; 404 Copper flow guide plate. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0025] Example 1: Construction of a full-process microfluidic chip

[0026] like Figure 1 The image shows a full-process microfluidic chip according to a preferred embodiment of the present invention, comprising: a front cover plate 1, a rear cover plate 2, a heating block 3, a Peltier device 4, a conduit 5, a magnet 6, a first aluminum block 201, and a second aluminum block 202.

[0027] The front cover 1 and the rear cover 2 are made of transparent polycarbonate material, extending parallel to each other in the vertical direction, and are bonded together by an adhesive. Figure 2 As shown, the surface of the front cover 100 facing the rear cover 2 has a length direction ( Figure 2 The five rectangular grooves arranged from left to right form an enrichment chamber 101, a thermal pyrolysis chamber 102, a first washing chamber 103, a second washing chamber 104, and a nucleic acid amplification reaction chamber 105 respectively when the front cover plate 1 and the rear cover plate 2 are fastened together. Before detection, the encapsulation reagent 107 can be filled in. All the chambers are open at the top and interconnected to store the oil phase 106 and isolate it from the external environment.

[0028] Combination Figure 2 , Figure 3As shown, the first aluminum block 201 and the second aluminum block 202 are respectively attached to the back of the rear cover plate 200 and aligned with the pyrolysis chamber 102 and the nucleic acid amplification reaction chamber 105. The first aluminum block 201 corresponds to the pyrolysis chamber 102, and its temperature can reach 75°C under the action of the heating block 300. The second aluminum block 202 corresponds to the nucleic acid amplification reaction chamber 105, and rapid heating and cooling are achieved using the Peltier device 400. The dimensions of the first aluminum block 201 and the second aluminum block 202 are slightly larger than those of the pyrolysis chamber 102 and the nucleic acid amplification reaction chamber 105, respectively.

[0029] like Figure 1 As shown, the catheter 5 includes an inlet catheter and an outlet catheter, used for sample entry and exit respectively, and is inserted into the enrichment chamber 101 from above the front cover plate 1 and the rear cover plate 2. The heating block 3 and the Peltier device 4 are respectively disposed on the back of the first aluminum block 201 and the second aluminum block 202; the magnet 6 is used to attract magnetic beads to realize the transfer of bacteria and / or nucleic acid molecules between the chambers.

[0030] like Figure 4 As shown, the heating block 300 includes a type K thermocouple 301 and a resistance heating rod 302; as Figure 5 As shown, the Peltier device 400 includes: a positive electrode 401, a negative electrode 402, a P / N type semiconductor 403, and a copper current-conducting plate 404; it should be understood that the heating block 300 and the Peltier device 400 are both prior art and can be obtained by purchase.

[0031] The collected samples are mixed with specific binding magnetic beads beforehand, and then introduced through catheter 5. Under the action of an external magnet 6, bacteria / nucleic acid can be transferred from front to back, and amplification is carried out in the last chamber. Quantitative detection can be achieved by fluorescence microscopy.

[0032] According to this embodiment, the first groove on the front cover plate 1 has a width of 1 mm, a length of 12 mm, and a depth of 16 mm, with a pre-packaged volume of 192 μL (i.e., enrichment chamber 101). The four subsequent grooves have the same dimensions: a width of 1 mm, a length of 10 mm, and a depth of 16 mm, with a pre-packaged volume of 160 μL (i.e., pyrolysis chamber 102, first washing chamber 103, second washing chamber 104, and nucleic acid amplification reaction chamber 105). The upper oil phase storage space has dimensions of 1 mm, a length of 74 mm, and a depth of 12 mm, with a pre-packaged volume of 888 μL. It should be understood that the above are theoretically packaged volumes, and the specific packaged volume depends on actual experiments. It should also be understood that this is only an example and not a limitation. The full-process microfluidic chip provided by this invention is not limited to this size and can be appropriately adjusted according to actual conditions.

[0033] Example 2: Detection of VBNC Escherichia coli in water

[0034] According to this embodiment, the detection of VBNC (Vitamin B Count) Escherichia coli in water using the full-process microfluidic chip provided in Embodiment 1 includes the following steps:

[0035] 1) Preparation:

[0036] Ultrapure water, TE buffer, washing solution 1 (70% ethanol), washing solution 2 (13% PEG-8000), and elution solution (PCR reaction reagent) are sequentially pre-encapsulated into enrichment chamber 101, pyrolysis chamber 102, first washing chamber 103, second washing chamber 104, and nucleic acid amplification reaction chamber 105. Then, mineral oil 106 is added to the top of the chambers to isolate them from interference from external factors.

[0037] 2) Accumulation of bacteria in water:

[0038] First, specific magnetic beads targeting VBNC E. coli need to be added to the collected water sample and mixed well. These magnetic beads are modified with streptavidin (available commercially) and can bind to the nucleic acid aptamers of VBNC E. coli through an amidation reaction. The modified magnetic beads can be used to enrich VBNC E. coli. After standing for 5-10 minutes, a magnet 6 is placed behind the enrichment chamber 101. The sample can be added into the enrichment chamber 101 through the conduit 5 driven by the pump. Under the action of the magnet 6, the bacteria bound to the magnetic beads are effectively enriched. The enrichment time is determined by the sample volume and flow rate.

[0039] 3) Bacterial thermal lysis:

[0040] Subsequently, under the drag of the external magnet 6, the bacteria bound to the magnetic beads will cross the upper oil phase from the enrichment chamber 101 and enter the adjacent thermal pyrolysis chamber 102. After the heating block 3 is powered on, the power is adjusted to 150W. With the help of the first aluminum block 201, heat transfer is fast, and the bacteria can be effectively lysed in 15 minutes. After the lysis is completed, the specific magnetic beads are separated from the target. Under the action of the external magnet 6, the specific magnetic beads can be transferred back to the enrichment chamber 101 or removed from the chip.

[0041] 4) Nucleic acid extraction and amplification:

[0042] Subsequently, 2 μL of nucleic acid extraction silica hydroxyl magnetic beads are added to the pyrolysis chamber 102 to adsorb the lysed nucleic acids. After standing for 5–10 min, the magnetic beads are dragged across the oil phase into the first washing chamber 103 using an external magnet 6. The mixture is stirred for 1–3 min, and this dragging is repeated three times between the pyrolysis chamber 102 and the first washing chamber 103. The same procedure is then repeated in the second washing chamber 104. Following this, under the action of the external magnet 6, the nucleic acid molecules enter the nucleic acid amplification reaction chamber 105. The magnetic beads are eluted by the amplification solution and can be returned to the previous chamber or removed from the chip. Under the rapid heating and cooling of the Peltier device 4, the nucleic acid molecules undergo in-situ PCR amplification. The fluorescence intensity can be observed under a fluorescence microscope to quantify VBNC E. coli in water.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A full-process microfluidic chip for rapid, accurate, and highly sensitive detection of water quality samples, characterized in that, include: The system includes a front cover plate, a rear cover plate, a first aluminum block, a second aluminum block, a conduit, a heating block, a Peltier device, and a magnet; the front cover plate and the rear cover plate are made of transparent polycarbonate material; wherein, The front cover plate has five rectangular grooves arranged sequentially along its length on the surface facing the rear cover plate. When the front cover plate and the rear cover plate extend parallel to each other in the vertical direction and are fastened together, they respectively form an enrichment chamber, a thermal pyrolysis chamber, a first washing chamber, a second washing chamber, and a nucleic acid amplification reaction chamber. All chambers have open tops and are interconnected, used to store the oil phase and isolate the external environment. The first aluminum block and the second aluminum block are respectively attached to the back of the rear cover plate and aligned with the thermal pyrolysis chamber and the nucleic acid amplification reaction chamber, respectively. The size of the first aluminum block and the second aluminum block is slightly larger than that of the thermal pyrolysis chamber and the nucleic acid amplification reaction chamber, respectively. The catheter is inserted into the enrichment chamber from above the front cover plate and the rear cover plate. The catheter includes an inlet catheter and an outlet catheter, which are used for sample entry and exit, respectively. A heating block and a Peltier device are respectively disposed on the back of the first aluminum block and the second aluminum block. After the heating block is powered on, it rapidly transfers heat through the first aluminum block to achieve effective lysis of bacteria in the pyrolysis chamber. The Peltier device can rapidly raise and lower the temperature to facilitate the in-situ PCR amplification of nucleic acid molecules. The magnet is used to attract magnetic beads to enable the transfer of bacteria and / or nucleic acid molecules through the oil phase at the top of the chamber between the chambers; Among them, by using oil sealing and magnetron sputtering technology, nucleic acid extraction and amplification reagents can be pre-embedded in the chip. After sampling, specifically bound magnetic beads are added, and enrichment is achieved under the action of magnetic sheets. Then, with the help of an external magnet, nucleic acid is transferred and reacted in different reagents to complete nucleic acid extraction and amplification. Finally, the quantitative detection of VBNC Escherichia coli in water samples is carried out by observing fluorescence intensity.

2. The end-to-end microfluidic chip according to claim 1, characterized in that, The front cover and the rear cover are bonded together with an adhesive.

3. The application of a full-process microfluidic chip according to any one of claims 1-2 in the detection of VBNC (Vol. VBNC) Escherichia coli in water samples.

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