Miniaturized portable PCR-SERS nucleic acid instant detection platform

Abstract

The invention discloses a miniaturized portable PCR-SERS nucleic acid instant detection platform, and belongs to the technical field of nucleic acid detection. The platform is composed of a miniaturized thermal cycler, a miniature PCR reaction cavity, a filling type detection pen and a lateral flow chromatography test strip, a core component of the miniaturized thermal cycler is an inner core capable of providing temperature required by PCR amplification reaction, and the miniature PCR reaction cavity is composed of an upper-layer heat insulation adhesive tape, a PCR amplification paper film and a lower-layer heat insulation adhesive tape. The PCR amplification paper membrane is composed of a cellulose acetate membrane and a reagent which is freeze-dried on the cellulose acetate membrane in advance and is required by a PCR reaction, and a surface carboxylation SERS probe which is activated by EDC / NHS and is coupled with targeted recognition molecules is sprayed on a combination pad of the lateral flow chromatography test strip. The miniature thermal cycler has the beneficial effects that the volume of the miniature thermal cycler is about 35cm < 3 >, so that the miniature thermal cycler is very convenient to carry; reagents required by the PCR reaction are freeze-dried on the cellulose acetate membrane in advance, so that reagent-free operation in the whole process is realized.

Description

TECHNICAL FIELD

[0001] The application relates to a nucleic acid detection platform, in particular to a miniaturized portable PCR-SERS nucleic acid instant detection platform, and belongs to the technical field of nucleic acid detection. BACKGROUND

[0002] The molecular diagnostic technology based on NAAT (nucleic acid amplification testing) is currently widely used in the fields of medical diagnosis, species identification, environmental monitoring, etc. PCR (polymerase chain reaction) is widely regarded as the gold standard for nucleic acid detection due to its excellent sensitivity, ability to tolerate nucleotide variations, and ability to perform multiplex detection. Due to the good specificity, sensitivity and repeatability of PCR molecular detection, the molecular diagnostic industry continues to be popular. At the same time, the application end also puts forward the requirements of faster, more portable and more accurate, and the development of miniaturized sample input and result output molecular instant detection (POCT) platform can greatly improve the accessibility of PCR.

[0003] In the aspect of miniaturized PCR, microchip PCR research occupies a dominant position. It integrates a complex fluid operation system and functional unit module on a chip of several square centimeters, integrates sample preparation, reaction, sorting, detection and other basic operations, and has the characteristics of small volume, few components, high efficiency, automation and integration. However, due to the complex manufacturing process of microchip PCR, and the difficulty in cleaning the microchip and the difficulty in verifying the effectiveness of cleaning, the microchip cannot be reused if it cannot meet the cleaning requirements, so the manufacturing cost and use cost of microchip PCR are relatively high. In addition, since microchip PCR involves fluid operation systems such as injection pumps, it is not convenient to carry, which limits its transformation to the POCT end. Therefore, developing a low-cost miniaturized portable thermal cycler instrument is a necessary condition to realize nucleic acid on-site detection. In addition, the reagents such as dNTP, enzyme and primer required by PCR need to be refrigerated, and the shelf life is relatively short, and there is also the possibility of cross contamination. In recent years, in order to meet the on-site and timeliness of POCT and facilitate transportation and storage, a common method adopted by some PCR reagent suppliers in laboratory experiments is to provide freeze-dried reagent premix beads.

[0004] For post-PCR detection, lateral flow assays (LFAs) are popular in POCT for their portability, visual readout, and low cost. The most typical low-cost and mass-producible POCT platform is colloidal gold strip. There is a method of combining colloidal gold strip with PCR technology to form PCR-nucleic acid strip. The method of detecting nucleic acid is as follows: firstly, a pair of PCR primers specific to the target gene is designed, and the primers are labeled with two different markers, such as biotin and fluorescein. Double-labeled double-stranded amplicon is produced by PCR amplification. Then, a strip is made. Specifically, a certain amount of complex of streptavidin and colloidal gold that can specifically bind to one of the markers of the amplicon is fixed on the conjugate pad. The T line is sprayed with a marker such as fluorescein antibody that can specifically bind to the other marker of the amplicon. The C line is sprayed with a substance such as biotin that can specifically bind to the complex on the conjugate pad. Finally, the colloidal gold strip is used to detect the amplicon produced by PCR. For positive samples, the double-labeled amplicon flows from the sample pad to the conjugate pad due to capillary force, and the biotin on the amplicon specifically binds to the streptavidin on the conjugate pad. The complex then flows to the T line, and the fluorescein on the double-labeled amplicon specifically binds to the fluorescein antibody on the T line. The excess streptavidin on the conjugate pad specifically binds to the biotin on the C line. Because the streptavidin on the conjugate pad is labeled with colloidal gold, the T line and the C line show red color at the same time. For negative samples, the streptavidin on the conjugate pad specifically binds to the biotin on the C line, so only the C line shows red color. If the C line does not show color, the result cannot be determined.

[0005] Nowadays, more and more DNA biosensors are used in POCT molecular diagnosis in the form of lateral chromatography. However, the PCR module of the PCR-nucleic acid test strip is still limited to the conventional laboratory PCR instrument, which greatly reduces its portability, and the long thermal cycling time of PCR also becomes a key problem restricting its detection speed. Therefore, it is necessary to develop a detection mode combining miniaturized PCR and colloidal gold immunochromatography together, so that the PCR-nucleic acid test strip can truly achieve the POCT mode. However, the traditional lateral flow assay (LFA) test strip usually has low sensitivity, which limits its diagnostic performance. In order to solve this problem, people developed a SERS-LFA platform based on surface-enhanced Raman spectroscopy (SERS), which combined noble metal nanostructures with Raman reporters to greatly improve the signal sensitivity. SERS is an ultra-sensitive, non-destructive and rapid optical detection method, which has attracted widespread attention in the field of biomedical detection. Compared with traditional LFA, SERS-LFA can improve the detection limit by more than two orders of magnitude, has molecular fingerprint recognition ability and anti-photobleaching ability. In addition, when used in combination with portable Raman spectrometers, they are very suitable for on-site genetic diagnosis. In the past research, most of the naked gold nanoparticles used in the label detection cannot meet the detection environment of the test strip. The requirements for the nanoparticle labels used on the test strip should include: (1) Colloidal stability in solution under various conditions; (2) Sensitivity in a large and useful dynamic range; (3) High conjugation efficiency and repeatability (without losing the integrity and activity of chemical and biological samples); (4) Lack of or very low non-specific binding properties (to ensure high signal-to-noise ratio); (5) Commercialization at low cost; (6) Easy-to-expand conjugation procedures.

[0006] Therefore, it is necessary to develop a SERS probe with high stability and high sensitivity in order to introduce PCR-SERS technology into the test strip. SUMMARY

[0007] In order to solve the problems of the prior art, the purpose of the present application is to provide a miniaturized portable PCR-SERS nucleic acid instant detection platform.

[0008] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows: A miniaturized portable PCR-SERS nucleic acid instant detection platform is composed of the following components: a miniaturized thermal cycler, a micro PCR reaction chamber adapted for the miniaturized thermal cycler, a refillable detection pen, and a lateral flow chromatography test strip for PCR-SERS detection, wherein: The miniaturized thermal cycler includes a housing, a core, and a power supply. The core, located inside the housing, is the core component that provides the temperature required for the PCR amplification reaction. It includes an electrically connected single-chip microcomputer, a digital potentiometer, a heating chip, and a display screen. The entire core utilizes a double-layer PCB structure, with the heating chip fixed to a reaction tank that runs through the upper layer, and the digital potentiometer, single-chip microcomputer, and display screen fixed to the lower layer. The micro PCR reaction chamber consists of an upper layer of thermal insulation tape, a PCR amplification paper membrane, and a lower layer of thermal insulation tape. The PCR amplification paper membrane is sandwiched between the upper and lower layers of thermal insulation tape and sealed by both. The PCR amplification paper membrane consists of a cellulose acetate membrane and reagents required for the PCR reaction. The reagents required for the PCR reaction are pre-lyophilized on the cellulose acetate membrane. The lateral flow chromatography test strip is composed of a backing pad, a chromatography pad, a conjugate pad, a sample pad and a water-absorbing pad. The conjugate pad is sprayed with a surface carboxyl-SERS probe activated by EDC / NHS and coupled to a target recognition molecule. The refillable detection pen consists of a pen case, a built-in card slot, a spring and a capillary glass needle. The lower end of the pen case is adapted to the detection pen socket of the miniaturized thermal cycler, and a gear switching button is provided on the side. The built-in card slot consists of a groove, a pressing rod and a gear lever. The pressing rod and the gear lever are both provided at the top of the groove. The gear lever is adapted to the gear switching button on the side of the pen case. The entire built-in card slot is filled in the pen case and pressed on the spring. The capillary glass needle is detachably provided at the bottom end of the built-in card slot.

[0009] Preferably, the housing of the miniaturized thermal cycler is in the shape of a cube and is approximately 35 cm in size. 3 , it is hollow inside, with a display expansion port on the front, a detection pen socket on the upper side, and a ventilation port on the back, among which the detection pen socket is pulled up.

[0010] Preferably, the miniaturized thermal cycler further comprises a fan, which is disposed at a vent of the housing and electrically connected to the core.

[0011] Preferably, the upper thermal insulation tape and the lower thermal insulation tape are made of KAPTON tape.

[0012] Preferably, the surface carboxylation SERS probe activated by EDC / NHS and coupled with the target recognition molecule is prepared by the following method: (1) Preparation of gold seeds: Using chloroauric acid, sodium citrate, and sodium borohydride as raw materials, stir continuously overnight at room temperature to form a gold seed solution; (2) coating the gold seeds with polyvinyl pyrrolidone to obtain polyvinyl pyrrolidone-coated gold seeds; (3) Preparation of gold nanostars: Gold nanostars were prepared by seed-mediated growth method using polyvinyl pyrrolidone, chloroauric acid and gold seeds coated with polyvinyl pyrrolidone as raw materials; (4) Preparation of naked SERS probe: Using gold nanostars and the SERS signal molecule sodium diethyldithiocarbamate as raw materials, the reaction was carried out at room temperature in the dark overnight to obtain a naked SERS probe; (5) Encapsulation: encapsulating the bare SERS probe with a polystyrene shell to obtain a polystyrene shell-encapsulated SERS probe; (6) Carboxylation treatment: The surface of the SERS probe encapsulated by the polystyrene shell is treated with hydrochloric acid to obtain a surface carboxylation SERS probe; (7) Activation treatment: The surface carboxylation SERS probe was activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain an EDC / NHS-activated surface carboxylation SERS probe; (8) Coupling of target recognition molecules: Target recognition molecules are added to the EDC / NHS-activated surface carboxylation SERS probe and coupled at room temperature to obtain a surface carboxylation SERS probe activated by EDC / NHS and coupled to the target recognition molecules.

[0013] More preferably, in step (5), the method for encapsulating the naked SERS probe with a polystyrene shell is as follows: water and ethanol are added to a mixture of styrene, divinylbenzene and polyvinylpyrrolidone, wherein the ratio of styrene, divinylbenzene and polyvinylpyrrolidone is 19 mL: 1 mL: 6 g, the reaction mixture is stirred at 70°C under a nitrogen atmosphere for 1 h, and then the initiator 2,2'-azo(2-methylpropylamidine) dihydrochloride is added. After 8 min, the naked SERS probe is added to the reaction mixture, and the reaction is continued for 10 min. Then, maleic anhydride is added, and the mixture is heated at 70°C under a nitrogen atmosphere for another 10 h to obtain a SERS probe encapsulated by a polystyrene shell.

[0014] Preferably, the C line of the chromatography pad is coated with rabbit anti-FAM antibody, and the T line is coated with biotinylated bovine serum albumin.

[0015] The present invention is beneficial in that: (1) The miniaturized thermal cycler provided by the present invention has a volume of approximately 35 cm 3 , is only the size of a ping-pong ball, very convenient to carry, providing the necessary conditions for on-site nucleic acid detection. In addition, its production process is simple and does not involve cleaning issues. Compared with microchip PCR, the production cost and use cost are significantly reduced; (2) The micro PCR reaction chamber provided by the present invention is compatible with a miniaturized thermal cycler. The reagents required for the PCR reaction are pre-lyophilized on a cellulose acetate membrane. After loading, the overall thickness is less than 2 mm. Compared with freeze-dried reagent premix beads, this achieves a reagent-free operation throughout the entire process. (3) The surface carboxylation SERS probe provided by the present invention, which is activated by EDC / NHS and coupled with a target recognition molecule, has high sensitivity and lays the foundation for the construction of a PCR-SERS nucleic acid instant detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the front structure of a miniaturized thermal cycler; Figure 2 This is a schematic diagram of the reverse structure of a miniaturized thermal cycler; Figure 3 This is a schematic diagram of the structure of the core of a miniaturized thermal cycler; Figure 4 It is a schematic diagram of the structure of a miniature PCR reaction chamber adapted for a miniaturized thermal cycler; Figure 5 This is a schematic diagram of sample loading and packaging of a miniature PCR reaction chamber adapted for a miniaturized thermal cycler; Figure 6 It is a schematic diagram of the composition of a lateral flow chromatography test strip; Figure 7 This is the synthetic route of surface carboxylation SERS probes; Figure 8 This is a schematic diagram of the structure of a refillable detection pen adapted for a miniaturized thermal cycler; Figure 9 1. It is a schematic structural diagram of the pen shell of the refillable detection pen; Figure 10 This is a schematic diagram of the structure of the built-in card slot of the refillable test pen.

[0017] The meaning of the reference numerals in the figures: 11-housing, 12-core, 13-power supply, 14-fan; 111-display screen expansion port, 112-test pen socket, 113-ventilation port; 121- single chip microcomputer, 122- digital potentiometer, 123- heating chip, 124- display screen; 21-upper thermal insulation tape, 22-PCR amplification paper film, 23-lower thermal insulation tape; 31-back plate pad, 32-chromatographic pad, 33-binding pad, 34-sample pad, 35-absorbent pad; 41-pen case, 42-built-in card slot, 43-spring, 44-capillary glass needle; 411 - test strip observation window, 412 - built-in card slot mounting port, 413 - needle outlet, 414 - button card slot, 415 - gear shift button; 421 - groove, 422 - pressing rod, 423 - gear rod; 4211 - needle insertion hole. DETAILED DESCRIPTION

[0018] The application will be specifically introduced below in combination with the drawings and specific embodiments.

[0019] The miniaturized portable PCR-SERS nucleic acid instant detection platform provided by the application is composed of the following components:

[0020] First part, miniaturized thermal cycler The miniaturized thermal cycler is used to provide the temperature required for PCR amplification reaction, as shown in Figure 1 , Figure 2 and Figure 3 , the miniaturized thermal cycler is composed of a shell 11, a core 12, a power supply 13 and a fan 14, wherein: The shell 11 is in the shape of a cube, about 35 cm 3 in size, hollow, with a display external expansion port 111 on the front side, a detection pen insertion port 112 on the upper side, and a ventilation port 113 on the rear side, wherein the detection pen insertion port 112 is adapted to the shape of the detection pen and is pulled up by 1 cm to facilitate the insertion of the detection pen; The core 12 is arranged inside the shell 11 and is the core component for providing the temperature required for PCR amplification reaction; The power supply 13 is arranged outside the shell 11 and is electrically connected with the core 12, for providing working power for the core 12; The fan 14 is arranged at the ventilation port 113 of the shell 11 and is electrically connected with the core 12, and its start and stop are controlled by the core 12.

[0021] The structure of the core 12 will be described in detail below.

[0022] As shown in Figure 3 , the core 12 mainly includes a single-chip microcomputer 121 (for example: STM32F103C8T6), a digital potentiometer 122 (for example: AD5293BRUZ-20-RL7), a heating chip 123 (for example: DN505-1210Y) and a display screen 124 (for example: OLED), electrically connected, wherein: The single-chip microcomputer 121 is used to realize the overall control logic and process scheduling, including: (1) initializing and controlling various peripherals (such as the digital potentiometer 122, the display screen 124, the fan 14, etc.); (2) Send resistance setting value to digital potentiometer 122 through SPI communication interface, so as to indirectly adjust the temperature of heating chip 123; (3) Realize PCR temperature control cycle control (such as 98°C denaturation, 55°C annealing, 72°C extension); (4) Real-time output temperature control state and progress prompt to display screen 124; (5) Perform time delay to ensure smooth temperature transition.

[0023] Digital potentiometer 122 is used to accept the instruction of single-chip microcomputer 121 to change the resistance value output to single-chip microcomputer 121, and the output resistance value controls the temperature setting resistance in the internal heating loop of heating chip 123.

[0024] Heating chip 123 is used for temperature control and heat source output, and its key characteristics include: (1) Set target temperature through external resistance; (2) Provide a maximum output power of 9W when powered at 15V; (3) Temperature control range from ambient temperature to 100℃ with a maximum temperature of 120℃; (4) Fast thermal response, suitable for PCR rapid heating requirements; (5) Electrically isolated ceramic structure, suitable for portable and high safety design.

[0025] OLED display screen 124 is used for visual feedback of real-time temperature control process and running state.

[0026] The whole kernel 12 adopts upper and lower double-layer PCB board structure, specifically: the upper layer board is provided with a through reaction tank (1.2cmx0.7cm), and the heating chip 123 is fixed in the reaction tank; the digital potentiometer 122, the single-chip microcomputer 121 and the display screen 124 are fixed on the lower layer board; the upper layer board and the lower layer board are fixed by bolts.

[0027] The working principle of the miniaturized thermal cycler is equivalent to that of kernel 12, that is: the single-chip microcomputer 121 controls the digital potentiometer 122 to send a specific resistance value to the heating chip 123, and the heating chip 123 changes the temperature by changing the resistance value, and realizes the temperature and change required by PCR amplification reaction (for example: temperature cycle of 98℃, 55℃, 72℃) by controlling the duration of resistance value.

[0028] The miniaturized thermal cycler has compact overall structure, which is almost the same size as a standard ping-pong ball, and is convenient for integration in a small-sized portable instant nucleic acid detection platform.

[0029] Second part, micro-PCR reaction cavity adapted to miniaturized thermal cycler As Figure 4As shown, the micro PCR reaction chamber adapted for a miniaturized thermal cycler is composed of an upper layer of thermal insulation tape 21, a PCR amplification paper film 22, and a lower layer of thermal insulation tape 23, wherein: PCR amplification paper membrane 22: composed of cellulose acetate membrane and reagents required for PCR reaction, the reagents required for PCR reaction are pre-lyophilized on the cellulose acetate membrane; The upper thermal insulation tape 21 and the lower thermal insulation tape 23 are used to sandwich the PCR amplification paper film 22 and seal it. They are sealed by mechanical bonding and can be cut. They have low thermal conductivity, high flexibility and good sealing properties. KAPTON tape with super high temperature resistance and high adhesion is preferred.

[0030] The composition of the reagents required for the PCR reaction is shown in Table 1.

[0031] Table 1 Reagents required for PCR reaction Reagent name Reagent dosage PCR mix 125 μl Forward primer 10 μl Reverse primer 10 μl Trehalose (30wt%) 40 μl Bovine serum albumin (BSA, 0.5wt%) 10 μl Polyethylene glycol 20000 (PEG20000, 2wt%) 10 μl Tween 20 (5wt%) 10 μl The method for pre-lyophilizing the reagents required for the PCR reaction on a cellulose acetate membrane is as follows: The PCR mix premix, forward primer, reverse primer, trehalose, BSA, PEG20000 and Tween 20 were mixed and added dropwise to the cut cellulose acetate membrane, pre-frozen at -20°C overnight, and then freeze-dried in a freeze dryer with the cold trap temperature set to -60°C, the sample temperature set to -40°C, the freeze-drying time set to 24 hours, and the vacuum degree set to 1000 Pa.

[0032] like Figure 5 As shown, in the sample loading stage, the PCR amplification paper membrane 22 is first placed on the cut lower insulation tape 23, and then the sample is added to the PCR amplification paper membrane 22. The sample addition amount is 5-10μl exceeding the fluid absorption saturation volume of the PCR amplification paper membrane 22. The added sample penetrates into the PCR amplification paper membrane 22 and partially dissolves the freeze-dried reagents required for the PCR reaction. Finally, it is sealed with the cut upper insulation tape 21 to form a micro PCR reaction chamber with free liquid. The overall thickness is less than 2mm, and the entire process is reagent-free.

[0033] The micro PCR reaction chamber containing free liquid is attached to the heating chip 123 of the miniaturized thermal cycler, and the PCR amplification reaction can be directly performed after the miniaturized thermal cycler is started.

[0034] Part III: Lateral flow chromatography test strips for PCR-SERS detection like Figure 6 As shown, the lateral flow chromatography test strip consists of a backing pad 31, a chromatography pad 32, a conjugate pad 33, a sample pad 34 and a water absorbent pad 35, wherein: Backboard pad 31: Made of PVC film without any chemical treatment; Chromatography pad 32: a nitrocellulose membrane is used as a substrate, C-line and T-line coating is performed on the substrate using a membrane gold spraying instrument, and the substrate is dried at room temperature overnight to obtain the chromatography pad 32, which is stacked on the middle part of the back pad 31 as a whole. Binding pad 33: a glass cellulose membrane is used as a substrate, the substrate is first soaked in the following optimized buffer system: 1wt% bovine serum albumin (BSA), 5wt% polyvinylpyrrolidone (PVP, molecular weight about 11000), 1× phosphate buffer (PBS), 1wt% Tween 20, 1wt% sucrose, 5wt% polyethylene glycol 20000 (PEG), then vacuum dried, and finally sprayed with surface carboxylated SERS probes activated by EDC / NHS and coupled with target recognition molecules (such as streptavidin, biotin, antibodies, nucleic acid probes) to obtain the binding pad 33, which is located on the side of the C-line, part of which is stacked on the chromatography pad 32, and the other part is stacked on the back pad 31. Sample pad 34: a glass cellulose membrane is used as a substrate, the substrate is first soaked in the following optimized buffer system: 1wt% bovine serum albumin (BSA), 5wt% polyvinylpyrrolidone (PVP, molecular weight about 11000), 1× phosphate buffer (PBS), 1wt% Tween 20, 1wt% sucrose, 5wt% polyethylene glycol 20000 (PEG), and then vacuum dried to obtain the sample pad 34, which is located on the side of the C-line, part of which is stacked on the binding pad 33, and the other part is stacked on the back pad 31, and the part of the binding pad 33 not covered by the sample pad 34 is flush with the surface of the sample pad 34 close to the side of the C-line. Water absorption pad 35: a coarse fiber filter paper membrane is used, without any chemical treatment, and the water absorption pad 35 is located on the side of the T-line, part of which is stacked on the chromatography pad 32, and the other part is stacked on the back pad 31.

[0035] The preparation method of the surface carboxylated SERS probes activated by EDC / NHS and coupled with target recognition molecules will be described in detail below.

[0036] First, the surface carboxylated SERS probes are prepared. As shown in Figure 7 The preparation method of the surface carboxylated SERS probes is as follows: (1) Preparation of gold seeds: 1mL of 1wt% chloroauric acid aqueous solution is diluted to 90mL, 2mL of 38.8mM sodium citrate aqueous solution is added, then 1mL of freshly prepared 0.075wt% sodium borohydride (NaBH4) aqueous solution is slowly added, and the mixture is continuously stirred at room temperature overnight to form a gold seed solution; (2) Coating gold seeds with polyvinylpyrrolidone (PVP): Add 10 mL of 0.94 mM PVP (K30) solution to 50 mL of gold seed solution and stir continuously at room temperature for 24 h to obtain a PVP-coated gold seed solution with a gold seed concentration of 36.25 PM. (3) Preparation of gold nanostars (Au NSs): 75 g of PVP (average molecular weight of 11,000) was dissolved in 75 mL of N,N-dimethylformamide (DMF), and 410 μL of 50 mM chloroauric acid (HAuCl4.4H2O) aqueous solution and 468.75 μL of PVP-coated gold seed solution with a gold seed concentration of 36.25 PM were added. The mixture was stirred at room temperature for 3 h. After the reaction, the reaction solution was centrifuged and washed twice with ethanol and water respectively to obtain Au NSs precipitate. The Au NSs precipitate was redispersed in 7.5 mL of water to obtain Au NSs redispersed solution.

[0037] (4) Preparation of bare SERS probe: Add 0.25 μM sodium diethyldithiocarbamate (DDTC, SERS signal molecule) ethanol solution to the Au NSs redispersion solution, with the volume ratio of Au NSs redispersion solution to DDTC ethanol solution being 10:1. Then, react overnight at room temperature in the dark, centrifuge to remove free molecules, and obtain a bare SERS probe. The bare SERS probe is redispersed in 15 mL of water to obtain a bare SERS probe solution. (5) Polystyrene (PS) shell encapsulation: 19.5 mL of water and 82.5 mL of ethanol were added to a mixture of styrene (ST, monomer, 0.95 mL), divinylbenzene (DVB, cross-linker, 0.05 mL), and polyvinylpyrrolidone (PVP, average molecular weight 58,000, 300 mg). The reaction mixture was stirred at 70 °C under nitrogen atmosphere for 1 h, and then 3 mL of 2,2'-azo(2-methylpropylamidine) dihydrochloride (AIBA, initiator) aqueous solution (1.7 wt%) was added. After 8 min, 15 mL of bare SERS probe solution was added to the reaction mixture and the reaction was continued for 10 min. Then, maleic anhydride (MA) was added with a molar ratio of MA to ST of 2:1. The mixture was heated at 70 °C under nitrogen atmosphere for another 10 h. Finally, the mixture was centrifuged in deionized water (to remove residual reactants) to obtain a PS shell encapsulated SERS probe. The PS shell encapsulated SERS probe was washed 5 times with deionized water for later use.

[0038] 100 μL of 0.1 M HCl solution was added to 1 mL of the PS shell-encapsulated SERS probe and stirred at room temperature for 1 h to obtain a surface carboxylation SERS probe with targeted coupling capability.

[0039] Then, the surface carboxylation SERS probe was activated with EDC / NHS and coupled with the target recognition molecule. Specifically: (1) EDC / NHS activation: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the surface carboxylation SERS probe. Specifically, the surface carboxylation SERS probe was centrifuged at 6000 rpm for 10 min, the supernatant was removed, and then 200 μL of 20 mg / mL EDC solution and 200 μL of 20 mg / mL NHS solution were added. The mixture was stirred at room temperature for 1 h to obtain the EDC / NHS-activated surface carboxylation SERS probe. (2) Target recognition molecule coupling: Add target recognition molecules (final concentration 1 mg / mL) to the EDC / NHS-activated surface carboxylation SERS probe, react at room temperature for 1 h (coupling), then centrifuge to remove excess target recognition molecules, and use a buffer solution containing 1×PBS+1wt% sucrose to concentrate to 1 / 10 the volume of the original solution to obtain a surface carboxylation SERS probe activated by EDC / NHS and coupled with the target recognition molecule, which was stored at 4°C for future use.

[0040] Part 4: Refillable Detection Pen for Miniaturized Thermal Cycler like Figure 8 As shown, the refillable detection pen adapted for a miniaturized thermal cycler is composed of a pen housing 41, a built-in card slot 42, a spring 43 and a capillary glass needle 44, wherein: like Figure 9 As shown, the pen housing 41 is rectangular in shape, hollow inside, and the lower end is adapted to the detection pen socket 112 of the miniaturized thermal cycler. A test strip observation window 411 is provided on the front side, a built-in card slot installation port 412 is provided on the upper side, a needle outlet 413 is provided on the lower side, a button card slot 414 is provided on the left or right side, a gear switching button 415 is provided at the button card slot 414, and a cover (not shown) is provided at the built-in card slot installation port 412; like Figure 10 As shown, the built-in card slot 42 is composed of a groove 421, a pressing rod 422 and a shift rod 423, wherein the groove 421 is used to load the lateral flow chromatography test strip for PCR-SERS detection ( Figure 6 ), the shape is adapted to the inner cavity of the pen housing 41, and a pinhole 4211 is formed at the lower end. The position of the pinhole 4211 matches the needle outlet 413 on the pen housing 41, and the aperture is just large enough to insert the capillary glass needle 44. The pressing rod 422 and the shift rod 423 are both arranged at the top of the groove 421. The shift rod 423 is adapted to the shift switch button 415 on the side of the pen housing 41. The entire built-in card slot 42 is filled in the pen housing 41; Spring 33: arranged at the bottom of the inner cavity of the pen shell 41, the upper end is in contact with the bottom of the built-in card slot 42, used to pop up the built-in card slot 42, so as to realize gear switching; Capillary glass needle 44: made of capillary glass tube by needle drawing instrument, with front end sharp, open tube cavity structure at the end, arranged at the bottom of the recess 421 in a detachable manner through the needle insertion hole 4211 on the recess 421, and the end is in contact with the sample pad of the lateral flow chromatography test strip in the recess 421, the release direction of the capillary glass needle 44 is consistent with the capillary chromatography direction of the lateral flow chromatography test strip, so that a stable capillary liquid flow channel can be formed.

[0041] The adsorption mechanism of the capillary glass needle 44 is: relying on capillary action, automatically absorbing liquid under the condition of no external force.

[0042] The release mechanism of the capillary glass needle 44 is: by contacting with the sample pad of the lateral flow chromatography test strip in the recess 421, the strong capillary force of the hydrophilic fiber in the sample pad actively drags the liquid, so as to realize the release without external force, which belongs to the contact-induced capillary release mechanism.

[0043] The use method of the small portable PCR-SERS nucleic acid instant detection platform provided by the application is: (1) sample adding Place the PCR amplification paper film 22 on the lower layer of heat insulation tape 23, then add 20-30 μL of sample to the PCR amplification paper film 22, and then seal the PCR amplification paper film 22 with the upper layer of heat insulation tape 21 to form a micro-PCR reaction cavity containing free liquid.

[0044] (2) assemble the detection pen Load the capillary glass needle 44 and the lateral flow chromatography test strip for PCR-SERS detection on the recess 421 of the built-in card slot 42, Figure 6 Then, the built-in card slot 42 is assembled into the pen shell 41, the gear switching button 415 on the side of the pen shell 41 is switched to OFF gear, at this time the spring 43 in the pen shell 41 pops up the built-in card slot 42, the entire capillary glass needle 44 is in the interior of the pen shell 41, and there is no exposure.

[0045] (3) PCR reaction Place the micro-PCR reaction cavity containing free liquid on the heating chip 123 of the small heat cycler, insert the assembled detection pen into the detection pen socket 112 of the small heat cycler, start the small heat cycler to perform thermal cycling, and the sample completes the PCR reaction in the micro-PCR reaction cavity.

[0046] (4) chromatography of PCR product After the thermal cycle, press the press rod 422 of the detection pen to make the blocking rod 423 be clamped in the button clamping groove 414, at this time the capillary glass needle 44 will pierce the micro-PCR reaction cavity, and the sample in the micro-PCR reaction cavity will be moved to the sample pad 34 of the lateral flow chromatographic test strip under the capillary action of the capillary glass needle 44, and the chromatography is carried out on the lateral flow chromatographic test strip. After the chromatography is completed, the result is read.

[0047] After the detection is completed, the lateral flow chromatographic test strip and the capillary glass needle are replaced, and the next detection is prepared. The lateral flow chromatographic test strip and the capillary glass needle are replaced every time, and zero cross contamination is achieved.

[0048] Application Case 1: Field monitoring of fecal indicator bacteria at a seawater bathing beach 1. Design and synthesis of targeted primers Targeted primers are designed according to the nucleotide sequence of Escherichia coli (GenBank: OQ719750.1, SEQ ID NO: 1), and a FAM group is modified at the 5' end of the forward primer, and a biotin is modified at the 5' end of the reverse primer. The length of the amplified fragment is 150-200 bp (Tm value 60±2℃).

[0049] The nucleotide sequences of the forward primer and the reverse primer before modification are as follows: Forward primer: 5'-AGCGGGGAGGAAGGGAGTAAAG-3' (SEQ ID NO: 2); Reverse primer: 5'-GACTCAAGCTTGCCAGTATCAGATG-3' (SEQ ID NO: 3).

[0050] The forward primer modified with a FAM group at the 5' end and the reverse primer modified with a biotin at the 5' end are synthesized, respectively.

[0051] 2. Preparation of PCR amplification paper film The reagents required for PCR reaction (Table 1) are pre-frozen on the cellulose acetate film to obtain the PCR amplification paper film.

[0052] 3. Preparation of lateral flow chromatographic test strip First, the binding pad is prepared according to the method provided in the third part by taking streptavidin as the targeted recognition molecule.

[0053] Then, the chromatographic pad is prepared according to the method provided in the third part, wherein the C line is coated with rabbit anti-FAM antibody (1.2 mg / mL), and the T line is coated with biotinylated bovine serum albumin (biotin-BSA, 1.0 mg / mL).

[0054] Finally, the sample pad, the binding pad, the chromatographic pad, the water absorption pad, and the back plate pad are laminated to prepare the lateral flow chromatographic test strip according to the method provided in the third part.

[0055] 4. Assemble the detection pen Assemble the lateral flow test strip into the test pen.

[0056] 5. Setting the Temperature and Variation of the Miniaturized Thermal Cycler Modify the microcontroller code to achieve the temperature and changes required for PCR amplification reaction by controlling the duration of the resistance value: temperature cycles of 98℃, 55℃, and 72℃.

[0057] 6. Collect and process samples Fifty seawater samples were collected from a bathing beach, and bacteria were enriched using a 0.22 μm filter membrane. The samples were then treated with a lysis buffer (containing 1% SDS + 20 mg / mL proteinase K) for 10 min.

[0058] 7. Add sample Take 30 μL of the treated sample and drop it onto the PCR amplification paper membrane, and seal the PCR amplification paper membrane with thermal insulation tape to form a micro PCR reaction chamber containing free liquid.

[0059] 8. Perform PCR reaction Place the micro PCR reaction chamber containing free liquid on the heating chip of the miniaturized thermal cycler, insert the assembled detection pen into the detection pen socket of the miniaturized thermal cycler, start the miniaturized thermal cycler for thermal cycling, and the sample completes the PCR reaction in the micro PCR reaction chamber.

[0060] 9. Chromatography of PCR products After the thermal cycle is completed, press the pressing rod of the detection pen, the capillary glass needle pierces the micro PCR reaction chamber, and the sample in the chamber moves upward to the lateral flow chromatography test strip through the capillary glass needle, and chromatographs are performed on the lateral flow chromatography test strip. After the chromatography is completed, the results are read.

[0061] 10. Analyze the results A portable Raman spectrometer was used to detect the T line of the lateral flow chromatography test strip and calculate the concentration of Escherichia coli in the seawater sample.

[0062] Calculation result: The concentration of Escherichia coli in the seawater sample is 10 CFU / mL.

[0063] In this test, the total time for single-sample detection is <30 min, the specificity is >95%, and the detection limit is 10 CFU / mL (10-100 times higher than the fluorescence method).

[0064] Application Case 2: Offshore Tamar Alexandrium bloom monitoring 1. Design and synthesize targeting primers Targeting primers were designed based on the complete nucleotide sequence of Alexandrium tamarense (GenBank: AB196556.1, SEQ ID NO: 4). The 5' end of the forward primer was modified with a FAM group, and the 5' end of the reverse primer was modified with biotin. The amplified fragment length was 150-200 bp (Tm value 60±2℃).

[0065] The nucleotide sequences of the forward primer and reverse primer before modification are as follows: Forward primer: 5′-TGATAGCACACAAGTACCATGAGG-3′ (SEQ ID NO: 5); Reverse primer: 5'-CAACACTCCCACCAAGCAAA-3' (SEQ ID NO: 6).

[0066] A forward primer with a 5' end modified with a FAM group and a reverse primer with a 5' end modified with biotin were synthesized respectively.

[0067] 2. Preparation of PCR amplification paper membrane The reagents required for the PCR reaction (Table 1) were pre-lyophilized on a cellulose acetate membrane to obtain a PCR amplification paper membrane.

[0068] 3. Preparation of lateral flow chromatography test strips First, streptavidin was used as the target recognition molecule and a conjugate pad was prepared according to the method provided in the third part.

[0069] Then, the chromatography pad was prepared according to the method provided in Section 3, wherein the C line was coated with rabbit anti-FAM antibody (1.2 mg / mL) and the T line was coated with biotinylated bovine serum albumin (biotin-BSA, 1.0 mg / mL).

[0070] Finally, according to the method provided in the third part, the sample pad, conjugate pad, chromatography pad, absorbent pad, and backing pad are stacked to prepare a lateral flow chromatography test strip.

[0071] 4. Assemble the detection pen Assemble the lateral flow chromatography test strip for Alexandrium tamarense into the test pen.

[0072] 5. Setting the Temperature and Variation of the Miniaturized Thermal Cycler Modify the microcontroller code to achieve the temperature and changes required for PCR amplification reaction by controlling the duration of the resistance value: temperature cycles of 98℃, 55℃, and 72℃.

[0073] 6. Collect and process samples Fifty samples of coastal seawater were collected, and bacteria were enriched using a 0.22 μm filter membrane. The samples were then treated with lysis buffer (containing 1% SDS + 20 mg / mL proteinase K) for 10 min.

[0074] 7. Sample loading Take 30 μL of the treated sample and drop it onto the PCR amplification paper film. Seal the PCR amplification paper film with a heat insulation tape to form a micro-PCR reaction chamber containing free liquid.

[0075] 8. Perform PCR reaction Place the micro-PCR reaction chamber containing free liquid on the heating chip of the miniaturized thermal cycler. Insert the assembled detection pen into the detection pen insertion port of the miniaturized thermal cycler. Start the miniaturized thermal cycler to perform thermal cycling. The sample completes the PCR reaction in the micro-PCR reaction chamber.

[0076] 9. Perform chromatography on the PCR product After the thermal cycling, press the pressing rod of the detection pen. The capillary glass needle pierces the micro-PCR reaction chamber. The sample in the chamber is moved upward to the lateral flow chromatography test strip through the capillary glass needle. Perform chromatography on the lateral flow chromatography test strip. Read the result after the chromatography is completed.

[0077] 10. Analyze the result Use the portable Raman spectrometer to detect the T line of the lateral flow chromatography test strip and calculate the concentration of Alexandrium tamarense in the seawater sample.

[0078] The calculation result is that the concentration of Alexandrium tamarense in the seawater sample is 100 cells / mL.

[0079] In this detection, the total time for single sample detection is < 30 min, the specificity is > 95%, and the detection limit reaches 100 cells / mL.

[0080] It should be noted that the above examples are merely examples for clearly illustrating the present application and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. It is impossible to exhaust all the embodiments here. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. A miniaturized portable PCR-SERS nucleic acid instant detection platform, characterized in that: The device is composed of the following components: a miniaturized thermal cycler, a micro PCR reaction chamber adapted for the miniaturized thermal cycler, a refillable detection pen, and a lateral flow chromatography test strip for PCR-SERS detection, wherein: The miniaturized thermal cycler comprises a housing (11), a core (12) and a power supply (13), wherein the core (12) is arranged inside the housing (11) and is a core component that provides the temperature required for the PCR amplification reaction, and comprises an electrically connected single-chip microcomputer (121), a digital potentiometer (122), a heating chip (123) and a display screen (124). The entire core (12) adopts an upper and lower double-layer PCB board structure, the heating chip (123) is fixed in a reaction tank that passes through the upper board, and the digital potentiometer (122), the single-chip microcomputer (121) and the display screen (124) are fixed on the lower board; The micro PCR reaction chamber is composed of an upper thermal insulation tape (21), a PCR amplification paper film (22) and a lower thermal insulation tape (23), wherein the PCR amplification paper film (22) is sandwiched between the upper thermal insulation tape (21) and the lower thermal insulation tape (23) and is sealed by the two, wherein the PCR amplification paper film (22) is composed of a cellulose acetate film and reagents required for the PCR reaction, and the reagents required for the PCR reaction are pre-lyophilized on the cellulose acetate film; The lateral flow chromatography test strip is composed of a backing pad (31), a chromatography pad (32), a conjugation pad (33), a sample pad (34) and a water-absorbing pad (35) stacked together, wherein the conjugation pad (33) is sprayed with a surface carboxylation SERS probe activated by EDC / NHS and coupled to a target recognition molecule; The refillable detection pen is composed of a pen housing (41), a built-in card slot (42), a spring (43) and a capillary glass needle (44), wherein the lower end of the pen housing (41) is adapted to the detection pen socket (112) of the miniaturized thermal cycler, and a gear switching button (415) is provided on the side. The built-in card slot (42) is composed of a groove (421), a pressing rod (422) and a gear rod (423), and the pressing rod (422) and the gear rod (423) are both provided at the top of the groove (421). The gear rod (423) is adapted to the gear switching button (415) on the side of the pen housing (41). The entire built-in card slot (42) is filled in the pen housing (41) and pressed on the spring (43). The capillary glass needle (44) is detachably provided at the bottom end of the built-in card slot (42).

2. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 1, characterized in that: The housing (11) of the miniaturized thermal cycler is in the shape of a cube and is approximately 35 cm in size. 3 The interior is hollow, a display external expansion port (111) is opened on the front side, a detection pen socket (112) is opened on the upper side, and a ventilation port (113) is opened on the rear side, wherein the detection pen socket (112) is pulled up.

3. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 1, characterized in that: The miniaturized thermal cycler further includes a fan (14), which is disposed at a vent (113) of the housing (11) and electrically connected to the core (12).

4. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 1, characterized in that: The upper thermal insulation tape (21) and the lower thermal insulation tape (23) are made of KAPTON tape.

5. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 1, characterized in that: The surface carboxylation SERS probe activated by EDC / NHS and coupled to the target recognition molecule is prepared by the following method: (1) Preparation of gold seeds: Using chloroauric acid, sodium citrate, and sodium borohydride as raw materials, stir continuously overnight at room temperature to form a gold seed solution; (2) coating the gold seeds with polyvinyl pyrrolidone to obtain polyvinyl pyrrolidone-coated gold seeds; (3) Preparation of gold nanostars: Gold nanostars were prepared by seed-mediated growth method using polyvinyl pyrrolidone, chloroauric acid and gold seeds coated with polyvinyl pyrrolidone as raw materials; (4) Preparation of naked SERS probe: Using gold nanostars and the SERS signal molecule sodium diethyldithiocarbamate as raw materials, the reaction was carried out at room temperature in the dark overnight to obtain a naked SERS probe; (5) Encapsulation: encapsulating the bare SERS probe with a polystyrene shell to obtain a polystyrene shell-encapsulated SERS probe; (6) Carboxylation treatment: The surface of the SERS probe encapsulated by the polystyrene shell is treated with hydrochloric acid to obtain a surface carboxylation SERS probe; (7) Activation treatment: The surface carboxylation SERS probe was activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain an EDC / NHS-activated surface carboxylation SERS probe; (8) Coupling of target recognition molecules: Target recognition molecules are added to the EDC / NHS-activated surface carboxylation SERS probe and coupled at room temperature to obtain a surface carboxylation SERS probe activated by EDC / NHS and coupled to the target recognition molecules.

6. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 5, characterized in that: In step (5), the method of encapsulating the bare SERS probe with a polystyrene shell is as follows: Water and ethanol were added to a mixture of styrene, divinylbenzene, and polyvinylpyrrolidone in a ratio of 19 mL: 1 mL: 6 g. The reaction mixture was stirred at 70°C under a nitrogen atmosphere for 1 hour, and then the initiator 2,2'-azo(2-methylpropylamidine) dihydrochloride was added. After 8 minutes, the naked SERS probe was added to the reaction mixture, and the reaction was continued for 10 minutes. Maleic anhydride was then added, and the mixture was heated at 70°C under a nitrogen atmosphere for another 10 hours to obtain a polystyrene shell-encapsulated SERS probe.

7. The miniaturized portable PCR-SERS nucleic acid instant detection platform according to claim 1, characterized in that: The C line of the chromatography pad (32) is coated with rabbit anti-FAM antibody, and the T line is coated with biotinylated bovine serum albumin.

Images