CN113156114A - Portable staphylococcus detection method and device based on microfluidic paper chip - Google Patents
Portable staphylococcus detection method and device based on microfluidic paper chip Download PDFInfo
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Abstract
The invention discloses a portable staphylococcus detection method and a device based on a microfluidic paper chip in the field of biomedical detection, wherein a sample solution is controlled to flow into a culture and color development unit, then an enterotoxin A specific antibody solution on staphylococcus aureus flows into the culture and color development unit, then a PBS (phosphate buffer solution) washes the surplus primary antibody solution which is not attached in the culture and color development unit, a peroxidase anti-enterotoxin A specific antibody solution flows into the culture and color development unit, the PBS buffer solution washes the surplus secondary antibody solution which is not attached in the culture and color development unit again, a peroxide substrate OPD solution flows into the culture and color development unit, an enzyme decomposition substrate on the surface of the secondary antibody which is combined with the surface of the staphylococcus aureus generates a colored reaction product, a stop solution destroys peroxidase after the reaction, and an enzyme-linked substrate is not decomposed again; the invention adopts the microfluidic paper-based chip technology and the ELISA detection technology, and HSV analysis can obtain the concentration value after photographing the color development area by the mobile phone.
Description
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a staphylococcus aureus detection device and a method based on an enzyme-linked immunosorbent assay (ELISA) technology and a combined microfluidic paper-based chip, which can be used for quickly and manually detecting the concentration of staphylococcus aureus in any solution.
Background
Staphylococcus aureus (Staphylococcus aureus) is a common main pathogenic bacterium for inducing daily diseases of human beings and animals, belongs to a gram-positive coccus, is widely distributed in skins of human bodies and animal bodies and cavities communicated with the outside, seriously threatens food safety, can cause food poisoning of human beings when concentration exceeds standard, and is always a key object for monitoring biological pollutants in food. Staphylococcus aureus can almost pollute all foods, particularly seriously threatens meat and products thereof, and is called as 'mesophilic bacteria'; the risk of the damage to food quality safety is mainly that a large amount of invasive substances such as enterotoxin and leukocidin are generated during amplification and then enter a human body through a food chain to cause severe food poisoning. Meanwhile, staphylococcus aureus also has the properties of high salt resistance, high temperature resistance, capability of growing under extreme conditions and the like, and the food safety is greatly influenced.
Microfluidic technology is a technology that operates, controls, and detects on the micrometer scale. The functions of the whole laboratory can be integrated on a tiny chip through the integration and the miniaturization of the analytical instrument. The microfluidic paper chip is characterized in that a fluid channel network and related control and analysis devices are constructed on filter paper through various micromachining technologies, and a 'micro laboratory on paper' is established. The micro-fluidic is particularly suitable for disposable accurate analysis sensors because of high sensitivity and extremely low cost of filter paper.
The traditional culture medium colony observation method has the defects of large environmental influence factor, low concentration measurement range, easy pollution in the detection process, low detection precision and the like, and meanwhile, the serious consequence of biological pollution is very easy to occur due to improper treatment after the culture medium is used. For example, the test paper used in the Chinese patent No. ZL200520120355.0 entitled test sheet for pathogenic microorganisms in food has high cost, needs to be matched with peripheral equipment such as a constant temperature incubator and the like, obtains the concentration by a method of counting bacterial colonies manually, and is not suitable for rapid and convenient household detection. And the laboratory ELISA measurement method with extremely high precision is complex in measurement process, expensive in instrument, and required to be in a professional sterile environment, so that the requirement of daily family use of people is obviously not met. The microfluidic paper chip has great advantages in this respect, and simultaneously meets the requirements of low cost, large detection range and high precision.
Disclosure of Invention
The invention aims to provide a portable staphylococcus detection method and device based on a microfluidic paper chip aiming at the detection requirement of the concentration of staphylococcus aureus in household food, and meets the requirements of low cost, wide detection range and high precision of daily household use of people.
The invention relates to a portable staphylococcus detection method based on a microfluidic paper chip, which adopts the technical scheme that the method comprises the following steps:
step 1) adding a sample solution to be detected into a sample adding hole, controlling an electrowetting valve of a channel corresponding to a sample introduction unit to open by an MCU module, enabling the sample solution to flow into a culture and color development unit, then opening an electrowetting valve of a channel corresponding to an anti-storage unit, enabling an enterotoxin A specific antibody solution on staphylococcus aureus to flow into the culture and color development unit, and enabling the enterotoxin A specific antibody solution to be combined with an enterotoxin A specific site on the staphylococcus aureus;
step 2) opening electrowetting valves of channels corresponding to the flushing fluid storage unit and the waste liquid storage unit, enabling the PBS buffer solution to flow through the culture and color development unit and be retained in the waste liquid storage unit, and flushing redundant primary antibody solution which is not attached in the culture and color development unit; after flushing, closing the electrowetting valves of the channels corresponding to the flushing liquid storage unit and the waste liquid storage unit;
step 3) opening an electrowetting valve of a channel corresponding to the secondary antibody storage unit, and closing the electrowetting valve after the peroxidase anti-enterotoxin A specific antibody solution flows into the culture and color development unit, so that the peroxidase anti-enterotoxin A specific antibody solution is combined with the anti-enterotoxin A specific antibody specific site;
step 4) opening the electrowetting valves of the channels corresponding to the flushing fluid storage unit and the waste liquid storage unit again, enabling the PBS buffer solution to flow through the culture and storage unit and be retained in the waste liquid storage unit, flushing the surplus secondary antibody solution which is not attached in the culture and color development unit by using the PBS buffer solution, and closing the electrowetting valves of the channels corresponding to the flushing fluid storage unit and the waste liquid storage unit after flushing;
step 4) opening an electrowetting valve on a channel corresponding to the enzyme-linked substrate storage unit, closing the electrowetting valve after a peroxide substrate OPD solution flows into the culture and color development unit, decomposing a substrate by enzyme on the surface of a secondary antibody bound to the surface of staphylococcus aureus to generate a colored reaction product, opening the electrowetting valve on a channel corresponding to the stop solution storage unit after reaction, closing the corresponding electrowetting valve after the stop solution flows into the culture and color development unit, destroying peroxidase by the stop solution, not decomposing the enzyme-linked substrate, and stabilizing color development;
step 5): and (4) photographing the reaction and color development unit, analyzing HSV color, and comparing the HSV color with pre-stored color data to obtain the concentration of staphylococcus aureus in the sample solution.
The invention relates to a portable staphylococcus detection device based on a microfluidic paper chip, which adopts the technical scheme that: the bottom layer of the device is a circuit layer, a paper base layer is arranged right above the circuit layer, the upper surface of the paper base layer is covered with a plastic sealing film, a sample adding hole is formed in the plastic sealing film, seven sample storage units, namely a sample introduction unit, a culture and color development unit, an anti-storage unit, a secondary antibody storage unit, a flushing fluid storage unit, a waste liquid storage unit, a stop solution storage unit, an enzyme-linked substrate storage unit and a bacterial lysate storage unit, are arranged on the paper base layer, the seven sample storage units are radially distributed around the culture and color development unit, the sample introduction unit, each sample storage unit and the culture and color development unit are connected through a corresponding micro-channel, each micro-channel is connected with an electrowetting valve, and a hydrophilic electrode and a corresponding hydrophobic electrode form the electrowetting valve; the sample introduction unit is arranged right below the sample addition hole and communicated with the sample addition hole, the primary antibody storage unit stores an enterotoxin A specific antibody solution on staphylococcus aureus, the secondary antibody storage unit stores a peroxidase anti-enterotoxin A specific antibody solution, the flushing fluid storage unit stores a PBS buffer solution, the stop solution storage unit stores a stop solution, the enzyme-linked substrate storage unit stores a peroxide substrate OPD solution, the bacteria lysate storage unit stores a RIPA cell lysate, and the culture and color development unit stores a broth culture medium liquid with NACL concentration of 7.5%; and the circuit layer is provided with an MCU module connected with each electrowetting valve to control the opening and closing of each electrowetting valve.
Compared with the prior method and technology, the invention has the following advantages:
1. the invention adopts the microfluidic paper-based chip technology combined with the ELISA detection technology, solves the problems of incapability of handheld measurement and poor detection precision, has low detection optical background, high precision, wide measurable linear range, strong anti-interference performance and low cost, and can obtain the concentration value by HSV analysis after photographing the color development area by a mobile phone.
2. The detection device can realize the detection of different biological samples by modifying the control time in the program or replacing the samples, and has strong expandability.
3. The invention does not need to be matched with a specific detection instrument and is controlled by an embedded MCU (microprogrammed control unit), no artificial interference is needed after sample adding, full-automatic detection and sample introduction are realized, the sample flowing and temperature control are automatically controlled, no artificial external operation is needed, and the operation is simple.
4. After the concentration monitoring is finished, the cell lysate channel is automatically opened, and the cell lysate is added to kill staphylococcus aureus in all channels, so that biological pollution caused by the traditional colony observation method is effectively prevented.
5. The electrodes in the paper base layer and the circuit layer adopt a special connection mode, so that the phenomena of short circuit caused by poor conductivity and control time sequence error caused by wetting of the paper chip are effectively prevented.
6. The special groove structure of the circuit layer can effectively realize the functions of water resistance and uniform heating, and the phenomenon of nonuniform heating in reaction in the traditional heating method is improved.
7. The composite structure of the paper base layer and the copper foil-coated epoxy glass cloth laminated board effectively solves the defect that a microfluidic paper base chip is soft and easy to damage.
8. The USB is adopted for integrated power supply, so that the power supply is convenient, and the use of a battery in a traditional paper chip is replaced in the aspect of the method, so that the environmental protection is facilitated.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a portable staphylococcus aureus detection device based on a combined microfluidic paper-based chip
FIG. 2 is a schematic view of the construction of the paper substrate 2 of FIG. 1;
FIG. 3 is an enlarged sectional view of the hydrophilic electrode 15 shown in FIG. 2;
FIG. 4 is a schematic diagram of the structure of the circuit layer 3 in FIG. 1;
FIG. 5 is a control schematic block diagram of the detection apparatus shown in FIG. 1;
fig. 6 is a control flowchart of the detection apparatus shown in fig. 1.
The serial numbers and designations of the various components in the drawings: 1: plastic sealing film; 2: a paper substrate layer; 3: a circuit layer; 4: a sample application hole; 5: a USB interface; 6: a sample introduction unit; 7: a resistive memory cell; 8: a secondary antibody storage unit; 9: a washing liquid storage unit; 10: a waste liquid area storage unit; 11: a stop solution storage unit; 12: an enzyme-linked substrate storage unit; 13: a bacteria lysate storage unit; 14: a culture and color development unit; 15: a hydrophilic electrode; 16: a hydrophobic electrode recess; 17: a pin header insertion hole; 18: a microchannel; 19: arranging needles; 20: an electrode filler; 21 a circuit layer substrate; 22: and a circuit groove.
Detailed Description
Referring to fig. 1, the portable staphylococcus aureus detection device based on the composite microfluidic paper-based chip is divided into three layers, namely a plastic sealing film 1, a paper base layer 2 and a circuit layer 3, wherein the circuit layer 3 is arranged at the bottommost layer and is made of a copper-clad epoxy glass cloth laminated board; the middle layer is a paper base layer 2, the paper base layer 2 is made of filter paper, and a thin plastic sealing film 1 is covered on the upper surface of the paper base layer 2 and is the plastic sealing film 1 on the uppermost layer. Wherein, the plastic sealing film 1 is provided with a sample adding hole 4, and a sample to be detected can be added for detection. And a USB interface 5 is arranged on the side surface of the circuit layer 3 and is used for connecting a mobile phone or other rechargeable equipment.
With reference to the paper substrate 2 shown in fig. 2, the paper substrate 2 is provided with a sample injection unit 6, a culture and color development unit 14, seven sample storage units, eight hydrophilic electrodes 15 and eight hydrophobic electrodes 16. The seven sample storage units are respectively a primary antibody storage unit 7, a secondary antibody storage unit 8, a flushing liquid storage unit 9, a waste liquid storage unit 10, a stop solution storage unit 11, an enzyme-linked substrate storage unit 12 and a bacteria lysate storage unit 13. The sample introduction unit 6, the culture and color development unit 14 and the seven sample storage units are all groove structures, and one hydrophilic electrode 15 and a corresponding hydrophobic electrode 16 form an electrowetting valve. The sample introduction unit 6 is arranged right below the sample introduction hole 4 and is communicated with the sample introduction hole 4, the sample introduction hole 4 is slightly smaller than the sample introduction unit 6, and sample liquid to be detected can be introduced into the sample introduction unit 6 through the sample introduction hole 4. The culture and color development unit 14 is arranged in the middle of the paper substrate 2, and seven sample storage units are arranged around the culture and color development unit 14 and are radially distributed by taking the culture and color development unit 14 as the center. The sample introduction unit 6 and each sample storage unit are connected with the culture and color development unit 14 through a corresponding micro-channel 18, so that the total number of the micro-channels 18 is eight, and all the micro-channels 18 have the same depth. One electrowetting valve is mounted on each microchannel 18, so that the structure of eight electrowetting valves is identical in total. The depth of the incubation and visualization unit 14 is slightly lower than the depth of the microchannel 18.
The sample introduction unit 6 is connected with the culture and color development unit 14 through a corresponding micro-channel 18, the micro-channel 18 is connected with a corresponding hydrophilic electrode 15 and a corresponding hydrophobic electrode 16, and the depth of the sample introduction unit 6 is the same as that of the micro-channel 18. The primary antibody memory unit 7 passes through the corresponding micro-channel 18 and is connected with the culture and color development unit 14 through the corresponding hydrophilic electrode 15 and hydrophobic electrode 16, and the depth of the primary antibody memory unit 7 is the same as that of the micro-channel 18. The secondary antibody memory unit 8 is connected with the culture and color development unit 14 through the corresponding micro-channel 18 and passing through the corresponding hydrophilic electrode 15 and hydrophobic electrode 16, and the depth of the secondary antibody memory unit 8 is the same as that of the micro-channel 18. The washing liquid storage unit 9 is connected to the incubation and color development unit 14 through a corresponding microchannel 18 and corresponding hydrophilic electrode 15 and hydrophobic electrode 16, and the depth of the washing liquid storage unit 9 is the same as that of the microchannel 18. The waste liquid storage unit 10 is connected with the culture and color development unit 14 through the corresponding micro-channel 18 and the corresponding hydrophilic electrode 15 and hydrophobic electrode 16, and the depth of the waste liquid storage unit 10 is slightly deeper than that of the micro-channel 18, so that the flushing waste liquid can be effectively retained in the waste liquid storage unit 10, and the waste liquid is effectively prevented from flowing backwards into the culture and color development unit 14. The stop solution storage unit 11 is connected with the culture and development unit 14 through a corresponding micro-channel 18 and a corresponding hydrophilic electrode 15 and hydrophobic electrode 16, and the depth of the stop solution storage unit 11 is the same as that of the micro-channel 18. The enzyme-linked substrate storage unit 12 is connected with the culture and color development unit 14 through a corresponding micro-channel 18 and a corresponding hydrophilic electrode 15 and a hydrophobic electrode 16, and the depth of the micro-channel 18 of the enzyme-linked substrate storage unit 12 is the same. The bacterial lysate storage unit 13 is connected with the culture and color development unit 14 through a corresponding micro-channel 18 and corresponding hydrophilic electrodes 15 and hydrophobic electrodes 16.
Before use, the sample injection unit 6 is not filled with any liquid, and the liquid to be detected can be dripped into the sample area 6 through the sample injection hole 4. The primary antibody storage unit 7 stores enterotoxin A specific antibody solution on staphylococcus aureus, the secondary antibody storage unit 8 stores peroxidase anti-enterotoxin A specific antibody solution, the washing liquid storage unit 9 stores PBS buffer solution, the stop solution storage unit 11 stores stop solution, the enzyme-linked substrate storage unit 12 stores peroxide substrate OPD solution, and the bacteria lysate storage unit 13 stores RIPA cell lysate. The culture and development unit 14 stores broth culture liquid with NACL concentration of 7.5%. Under the high salt of NACL concentration of 7.5%, the staphylococcus aureus can still better reproduce, so that the specific amplification culture can be carried out by the method.
Referring to fig. 2 and 3, the hydrophilic electrode 15 and the hydrophobic electrode 16 have the same structure, and the hydrophilic electrode 15 is taken as an example. The hydrophilic electrode 15 and the hydrophobic electrode 16 are formed by a groove formed in the paper base layer 2 and an electrode filler 20 filled in the groove, and each of the hydrophilic electrode 15 and the hydrophobic electrode 16 is provided with a pin header insertion through hole 17, and the pin header insertion through hole 17 faces vertically downward. The lower circuit layer 3 projects upward with pins 19, and each pin 19 is inserted into a corresponding one of the pin insertion through holes 17. The filling material of the electrode filler 20 of the hydrophilic electrode 15 is conductive silver paste mixed with a hydrophilic material, that is, conductive silver paste mixed with deoxidized anhydrous ethanol, and the filling material of the electrode filler 20 of the hydrophobic electrode 16 is conductive silver paste mixed with a hydrophobic material, that is, conductive silver paste mixed with perfluorodecyl mercaptan (PFDT). Electrode fill 20 is filled to be flush with the inner walls of microchannel 18 and to fit laterally into microchannel 18. The electrode fill 20 should fill as much of the pin insertion opening 17 as possible to prevent poor circuit contact with the underlying circuit layer 3, while the electrode fill 20 should be kept from being over-saturated so as to block the micro-channel 18 and pin insertion opening 17. After the conductive silver paste is filled and cured, the voltage of the hydrophilic electrode 15 and the hydrophobic electrode 16 can be controlled by means of the conductivity of the conductive silver paste.
Referring to fig. 1 and 4, a circuit layer substrate 21 is disposed on the bottom surface of the circuit layer 3, a circuit groove 22 is formed on the upper surface of the circuit layer substrate 21, a PCB integrated circuit is constructed in the circuit groove 22 by a series of PCB processing processes such as screen printing, and a corresponding circuit module is soldered. The circuit layer substrate 21 is made of a copper clad epoxy glass cloth laminated board, and other gaps of the circuit groove 22 are filled with heat conduction and insulation materials (heat conduction gel), so that the measurement accuracy problem caused by nonuniform heating of the chip and the measurement problem that the chip cannot be applied to a special temperature environment are solved. Wherein the height of the pin header 19 in the circuit groove 22 should be slightly higher than the height of the upper surface of the circuit layer substrate 21 so as to be able to be inserted upwards into the corresponding pin header insertion hole 17, and also play a role of fixing the relative position of the paper base layer 2 and the circuit layer 3. The side of the circuit layer substrate 21 is provided with a USB interface 5 for external connection.
Referring to fig. 1, 2 and 4, a layer of waterproof material is sprayed on the lower surface of the paper base layer 2, a thin layer of adhesive material, namely silica gel, is brushed on the upper surface of the circuit layer 3, and the paper base layer 2 and the circuit layer 3 are integrated through hot pressing. The press-fit chip is a composite structure of the paper base layer 2 and the copper foil-coated epoxy glass cloth laminated board, and the strength of the copper foil-coated epoxy glass cloth laminated board is high, so that the composite structure well overcomes the defect that the paper chip is soft and easy to damage. And adding corresponding sample liquid into all the sample storage units, and sealing the surface by using a transparent plastic sealing film 1 to leave sample adding holes 4. The sample storage unit 14 can be photographed by an upper computer, and the concentration value can be obtained by HSV color analysis.
Referring to fig. 4 and 5, the MCU module, the temperature monitoring module, the heating module, the power module and the USB interface 5 are disposed in the circuit recess 22. The power supply module is connected with the outside through a USB interface 5 and supplies power to the MCU module and other modules. The MCU module is respectively connected with the temperature monitoring module, the heating module, the power supply module, the hydrophilic electrode 15 and the hydrophobic electrode 16 and controls the opening and closing of each electrowetting valve. The temperature monitoring module provides real-time chip temperature data to the MCU module, the MCU module reads data and analyzes whether the current temperature is a preset value or not, and when the temperature is lower than the preset temperature, the MCU module controls the heating module to work and heat the chip. Wherein, the heating module selects four heating resistors to be distributed around the circuit groove 22 so as to supply heat uniformly. Meanwhile, the MCU module is connected with the paper base layer 2 through the pin 19, the voltage of the hydrophilic electrode 15 and the voltage of the hydrophobic electrode 16 are controlled, when the voltage of the hydrophilic electrode 15 and the voltage of the hydrophobic electrode 16 controlled by the MCU module are both 0V, the micro channel 18 is disconnected, liquid cannot pass through the hydrophobic electrode 16, when the MCU module outputs high level to the hydrophilic electrode 15 and outputs low level to the hydrophobic electrode 16, the hydrophobic electrode 16 is hydrophilized, the micro channel 18 is opened, and liquid freely flows through the hydrophobic electrode 16.
Referring to fig. 1-5 and fig. 6, when the detection device of the present invention works, after the USB interface 5, the MCU starts to work, the electrowetting valves of the channels are sequentially opened according to a time sequence, when the potential difference between the hydrophilic electrode 15 and the hydrophobic electrode 16 is 0, the micro channel 18 is closed, the liquid cannot pass through the hydrophobic electrode 16, when the potential difference between the hydrophilic electrode 15 and the hydrophobic electrode 16 is 5V, the hydrophobic electrode 16 is hydrophilized, the micro channel 18 is opened, the liquid freely flows through the hydrophobic electrode 16, and the MCU automatically controls the reaction temperature, the channel on-off and the reaction time in the whole process. The specific detection steps are as follows:
the method comprises the following steps: the sample liquid to be measured is manually added into the sample adding hole 4 until the sample adding unit 6 is fully dripped, and the sample area needs to be statically filled, otherwise, the measurement precision is influenced. The USB interface 5 is manually inserted to start power supply, the temperature monitoring module automatically detects the current temperature value and transmits the temperature value to the MCU module, the MCU module compares the current temperature value with the ideal cell amplification temperature of 45 degrees and controls the heating module to start working, the MCU module outputs high level to enable the heating module resistor to generate heat and adjust the temperature to 45 degrees, and at the moment, all liquids can freely flow in the circular storage area but cannot pass through the corresponding hydrophobic electrode. When the sample is heated to 45 ℃, the voltage of the hydrophobic electrode 16 corresponding to the sample introduction unit 6 is controlled to be 5V by the MCU module, at the moment, the hydrophobic electrode 16 is changed into a hydrophilic state, the sample liquid smoothly flows through the hydrophobic electrode 16 and flows into the culture and color development unit 14, then the voltage of the hydrophobic electrode 16 is controlled to be restored to be 0V by the MCU module, and the hydrophobic electrode 16 is restored to be in a hydrophobic state. In the subsequent steps, the working method of the electrowetting valve is the same when opening and closing the microchannel 18, and the description is omitted.
Bacteria in the sample solution flowing into the culture and color development unit 14 are amplified, and since the sodium chloride concentration of the culture and color development unit 14 is 7.5% in a high-salt environment, the temperature is 45 ℃ and the temperature is higher, no large amount of common strains can be amplified in the environment, and staphylococcus aureus can propagate in the environment in a large amount and attach to the bottom of the culture and color development unit 14. The MCU module carries out timing culture, after the set time (30 min), an electrowetting valve of a channel corresponding to the primary anti-storage unit 7 is opened, and the enterotoxin A specific antibody solution on staphylococcus aureus in the primary anti-storage unit 7 flows into the culture and color development unit 14. The enterotoxin a-specific antibody solution was allowed to bind to the enterotoxin a-specific site on staphylococcus aureus. The MCU module is used for timing, after the set time (2 min), the electrowetting valves of the channels corresponding to the washing liquid storage unit 9 and the waste liquid storage unit 10 are opened, the PBS buffer liquid pre-stored in the washing liquid storage unit 9 flows through the micro-channel 18 and the hydrophobic electrode 16 under the capillary action of paper, flows through the culture and color development unit 14 and flows to the waste liquid storage unit 10, the depth of the waste liquid storage unit 10 is lower than that of the micro-channel 18, so that the waste liquid is retained in the waste liquid storage unit 10, and the PBS buffer liquid can wash the surplus primary antibody solution which is not attached in the culture and color development unit 14. And after the set time (2 min) of flushing, closing the electrowetting valves of the channels corresponding to the flushing liquid storage unit 9 and the waste liquid storage unit 10. The MCU module controls to open an electrowetting valve of a channel corresponding to the secondary antibody storage unit 8, peroxidase anti-enterotoxin A specific antibody solution in the secondary antibody storage unit 8 flows into the culture and color development unit 14, and then the valve is closed, so that the peroxidase anti-enterotoxin A specific antibody solution is combined with anti-enterotoxin A specific antibody specific sites. Then the MCU module opens the channel electrowetting valves corresponding to the washing liquid storage unit 9 and the waste liquid storage unit 10 again, after the set time (2 min) is timed, the PBS buffer liquid pre-stored in the washing liquid storage unit 9 flows through the micro channel 18 and the hydrophobic electrode 16 under the capillary action of paper, flows through the culture and storage unit 14 and flows to the waste liquid storage unit 10. And (3) washing the excess secondary antibody solution which is not attached in the culture and color development unit 14 by using the PBS buffer solution, closing electrowetting valves of channels corresponding to the washing liquid storage unit 9 and the waste liquid storage unit 10 after washing for a set time (2 min), and completing enzyme attachment.
Step two: an electrowetting valve on the channel of the enzyme-linked substrate storage unit 12 is opened, and the valve is closed after the peroxide substrate OPD solution in the enzyme-linked substrate storage unit 12 flows into the culture and color development unit 14. The enzyme on the surface of the secondary antibody combined with the surface of the staphylococcus aureus in the culture and color development unit 14 decomposes a substrate to generate a colored reaction product, the MCU module controls the reaction to reach a set time (5 min), then an electrowetting valve on a channel of the stop solution storage unit 11 is opened, the stop solution in the enzyme-linked substrate storage unit 12 flows into the culture and color development unit 14 and then closes the valve, the stop solution destroys peroxidase, the enzyme-linked substrate is not decomposed, and the color development is stable.
Step three: manually taking a picture of the reaction and color development unit 14 by a mobile phone, analyzing HSV color, and comparing the HSV color with prestored color data to obtain the concentration of staphylococcus aureus in the sample liquid. Then, the MCU module controls to open all the electrowetting valves, so that the RIPA cell lysate in the bacteria lysate storage unit 13, other sample storage units and liquid in the micro-channel 18 are fully mixed, staphylococcus aureus in the micro-channel 18 and each sample storage unit is killed, and pollution is avoided.
Claims (10)
1. The utility model provides a portable staphylococcus detection device based on micro-fluidic paper chip, bottommost layer are circuit layer (3), are paper substrate (2) directly over circuit layer (3), and paper substrate (2) upper surface covers plastics and seals membrane (1), and it has one application hole (4), characterized by to open on plastics seal membrane (1): the paper base layer (2) is provided with seven sample storage units, namely a sample introduction unit (6), a culture and color development unit (14), a first anti-storage unit (7), a second anti-storage unit (8), a flushing fluid storage unit (9), a waste liquid storage unit (10), a stop solution storage unit (11), an enzyme-linked substrate storage unit (12) and a bacteria lysate storage unit (13), wherein the seven sample storage units are radially distributed around the culture and color development unit (14), the sample introduction unit (6), each sample storage unit and the culture and color development unit (14) are connected through a corresponding microchannel (18), each microchannel (18) is connected with an electrowetting valve, and a hydrophilic electrode (15) and a corresponding hydrophobic electrode (16) form the electrowetting valve; the sample introduction unit (6) is arranged right below the sample addition hole (4) and communicated with the sample addition hole (4), a primary antibody storage unit (7) stores an enterotoxin A specific antibody solution on staphylococcus aureus, a secondary antibody storage unit (8) stores a peroxidase anti-enterotoxin A specific antibody solution, a flushing fluid storage unit (9) stores a PBS buffer solution, a stop solution storage unit (11) stores a stop solution, an enzyme-linked substrate storage unit (12) stores a peroxide substrate OPD solution, a bacteria lysate storage unit (13) stores a RIPA cell lysate, and a culture and color development unit (14) stores a broth culture medium liquid with NACL concentration of 7.5%; and the circuit layer (3) is provided with an MCU module connected with each electrowetting valve to control the opening and closing of each electrowetting valve.
2. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 1, wherein the portable staphylococcus detecting device comprises: the paper base layer (2) is provided with a groove and electrode fillers (20) filled in the groove to form a hydrophilic electrode (15) and a hydrophobic electrode (16), each hydrophilic electrode (15) and each hydrophobic electrode (16) are provided with a pin row insertion through hole (17) which is vertical downwards, the circuit layer (3) protrudes upwards to form a pin row (19), and one pin row (19) is inserted into the corresponding pin row insertion through hole (17).
3. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 2, wherein the portable staphylococcus detecting device comprises: the electrode filler 20 of the hydrophilic electrode 15 is conductive silver paste mixed with deoxidized anhydrous ethanol, and the electrode filler 20 of the hydrophobic electrode 16 is conductive silver paste mixed with perfluorodecyl mercaptan.
4. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 2, wherein the portable staphylococcus detecting device comprises: the electrode filling (20) is flush with the inner wall of the microchannel (18).
5. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 2, wherein the portable staphylococcus detecting device comprises: the bottom surface of the circuit layer (3) is a circuit layer substrate (21), the circuit layer substrate (21) adopts a copper-clad epoxy glass cloth laminated board, the upper surface of the circuit layer substrate (21) is provided with a circuit groove (22), an MCU module, a temperature monitoring module, a heating module, a power supply module and a USB interface (5) are arranged in the circuit groove (22), the power supply module is connected with the outside through the USB interface (5), and the MCU module is respectively connected with the temperature monitoring module, the heating module, the power supply module, a hydrophilic electrode (15) and a hydrophobic electrode (16).
6. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 1, wherein the portable staphylococcus detecting device comprises: the sample introduction unit (6), the culture and color development unit (14) and the seven sample storage units are all groove structures, the depths of the sample introduction unit (6), the primary antibody storage unit (7), the secondary antibody storage unit (8), the flushing liquid storage unit (9), the stop solution storage unit (11) and the enzyme-linked substrate storage unit (12) are the same as the depth of the micro-channel (18), and the depths of the culture and color development unit (14) and the waste liquid storage unit (10) are lower than the depth of the micro-channel (18).
7. The portable staphylococcus detecting device based on the microfluidic paper chip as claimed in claim 1, wherein the portable staphylococcus detecting device comprises: the lower surface of the paper base layer (2) is sprayed with a layer of waterproof material, the upper surface of the circuit layer (3) is brushed with a layer of bonding material, and the paper base layer (2) and the circuit layer (3) are integrated by hot pressing.
8. The detection method of the portable staphylococcus detection device based on the microfluidic paper chip as claimed in claim 1, wherein the detection method comprises the following steps:
step 1) adding a sample solution to be detected into a sample adding hole (4), controlling an electrowetting valve of a channel corresponding to a sample introduction unit (6) to open by an MCU module, enabling the sample solution to flow into a culture and color development unit (14), then opening an electrowetting valve of a channel corresponding to an anti-storage unit (7), enabling an enterotoxin A specific antibody solution on staphylococcus aureus to flow into the culture and color development unit (14), and enabling the enterotoxin A specific antibody solution to be combined with an enterotoxin A specific site on staphylococcus aureus;
step 2) opening electrowetting valves of channels corresponding to the flushing liquid storage unit (9) and the waste liquid storage unit (10), enabling a PBS (phosphate buffer solution) to flow through the culture and color development unit (14) and stay in the waste liquid storage unit (10), and flushing surplus primary antibody solution which is not attached in the culture and color development unit (14); after flushing, closing the electrowetting valves of the channels corresponding to the flushing liquid storage unit (9) and the waste liquid storage unit (10);
step 3) opening an electrowetting valve of a channel corresponding to the secondary antibody storage unit (8), and closing the electrowetting valve after the peroxidase anti-enterotoxin A specific antibody solution flows into the culture and color development unit (14), so that the peroxidase anti-enterotoxin A specific antibody solution is combined with the anti-enterotoxin A specific antibody specific site;
step 4), opening the electrowetting valves of the channels corresponding to the flushing liquid storage unit (9) and the waste liquid storage unit (10) again, enabling the PBS buffer solution to flow through the culture and storage unit (14) and be retained in the waste liquid storage unit (10), flushing the residual secondary antibody solution which is not attached in the culture and color development unit (14) by the PBS buffer solution, and closing the electrowetting valves of the channels corresponding to the flushing liquid storage unit (9) and the waste liquid storage unit (10) after flushing;
step 4) opening an electrowetting valve on a channel corresponding to the enzyme-linked substrate storage unit (12), closing the electrowetting valve after a peroxide substrate OPD solution flows into the culture and color development unit (14), decomposing a substrate by enzyme on the surface of a secondary antibody bound to the surface of staphylococcus aureus to generate a colored reaction product, opening the electrowetting valve on a channel corresponding to the stop solution storage unit (11) after reaction, closing the corresponding electrowetting valve after the stop solution flows into the culture and color development unit (14), destroying peroxidase by the stop solution, not decomposing the enzyme-linked substrate, and stabilizing color development;
step 5): and (3) photographing the reaction and color development unit (14), analyzing HSV color, and comparing the HSV color with pre-stored color data to obtain the concentration of staphylococcus aureus in the sample solution.
9. The detection method according to claim 8, wherein: and 5) opening all electrowetting valves to fully mix RIPA cell lysate in the bacteria lysate storage unit (13) with other sample storage units and liquid in the micro-channel (18) so as to kill staphylococcus aureus in the micro-channel (18) and each sample storage unit.
10. The detection method according to claim 8, wherein: and the MCU module controls the potential difference between the hydrophilic electrode (15) and the hydrophobic electrode (16) to be 0, the micro channel (18) is closed, and when the potential difference between the hydrophilic electrode (15) and the hydrophobic electrode (16) is 5V, the micro channel (18) is opened.
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