CN111879922A - Integrated paper-based chip structure suitable for synchronous detection of nucleic acid and immunity and manufacturing method thereof - Google Patents
Integrated paper-based chip structure suitable for synchronous detection of nucleic acid and immunity and manufacturing method thereof Download PDFInfo
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Abstract
The integrated paper-based chip structure is suitable for the integrated paper-based chip structure for synchronously detecting nucleic acid and immunity, and the integrated paper-based chip sequentially comprises a paper micro-fluidic chip, a thermal resistance flexible heating sheet and a metal substrate from top to bottom. The flexible heating sheet of the thermal resistor and the paper chip as well as the heating sheet and the substrate can be bonded by double-sided adhesive tapes. The paper chip comprises at least 1 sample solution introducing part, a solution conveying flow channel and a separately arranged immunity and nucleic acid detection unit. After being introduced, the sample flows into the nucleic acid and the immunity detection unit through the solution transportation flow channel. The flexible heating sheet of the thermal resistor adopts a flexible material as a substrate, and at least 1 thermal resistor heating unit powered by a battery is integrated on the substrate through a micro-nano processing technology. The heating unit is only positioned right below the paper chip nucleic acid detection unit and is intended to provide the required temperature for nucleic acid reaction. The design can keep the paper chip at a relatively low temperature in the area except the nucleic acid detection unit so as to synchronously carry out immune reaction.
Description
Technical Field
The invention relates to the field of integrated paper-based chips. In particular to an integrated paper-based chip suitable for synchronously detecting nucleic acid and immunity and a manufacturing method thereof.
Background
The microfluidic chip laboratory can realize flexible combination and scale integration of various unit technologies on a tiny controllable platform, has the advantages of simple operation, low cost and the like, and can be used as important supplement of instrument methods. Since the early birth of the 90 s of the last century, microfluidic chips developed by groups of various countries have been successfully applied to detection of SARS, H1N1, MERS, HIV, hepatitis and Ebola virus and diagnosis and treatment of related serious infectious diseases. Paper-based microfluidic chips were originally proposed by scholars such as g.whitesides, harvard university, usa in 2007, and generally use conventional filter paper as a matrix material to realize self-driving of fluid by virtue of a capillary fiber structure inside the matrix material. Nucleic acid detection chips and immunoassay chips based on paper microfluidic technology have been developed separately. The chip comprises a paper-based polymerase chain reaction chip, a paper-based loop-mediated isothermal nucleic acid amplification chip and a paper-based immunodetection chip suitable for various infectious diseases. The two detection methods have respective advantages and disadvantages. Nucleic acid detection, which is a diagnostic method by detecting the genetic material nucleic acid of a virus, is a gold standard for infectious disease diagnosis, but has high nucleic acid detection sensitivity, extremely high requirements for sample collection and laboratories, technical level requirements of laboratory staff, and long detection time. Immunodetection detects infection by detecting viral proteins (antigens) in the body or antibodies specific for viral proteins in the body by the principle of specific reaction between antibody antigens, but antibody detection may not be a screening tool for early asymptomatic contacts.
In the COVID-19 epidemic situation, the nucleic acid detection and the immunoassay are directly applied to the rapid detection of the virus. However, in the prior art, the virus nucleic acid detection kit has extremely high omission factor, three or four samples are usually collected for accurate diagnosis, so that the infection risk of medical workers is easily increased, and the immunoassay is not suitable for being used as an independent screening index of the novel coronavirus. Therefore, the most recent treatment schemes consider that immunoassay can be used as a supplement to nucleic acid detection, and particularly, the immunoassay has a great auxiliary diagnosis effect on suspected cases which show similar clinical symptoms and are negative in nucleic acid detection.
Disclosure of Invention
To solve the above existing problems. The invention provides an integrated paper-based chip suitable for synchronous detection of nucleic acid and immunity and a manufacturing method thereof, wherein the integrated chip uses a micro-nano processing technology to manufacture a thermal resistor (MEMS) flexible heating sheet, and heats the local part (namely a nucleic acid detection unit area) of the paper-based chip to enable the paper-based chip to reach the temperature condition (constant temperature of 65 ℃) required by loop-mediated isothermal nucleic acid amplification. And the paper chip immunodetection unit area keeps room temperature due to the good heat conductivity of the metal substrate, and immune reaction is synchronously carried out. The invention aims to provide a portable nucleic acid and immune synchronous detection chip, provides a new method for detecting infectious diseases in China, and supports virus detection in areas lacking medical equipment. Meanwhile, the method can be used as a technology for diagnosing, treating, preventing, controlling and storing potential serious public health diseases in the future. To achieve this object:
the invention provides an integrated paper-based chip suitable for nucleic acid and immune synchronous detection, which comprises a paper chip, a thermal resistor flexible heating sheet and a hole digging metal substrate, wherein the integrated paper-based chip sequentially comprises the paper chip, the thermal resistor flexible heating sheet and the hole digging metal substrate from top to bottom, the thermal resistor flexible heating sheet and the paper chip are bonded by double faced adhesive tapes, the thermal resistor flexible heating sheet is placed on the hole digging metal substrate, paper-based material selection qualitative filter paper of the paper chip comprises at least 1 sample solution introducing part, a solution transportation flow channel and separately arranged detection units, the separately arranged detection units comprise an immune detection unit and a nucleic acid detection unit, the sample solution introducing part is connected with the separately arranged detection units through the solution transportation flow channel, the thermal resistor flexible heating sheet adopts a flexible material as a substrate, and the metal heating unit is arranged on the upper part of the substrate of the thermal resistor flexible heating sheet, electrodes are arranged on two sides of the heating unit and powered by a power supply, the heating unit is positioned under the nucleic acid detection unit of the paper chip and is intended to provide required temperature for nucleic acid reaction, the metal substrate adopts a hole digging design, so that the heating unit is positioned right above the hole, the circle center of the nucleic acid detection unit of the paper chip is positioned on a straight line perpendicular to the plane of the substrate, and the center of the heating unit and the circle center of the hole digging metal substrate are positioned on the straight line.
As a further improvement of the integrated paper-based chip, the paper-based material of the paper chip is Whatman Grade 1 qualitative filter paper, and the paper-based material can also be other types of qualitative filter paper according to actual conditions.
As a further improvement of the integrated paper-based chip, the flexible heating sheet of the thermal resistor adopts polyimide as a substrate, and the substrate can also adopt other materials.
As a further improvement of the integrated paper-based chip, the parts of the heating unit, including the electrodes and the heating wires, adopt gold and semiconductors as materials, and the heating unit can also adopt other metal or semiconductor materials.
The invention provides a manufacturing method of an integrated paper-based chip suitable for synchronous detection of nucleic acid and immunity, which comprises the following specific steps;
step 1: temporarily attaching a polyimide film to a silicon wafer;
preparing a monocrystalline silicon wafer with the diameter of 100mm, thoroughly cleaning the wafer, then preparing 4mL of polydimethylsiloxane solution PDMS, wherein the ratio of PDMS solution base to curing agent is 10:1, placing the liquid PDMS in a vacuum cavity after the proportioning is finished, decompressing and removing bubbles, next, uniformly coating the PDMS on the silicon wafer by using a spin coater to serve as an adhesive, attaching a polyimide film to the silicon wafer by using the adhesive, finally, placing the silicon wafer in an oven, and baking the silicon wafer overnight at 80 ℃;
step 2: photoetching and patterning to determine the position of the electrode and form a metal thermal resistance structure;
after the polyimide film is thoroughly cleaned, the whole wafer is coated with positive photoresist in a spinning mode, the positive photoresist is used in the subsequent steps, after photoetching and patterning, the metal resistance wire can be evaporated on the polyimide film by thermal evaporation, and the residual photoresist is removed by a Lift-off method;
and step 3: mechanically stripping the flexible heating sheet of the thermal resistor;
after the resistance wire heating is finished, the flexible heating sheet of the thermal resistance is peeled off from the carrier silicon wafer by using a mechanical peeling method;
3) and adhering the manufactured flexible heating sheet of the thermal resistor and the manufactured paper chip by using double-sided adhesive tape, and placing the flexible heating sheet of the thermal resistor on a substrate to ensure that the center of the heating unit is positioned right below the nucleic acid detection unit of the paper chip. Namely, the circle center of the paper chip nucleic acid detection unit, the center of the heating unit and the circle center of the digging hole are positioned on a straight line vertical to the plane of the substrate.
6. The method for manufacturing an integrated paper-based chip suitable for simultaneous detection of nucleic acids and immunity according to claim 1, wherein: in the step 1), the paper chip is manufactured by adopting wax-based ink jet printing or ultraviolet lithography or laser cutting.
As a further improvement of the manufacturing method of the invention, the manufacturing of the paper chip in the step 1) adopts wax-based ink jet printing or ultraviolet photoetching or laser cutting.
The invention provides an integrated paper-based chip suitable for synchronous detection of nucleic acid and immunity and a manufacturing method thereof, and the integrated paper-based chip has the following advantages:
1) the detection accuracy is improved. For the rapid detection of infectious diseases, the nucleic acid detection or the immunoassay alone may result in low detection accuracy and easily cause false negative. Therefore, the nucleic acid and immune synchronous detection provided by the method has the capability of improving the detection accuracy. Meanwhile, the chip detection flux can be flexibly designed according to requirements, a plurality of sample information can be provided through single analysis, and the clinical diagnosis efficiency is greatly improved.
2) Without relying on external detection equipment. After the paper-based chip is integrated with the thermal resistance heating sheet, the paper-based chip can be carried conveniently without depending on external detection equipment during nucleic acid detection. In addition, the cost of the paper chip can be reduced to be within 1 yuan RMB, the disposable use condition is met, and cross infection is avoided.
3) And the tedious operation is avoided. The paper filtering chip does not introduce complicated energy, optical, electronic and mechanical systems of instruments, does not relate to the technical requirements of equipment operation and maintenance and the like, and greatly simplifies the experimental flow and the operation difficulty.
4) The reaction conditions are simplified. The precise temperature control is a necessary condition for in vitro nucleic acid amplification reaction, the temperature control in the conventional nucleic acid detection is generally realized by a desktop-level temperature control box or a built-in heating/cooling pool, and the temperature control based on the heating of a thermal resistance film is more convenient, so that the nucleic acid detection condition is further simplified.
5) Application value is as follows: compared with the existing single nucleic acid detection and single immune detection paper-based chips, the novel paper-based chip suitable for synchronous detection of nucleic acid and immunity is developed, and has the advantages of improving the detection accuracy, avoiding the complicated operation, being simple in process and the like. The paper-based chip is taken as a carrier and combined with a micro-nano processing technology, so that a rapid detection platform can be provided for COVID-19 and other infectious diseases, and no special instrument and equipment is required. Research work provides a new platform for infectious disease detection in China and supports rapid virus detection in areas with medical equipment defects.
Drawings
FIG. 1 is a first schematic diagram of the structural design of the paper-based chip of the present invention;
FIG. 2 is a second schematic diagram of the structural design of the paper-based chip of the present invention;
FIG. 3 is a third schematic diagram of the structural design of the paper-based chip of the present invention;
FIG. 4 is a schematic diagram of a polyimide film attachment process in accordance with the present invention;
FIG. 5 is a schematic view of the process of manufacturing the resistance wire of the present invention;
FIG. 6 is a schematic diagram of a mechanical stripping process for flexible heating strips of a thermal resistor according to the present invention;
illustration of the drawings:
1. a paper chip; 1-1, a sample solution introducing portion; 1-2, a solution transportation flow channel; 1-3, an immunodetection unit; 1-4, a nucleic acid detection unit; 2. a thermal resistance flexible heating sheet; 2-1 electrode; 2-2, a heating unit; 3. and digging a hole in the metal substrate.
Detailed Description
The invention is described in further detail below with reference to the following detailed description and accompanying drawings:
the invention provides an integrated paper-based chip suitable for synchronous detection of nucleic acid and immunity and a manufacturing method thereof, wherein the chip is used for preparing a thermal resistance flexible heating sheet by utilizing a micro-nano processing technology, and heating a nucleic acid detection unit of a paper chip to reach the temperature condition (constant temperature of 65 ℃) required by loop-mediated isothermal nucleic acid amplification. And because of the good heat conductivity of the metal substrate, the paper-based immunoassay unit is kept at room temperature for immunoassay. The invention aims to provide a portable nucleic acid and immune synchronous detection chip, provides a new method for detecting infectious diseases in China, and supports virus detection in areas lacking medical equipment. Meanwhile, the technology is a potential major public health disease diagnosis, treatment, prevention, control and storage technology in the future;
the chip of the application is designed as follows;
since the paper chip has the advantage of being capable of rapidly detecting suspected patients at the disease source, the portability and the simplicity of operation need to be considered. Paper based material selection
Grade 1 qualitative filter paper, paper chip may contain one or more inlets for sample solution introduction. Besides the inlet, the chip comprises a solution transportation flow channel, nucleic acid and an immunoassay unit which are separately arranged. The flexible heating sheet of the thermal resistor can adopt polyimide (or other common flexible substrate materials) as a base, and gold or other metal and semiconductor materials as a heating unit to heat the paper chip nucleic acid detection unit. The flexible heating sheet of the thermal resistor and the paper chip can be bonded by double-sided adhesive tape. The flexible heating sheet of the thermal resistor is arranged on the hole digging metal substrate, so that a non-support film structure is arranged below the nucleic acid detection unit of the paper chip. The battery can be used for supplying power to the thermal resistance heating plate, so that the chip is convenient to transport and carry out field test. The design scheme can highly localize the heat provided by the heating sheet, so that the temperature of the paper chip nucleic acid detection unit is raised to the condition required by the loop-mediated isothermal nucleic acid amplification. Due to the good thermal conductivity of the metal substrate, the heat conduction can be accelerated, so that the temperature of the immunoassay unit is kept unchanged. The design scheme combines the paper chip and the micro-nano manufacturing technology, and the paper-based chip is applied to the synchronous detection of nucleic acid and an immunity method for the first time. The chip structure is shown in fig. 1, 2 and 3.
The chip of the present application is manufactured as follows;
the chip manufacturing is mainly divided into 2 parts, paper chip manufacturing and thermal resistance heating plate manufacturing.
Firstly, processing a paper-based chip;
the paper-based chip is simpler to process, and the key is to construct a hydrophobic structure on a common hydrophilic filter paper base material to selectively cut off the flow of fluid in the filter paper or directly construct a hydrophilic channel network. Wax-based ink-jet printing, uv lithography, laser cutting, and the like are common processing techniques.
The paper chip provided by the application is manufactured in a laser cutting mode, the cost of the consumable material of the single paper-based chip is as low as 0.8 yuan RMB, and the processing period is about 3 minutes per chip. And the yield of the chips is close to 100 percent by debugging the processing parameters. The process has the advantages of simplicity, rapidness, stable finished product and the like, has low requirements on processing environment and conditions, and has low equipment price. In addition, the processing process does not involve any chemical reagent, and is more environment-friendly than methods such as ultraviolet photoetching and the like.
The manufacturing of the thermal resistance heating plate comprises the following steps:
step 1: temporarily attaching a polyimide film to a silicon wafer;
a single crystal silicon wafer having a diameter of 100mm was prepared. The wafer is cleaned thoroughly as shown in fig. 4. Subsequently, 4mL of polydimethylsiloxane solution (PDMS) was prepared, where the ratio of PDMS liquid base to curing agent was 10: 1. After the proportioning is finished, the liquid PDMS is placed in a vacuum cavity and is decompressed to remove bubbles. Next, PDMS was spun onto a silicon wafer using a spin coater, and a polyimide film was attached to the silicon wafer. Finally, the silicon wafer is placed in an oven and baked at 80 ℃ overnight. Polyimide films as well as PDMS are available directly.
Step 2: photoetching and patterning to determine the position of the electrode and form a thermal resistance thin film heater structure;
after the polyimide film is thoroughly cleaned, the whole wafer is coated with the positive photoresist in a spinning mode, the subsequent steps are used, after photoetching patterning, metal resistance wires can be evaporated on the polyimide film by thermal evaporation, as shown in figure 5, and the residual photoresist is removed by a Lift-off method
And step 3: mechanically stripping the flexible heating sheet of the thermal resistor;
after the resistance wire heating is completed, the flexible heating sheet of the thermal resistance is peeled off from the carrier silicon wafer by using a mechanical peeling method, and the peeling is shown in fig. 6.
And adhering the manufactured flexible heating sheet of the thermal resistor and the manufactured paper chip by using double-sided adhesive tape, and placing the flexible heating sheet of the thermal resistor on a substrate to ensure that the center of the heating unit is positioned right below the nucleic acid detection unit of the paper chip. Namely, the circle center of the paper chip nucleic acid detection unit, the center of the heating unit and the circle center of the digging hole are positioned on a straight line vertical to the substrate plane, in order to prove the engineering reliability of the method, the applicant manufactures the paper chip and carries out the nucleic acid reaction based on the paper-based chip. In this reaction, temperature affects the activity of enzymes in nucleic acid reaction reagents and directly determines whether nucleic acid amplification is effective, but in experiments, particularly under high temperature conditions, there are problems of reagent evaporation, adsorption of polymer structure surface molecules, and the like. Therefore, in the experiment, the applicant uses colorless and transparent paraffin droplets with extremely strong chemical inertness to package the paper chip nucleic acid detection unit and tests the liquid sealing effect. (ii) a The test shows that: heating the ink solution or pure water (0.5 mu l) in the paper-based unit without liquid seal treatment for 20 minutes by a hot plate at 65 ℃ and then completely evaporating; after the paraffin liquid seal treatment, pure water or ink in the reaction unit is heated by a hot plate at 65 ℃ for 60 minutes (or heated by a hot plate at 95 ℃ for 45 minutes), and still keeps liquid, thereby proving the engineering reliability of the method.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, but any modifications or equivalent variations made according to the technical spirit of the present invention are within the scope of the present invention as claimed.
Claims (6)
1. Integrated paper base chip suitable for nucleic acid and immune synchronous detection, including paper chip (1), flexible heating plate of thermal resistance (2) and dig hole metal substrate (3), its characterized in that: the integrated paper-based chip is sequentially provided with a paper chip (1), a thermal resistor flexible heating sheet (2) and a hole digging metal substrate (3) from top to bottom, the thermal resistor flexible heating sheet (2) is bonded with the paper chip (1) through double faced adhesive tapes, the thermal resistor flexible heating sheet (1) is placed on the hole digging metal substrate (3), paper-based material selective qualitative filter paper of the paper chip (1) comprises at least 1 sample solution introducing part (1-1), solution transportation flow channels (1-2) and separately arranged detection units, each separately arranged detection unit comprises an immunity detection unit (1-3) and a nucleic acid detection unit (1-4), each sample solution introducing part (1-1) is connected with the separately arranged detection units through the solution transportation flow channels (1-2), and the thermal resistor flexible heating sheet (2) adopts a flexible material as a base, the heating device is characterized in that a metal heating unit (2-2) is arranged on the upper portion of a base of the thermal resistance flexible heating sheet (2), electrodes (2-1) are arranged on two sides of the heating unit (2-2) and are powered by a power supply, the heating unit (2-1) is located under a nucleic acid detection unit (1-4) of a paper chip and is supposed to provide required temperature for nucleic acid reaction, a hole digging design is adopted for the metal substrate (3), the heating unit is located right above the hole, the circle center of the nucleic acid detection unit (1-4) of the paper chip is located at the center of the heating unit (2-2) and the circle center of the hole digging metal substrate (3) is located on a straight line perpendicular to a substrate plane.
2. The integrated paper-based chip suitable for nucleic acid and immune synchronous detection according to claim 1, characterized in that: the paper base material of the paper chip (1) is Whatman grade 1 qualitative filter paper.
3. The integrated paper-based chip suitable for nucleic acid and immune synchronous detection according to claim 1, characterized in that: the flexible heating sheet (2) of the thermal resistor adopts polyimide as a substrate.
4. The integrated paper-based chip suitable for nucleic acid and immune synchronous detection according to claim 1, characterized in that: the heating wire part of the heating unit (2-1) adopts metal and semiconductor as thermal resistance materials.
5. The manufacturing method of the integrated paper-based chip suitable for synchronous detection of nucleic acid and immunity comprises the following specific steps:
1) manufacturing a paper chip, namely constructing a hydrophobic structure on a hydrophilic filter paper substrate material, selectively blocking fluid flow in filter paper, or directly constructing a hydrophilic channel network;
2) manufacturing a flexible heating sheet of a thermal resistor;
step 1: temporarily attaching a polyimide film to a silicon wafer;
preparing a monocrystalline silicon wafer with the diameter of 100mm, thoroughly cleaning the wafer, then preparing 4mL of polydimethylsiloxane solution PDMS, wherein the ratio of PDMS solution base to curing agent is 10:1, placing the liquid PDMS in a vacuum cavity after the proportioning is finished, decompressing and removing bubbles, next, uniformly coating the PDMS on the silicon wafer by using a spin coater to serve as an adhesive, attaching a polyimide film to the silicon wafer by using the adhesive, finally, placing the silicon wafer in an oven, and baking the silicon wafer overnight at 80 ℃;
step 2: photoetching and patterning to determine the position of the electrode and form a metal thermal resistance structure;
after the polyimide film is thoroughly cleaned, the whole wafer is coated with positive photoresist in a spinning mode, the positive photoresist is used in the subsequent steps, after photoetching and patterning, the metal resistance wire can be evaporated on the polyimide film by thermal evaporation, and the residual photoresist is removed by a Lift-off method;
and step 3: mechanically stripping the flexible heating sheet of the thermal resistor;
after the resistance wire heating is finished, the flexible heating sheet of the thermal resistance is peeled off from the carrier silicon wafer by using a mechanical peeling method;
3) and adhering the manufactured flexible heating sheet of the thermal resistor and the manufactured paper chip by using double-sided adhesive tape, and placing the flexible heating sheet of the thermal resistor on a substrate to ensure that the center of the heating unit is positioned right below the nucleic acid detection unit of the paper chip. Namely, the circle center of the paper chip nucleic acid detection unit, the center of the heating unit and the circle center of the digging hole are positioned on a straight line vertical to the plane of the substrate.
6. The method for manufacturing an integrated paper-based chip suitable for simultaneous detection of nucleic acids and immunity according to claim 1, wherein: in the step 1), the paper chip is manufactured by adopting wax-based ink jet printing or ultraviolet lithography or laser cutting.
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