An in situ spatial imaging method for single molecules or single cells on the surface of solid samples

Abstract

The present invention discloses an in-situ spatial imaging method for single molecules or single cells on the surface of solid samples, belonging to the field of biotechnology. The method comprises: (1) adding a hydrogel monomer to a nucleic acid amplification reaction system without an amplification template, wherein the reaction system comprises a specific primer and a fluorescent dye, and adding the hydrogel monomer to a gel-forming mold to form a hydrogel sheet with a thickness of 200 to 800 μm; (2) during detection, directly attaching the flexible hydrogel sheet to the surface of the solid sample to be tested, and then placing the sample attached to the hydrogel sheet under nucleic acid amplification conditions for nucleic acid amplification reaction; (3) using fluorescence imaging technology to analyze the fluorescent signal on the hydrogel sheet, calculate the amount of contamination, and determine the spatial distribution position of the contamination. The present invention uses a flexible hydrogel sheet to realize in-situ interface imaging of nucleic acids or microorganisms on a variety of solid samples, and the positions before and after amplification are consistent, thereby realizing in-situ spatial imaging of a single nucleic acid or microorganism on the surface of a solid sample.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an in-situ spatial imaging method for single molecules or single cells on the surface of solid samples. Background Art

[0002] With the development of technology, the demand for precise analysis of microorganisms is also increasing. In situ imaging and tracing of microorganisms on solid surfaces such as biological samples, analysis of their types, quantities and spatial distribution, and identification of contaminated locations on the sample surface can help discover the source of contamination more quickly and reduce economic losses and the spread of disease.

[0003] However, most of the existing nucleic acid detection technologies for biological samples use a surface smearing method to collect pathogens on solid samples for nucleic acid detection. This method is not only time-consuming and labor-intensive, but also cannot reflect the in situ spatial distribution of nucleic acids or cells on solid samples. Fluorescence in situ hybridization (FISH) is often used for in situ imaging of nucleic acid molecules or microorganisms. This technology first fixes the target in situ with a fixative, and then uses fluorescently labeled probes to complementarily hybridize with the target gene to achieve in situ detection of the molecule. Due to the lack of signal amplification process, this technology has difficulty in imaging low-concentration targets, especially in situ imaging of single-copy nucleic acid molecules. In situ PCR and in situ LAMP have been used to improve the sensitivity of in situ detection ( Nat. Methods , 2009, 6(2): 139-141. Malar J , 2018, 17(1): 235). This type of method relies on the confinement effect inside the cell, but it places particularly harsh and complex requirements on the permeability of the cell. The PCR and LAMP nucleic acid amplification products in aqueous solution are easy to diffuse, making it impossible to detect pathogens in situ.

[0004] In order to effectively prevent the diffusion of nucleic acid amplification products in aqueous solutions, surface probe-modified rolling circle amplification (RCA) or special branched structures (Y-shaped nucleic acids) have been applied to in situ molecular detection ( Chem. Sci. , 2017, 8, 3668, Angew. Chem. Int. Ed. 2018, 57, 9739-9743). Among them, the RCA product will not disperse, and the generated amplification product will extend into a long chain, and then the nucleic acid amplification product will be captured in situ by the solid surface probe; the Y-shaped nucleic acid is through the confined space formed by the special Y-shaped branch to restrict the free movement of the nucleic acid amplification product in situ. However, these technologies require cumbersome and long-term pre-treatment of solid samples to fix the target in situ and elute the fixative in advance, which often causes a large amount of target nucleic acid loss, easily produces false negative results, and cannot perform in situ spatial imaging of all targets on the solid sample. At the same time, these steps will also take a lot of time.

[0005] Therefore, how to establish an in situ spatial imaging method on the surface of solid samples to reveal the types, quantities and spatial distribution of nucleic acids and microorganisms at the single-molecule or single-cell resolution level is a problem that technicians in this field need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide an in situ spatial imaging method for nucleic acids or microorganisms on the surface of solid samples for the purpose of non-diagnosis of diseases. No sample collection or pretreatment is required. Nucleic acids, viruses or pathogenic microorganisms can be directly detected in situ on the surface of food, packaging materials (such as metals, plastics, ceramics) and biological tissue samples to clarify the spatial distribution of contamination.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides an in-situ spatial imaging method for a single molecule or a single cell on a solid sample surface, comprising the following steps:

[0009] (1) adding a hydrogel monomer to a nucleic acid amplification reaction system without an amplification template to obtain a mixed solution, wherein the reaction system includes primers for specifically amplifying a target nucleic acid DNA fragment and a fluorescent dye, and adding the mixed solution to a gel-forming mold to form a hydrogel sheet with a thickness of 200 to 800 μm as a flexible and conformable detection device;

[0010] (2) During the test, the flexible hydrogel sheet is directly attached to the surface of the solid sample to be tested, and the hydrogel sheet is pressed to make full contact with the surface of the solid sample, and then the solid sample attached to the hydrogel sheet is placed under nucleic acid amplification conditions to perform a nucleic acid amplification reaction;

[0011] (3) Fluorescence imaging technology is used to analyze the fluorescence signal on the hydrogel sheet after the nucleic acid amplification reaction, calculate the amount of nucleic acid or microbial contamination on the surface of the solid sample, and determine the spatial distribution location of the contamination.

[0012] The solid sample may be, but is not limited to, an inorganic solid sample of various packaging materials such as glass, plastic, metal, and ceramic, or a plant solid sample such as vegetables, fruits, and wood, or an in vitro biological tissue slice sample.

[0013] The target detected by the present invention may be a microorganism (including bacteria, fungi, viruses), or a nucleic acid molecule such as DNA, RNA, etc. Specifically, the microorganism may be, but is not limited to, foodborne pathogens such as Escherichia coli and Listeria monocytogenes.

[0014] The nucleic acid amplification reaction may be, but is not limited to, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or rolling circle nucleic acid amplification (RCA).

[0015] In step (1), matching primers are designed according to the target nucleic acid or target DNA fragment of the microorganism to be detected, and a nucleic acid amplification reaction system is prepared. The reaction system forms a hydrogel system by adding hydrogel monomers to cross-link and gel. Since the hydrogel has good flexibility and adhesion properties, it can not only cover the flat surface of the solid sample, but also fit tightly to the solid sample surface with curvature and roughness, so that the nucleic acid or microorganism attached to the surface of the solid sample is fixed in situ at the interface layer between the hydrogel sheet and the solid sample.

[0016] The hydrogel may be, but is not limited to, polyethylene glycol-based hydrogel or polyacrylamide-based hydrogel.

[0017] Furthermore, the present invention uses polyethylene glycol-based hydrogels, wherein the hydrogel monomers include four-arm ethylene glycol acrylate and mercapto-polyethylene glycol-mercapto. The above two monomers are mixed in an aqueous solution and gelled at room temperature for 1 to 5 minutes. In addition to the above hydrogel monomers, the present invention can also use other monomers and crosslinking agents that can form hydrogels.

[0018] Furthermore, the molecular weight of four-arm ethylene glycol acrylate is 10000 MW, the molecular weight of thiol-polyethylene glycol-thiol is 3400 MW, and the molar ratio of the two is 1: 2. The addition amount of four-arm ethylene glycol acrylate in the reaction system is 0.064~0.128 mg / μL, and the addition amount of thiol-polyethylene glycol-thiol is 0.044~0.088 mg / μL.

[0019] The present invention shows that the hydrogel sheet formed by using the above-mentioned ratio of hydrogel monomers has good bending and stretching capabilities, and can be closely attached to the uneven surface of solid samples. It also has good thermal stability and can maintain integrity under nucleic acid amplification reaction conditions. Its light transmittance meets the requirements of fluorescence imaging analysis and can be used as a flexible and conformable sensing device.

[0020] The research of the present invention shows that the in situ amplification effect of the curved and rough sample surface is affected by the thickness of the hydrogel sheet. For flat solid samples, a hydrogel patch with a thickness of 200-400 μm can achieve nucleic acid; for non-flat solid samples such as those with curved and rough surfaces, a hydrogel patch with a thickness of 400-800 μm is used.

[0021] Preferably, the hydrogel monomer and the nucleic acid amplification reagent are mixed and added to the plasma-treated gelling mold, and then placed at room temperature for cross-linking to form a hydrogel sheet. The hydrogel sheet is prepared in the plasma-treated gelling mold so that the complete hydrogel sheet can be taken out. Specifically, the gelling mold can adopt a commercial sealed chamber.

[0022] Preferably, the plasma treatment is to place the gel-forming mold in a vacuum environment and perform plasma treatment for 2 to 5 minutes.

[0023] The present invention selects corresponding reagents to prepare a nucleic acid amplification reaction system according to a specific nucleic acid amplification reaction mode. When the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, the reaction system is prepared as follows:

[0024] When the target is DNA, the reaction system I consists of: 1× isothermal amplification buffer, 6 mM MgSO 4 , 1.4 mM dNTPs, 640 U / mL Bst 2.0 DNA polymerase, fluorescent dye, 0.064 mg / μL four-arm ethylene glycol acrylate, 0.044 mg / μL thiol-polyethylene glycol-thiol, and 1× LAMP primer mix, the LAMP primer mix is ​​1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.4 μM LF and LB.

[0025] When the detection target is bacteria, 1.0 mg / mL BSA and 0.1 mg / mL lysozyme are added to reaction system I.

[0026] When the detection target is virus or RNA, 300 U / mL reverse transcriptase is added to reaction system I.

[0027] The fluorescent dye is any one of Eva Green, STBR Green, Syto 9 and SYTOX Orange.

[0028] In step (2), the hydrogel sheet after cross-linking at room temperature is covered on the surface of the solid sample to be tested to fix the target object (microorganism or nucleic acid molecule) in situ. Specifically, it is pressed for 1-3 minutes to make the hydrogel patch fully contact with the solid substrate. Then the hydrogel reaction system is sealed to perform interfacial nucleic acid amplification. Under the three-dimensional nano-confinement effect of the hydrogel, the target detection object undergoes nucleic acid amplification at the original position, and the amplification product is also confined to the original position. The fluorescent dye stains the nucleic acid amplification product to form a single fluorescent spot.

[0029] In the present invention, the hydrogel reaction system is placed in a sealed space to prevent water evaporation to ensure the accuracy of in-situ detection of different types of targets.

[0030] Furthermore, when the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, in step (2), the entire solid sample with the hydrogel sheet attached is placed under a constant temperature condition for incubation, and the reaction condition is heated at 63-68° C. for 10-20 min. The time of isothermal amplification is adjusted for different types of targets.

[0031] Specifically, for flat inorganic samples such as glass and plastic lunch boxes, a 200 μm thick hydrogel sheet was heated at 65°C for 10 min; for flat plant samples such as tea leaves and holly leaves, a 200 μm thick hydrogel sheet was heated at 65°C for 20 min; for non-flat plant samples such as lettuce, grapefruit leaves, apples, cherry tomatoes, and dates, a 400 μm thick hydrogel sheet was heated at 65°C for 20 min.

[0032] In step (3), the hydrogel in-situ reaction system on the solid sample is imaged under the excitation light source corresponding to the fluorescent dye. The hydrogel of the present invention is a three-dimensional network structure, and the pathogenic microorganisms or nucleic acid molecules are confined in the pores of the interface hydrogel layer. After the amplification reaction, a single fluorescent bright spot is formed in situ, and the original position and spatial distribution of the target object to be detected on the surface of the solid sample are retained. It is a universal method for in-situ detection of different pathogenic microorganisms.

[0033] The software or manual counting is used to count the fluorescence points, and the in-situ absolute quantitative detection of pathogenic microorganisms or nucleic acid molecules is achieved according to the number of amplification points. Manual counting can achieve the counting of fluorescence points at lower concentrations. When the number of template molecules is large, software is also needed to count them.

[0034] Preferably, the in situ hydrogel imaging images are binarized using Image J software, and the adjacent amplification points are segmented and accurately counted using the watershed function.

[0035] Furthermore, for solid samples with strong autofluorescence interference or large curved surfaces, in step (3), the hydrogel sheet is torn off to observe the fluorescence spots by in situ imaging to determine the spatial contamination distribution of nucleic acids or microorganisms on the surface of the solid sample.

[0036] The present invention has the following beneficial effects:

[0037] (1) The present invention provides a method for in-situ detection of nucleic acids or pathogenic microorganisms on the surface of solid samples such as food and packaging materials using a hydrogel sheet. The flexible hydrogel sheet has good bending, stretching and adhesion properties. After the prefabricated hydrogel sheet is covered on the surface of solid samples of different forms, it can fit closely with it. Under the three-dimensional nano-confinement effect of the hydrogel, the larger template molecules are confined to the initial position on the surface of the solid sample, while the smaller molecules such as primers can move freely. After the interfacial nucleic acid amplification, the nucleic acid product is confined in situ to form a single fluorescent bright spot under the excitation light source, and the spatial distribution of the target object is reflected by the position of the fluorescent spot. The in-situ imaging method provided by the present invention is versatile, and the types of solid samples that can be detected are particularly wide, including inorganic and organic material samples commonly found in life such as glass, polypropylene plastic, ceramics and stainless steel metal, as well as fruits such as apples, cherry tomatoes and dates, edible biological samples such as tea and lettuce, or in vitro biological tissue slices.

[0038] (2) The method provided by the present invention can in situ image the "coffee ring" phenomenon of nucleic acid contamination in aerosol droplets on the surface of solid samples, indicating that the positions before and after amplification are consistent. Hydrogel interface amplification amplifies the target signal in situ, and the fluorescence signal of the amplified product reflects the position distribution of the target on the solid sample, which simply and intuitively reflects the contamination law of the target on the surface of the solid sample by aerosol droplet contamination.

[0039] (3) The universal in situ imaging method provided by the present invention can detect different pathogenic bacteria, viruses and nucleic acid molecules. By selecting suitable specific amplification primers, different types of targets can be imaged in situ. This method can be applied to fields involving various solid sample detection, such as food, environment, clinical, and agriculture, to achieve rapid digital quantitative detection of different pathogenic microorganisms or nucleic acid molecules to be tested on the surface, without the need for pretreatment such as surface smearing, and is suitable for in situ imaging of pathogenic microorganisms on site or in resource-poor areas. It has extremely high application value in the fields of in situ detection of solid samples and traceability of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Comparison of monomer content of hydrogel patches. Rheological modulus (a), gelation effect of 20% and 10% hydrogel monomer (b).

[0041] Figure 2 Characterization of the flexibility of the hydrogel patch. Flexibility (a), stretchability (b), light transmittance (c), and thermal stability (d) of the hydrogel patch.

[0042] Figure 3 Comparison of hydrogel patch interfacial amplification for inorganic solid samples, flat and rough biological samples.

[0043] Figure 4Absolute quantitative endpoint diagram (a) and spike statistics (b) of in situ LAMP on hydrogel patches at different template concentrations.

[0044] Figure 5 The in situ detection performance of hydrogel LAMP for the target. The in situ imaging position before and after isothermal amplification.

[0045] Figure 6 The hydrogel patch image (a), amplification point image and laser confocal image (b) of the hydrogel on the curved leaf surface.

[0046] Figure 7 In situ imaging of nucleic acids and viruses in aerosol droplets on solid sample surfaces using hydrogel patches.

[0047] Figure 8 In situ imaging of hydrogel patches of SARS-CoV-2 (a), Escherichia coli (b), and Listeria monocytogenes (c) on the surface of inorganic solid samples.

[0048] Fig. 9 In situ imaging images of hydrogel patches of tea leaves (a), Escherichia coli on the surface of fruits (b), and Listeria monocytogenes on the surface of lettuce (c). DETAILED DESCRIPTION

[0049] In order to further explain the present invention in detail, the following specific embodiments are used for explanation. The embodiments herein are not intended to limit the scope of application of the present invention. Without departing from the essence and spirit of the present invention, any modification or replacement of the technical solution of the present invention shall fall within the protection scope of the present invention.

[0050] In the following embodiments, the pathogenic virus is represented by the new coronavirus, the pathogenic Gram-negative bacteria are represented by Escherichia coli, and the pathogenic Gram-positive bacteria are represented by Listeria monocytogenes.

[0051] The bacterial genomic DNA was extracted by the kit and stored at -20°C for future use.

[0052] The sequences of the novel coronavirus LAMP amplification primers are as follows:

[0053] F3: 5'-GTTCCTCATCACGTAGTCG-3';

[0054] B3: 5'-GTTTGGCCTTGTTGTTGTT-3';

[0055] FIP: 5'-GCCAGCCATTCTAGCAGGAGCAACAGTTAAGAAATTCAACTCC-3';

[0056] BIP: 5'-GATGCTGCTCTTGCTTTGCTACCAGACATTTTGCTCTCAA-3';

[0057] LB: 5'-GCTGCTTGACAGATTGAACCAG-3';

[0058] The sequences of primers and probes for E. coli LAMP amplification are as follows:

[0059] F3: 5'-GCCATCTCCTGATGACGC-3';

[0060] B3: 5'-ATTTACCGCAGCAGACG-3';

[0061] FIP: 5'-CATTTTGCAGCTGTACGTCTCGCAGCCCATCATGAATGTTGCT-3';

[0062] BIP: 5'-CTGGGGCGAGGTCGTGGTATTCCGACAAACACCACGAATT-3';

[0063] LF: 5'-CTTTGTAACAACCTGTCATCGACA-3';

[0064] LB: 5'-ATCAATCTCGATATCCATGAAGGTG-3';

[0065] The sequences of primers and probes for Listeria monocytogenes LAMP amplification are as follows:

[0066] F3: 5'-AAGCTGCTTTTGATGCTG-3';

[0067] B3: 5'-TCGATTAAAAGTAGCGCCTT-3';

[0068] FIP: 5'-CGGCTTTGAAGGAAGAATTTTTGATCGTAAGCGGAAAATCCTGT-3';

[0069] BIP: 5'-TACGGAGGTTCCGCAAAAGATTTTCAAAATATCGCGTAAGTCT-3';

[0070] LB: 5'-TGAAGTTCAAATCATCGACGGC-3';

[0071] All LAMP primers, probes, and SARS-CoV-2 RNA were synthesized by Shanghai Bioengineering Co., Ltd. SARS-CoV-2 pseudovirus was purchased from Xiamen Zhishan Biotechnology Co., Ltd. LAMP amplification reagents were purchased from New England Biolab. 4 Arm-PEG-acrylate (molecular weight 10,000) and SH-PEG-SH (molecular weight 3,400) were purchased from Xi'an Kaixin Biological Co., Ltd. Sealed chambers were purchased from BIO-RAD.

[0072] Example 1

[0073] 1. Comparison of the content of gelling monomers in hydrogel patch in-situ devices

[0074] In this example, the storage modulus and loss modulus of three different contents of hydrogel monomers in the in-situ device system were measured under a rheometer to determine whether the two hydrogel monomers had gelation potential. Hydrogels with different monomer contents were prepared at the same time to select the best patch composition.

[0075] 1. Prepare the mother solution: dissolve 64 mg 4 Arm-PEG-acrylate in sterile water and dilute to 100 μL to obtain 4 Arm-PEG-acrylate mother solution; dissolve 44 mg SH-PEG-SH in sterile water and dilute to 100 μL to obtain SH-PEG-SH mother solution for subsequent addition.

[0076] 5% monomer content hydrogel: Take 2.5 μL of 4-Arm-PEG-acrylate stock solution and 2.5 μL of SH-PEG-SH stock solution, and make up to 100 μL with sterile water;

[0077] 10% monomer content hydrogel: Take 5 μL of 4-Arm-PEG-acrylate stock solution and 5 μL of SH-PEG-SH stock solution, and make up to 100 μL with sterile water;

[0078] 20% monomer content hydrogel: Take 10 μL of 4-Arm-PEG-acrylate stock solution and 10 μL of SH-PEG-SH stock solution, and make up to 100 μL with sterile water.

[0079] like Figure 1 As shown in a, the storage modulus of the hydrogels with 5%, 10% and 20% monomer content are all higher than the loss modulus, indicating that the two hydrogel monomers have the potential to gel at room temperature.

[0080] 2. Prepare hydrogel patches with 10% and 20% monomer content in a sealed chamber, seal and let stand at room temperature for 5 min. Figure 1As shown in b, both 10% and 20% hydrogels have become gelled, but the 20% hydrogel has formed a patch and can be completely removed, while the 10% hydrogel is only in a viscous gel state. By comparing the gelling states of the two with different contents, the hydrogel patch with 20% monomer content was selected as the in-situ device.

[0081] 2. Characterization of flexible hydrogel patch in situ devices

[0082] The flexibility, light transmittance and thermal stability characteristics of the hydrogel patch with 20% monomer content were investigated.

[0083] like Figure 2 As shown in a, the hydrogel patch of this embodiment has excellent bending, stretching and adhesion properties. There is no residue when peeling off the solid sample, and all are in a complete patch state.

[0084] like Figure 2 As shown in b and c, the hydrogel patch was measured using a universal material testing machine and a spectrophotometer to have good tensile strength and light transmittance, indicating that the hydrogel can be flexibly adhered to the uneven solid sample and will not hinder the imaging of the amplification point between the hydrogel patch and the solid sample.

[0085] like Figure 2 As shown in Figure d, the hydrogel in the sealed chamber remained intact after being placed at 65°C and 25°C for 60 min, and could be taken out intact in the form of a gel patch, indicating that the patch had good thermal stability at room temperature and amplification heating temperature.

[0086] The above results show that the hydrogel patch provided by the present invention has good flexibility, has high adhesion to the surface of solid samples, and maintains good integrity at the amplification temperature.

[0087] 3. Comparison of in situ imaging of solid inorganic samples and hydrogel patch amplification of biological samples

[0088] 1. Use a glass slide as a flat inorganic solid sample, flat Pittosporum tobira leaves and rough pomelo leaves as biological solid samples, and the detection object is represented by the nucleic acid molecules of the new coronavirus. Apply the new coronavirus RNA to the surface of the solid sample to be tested and use it for detection after drying.

[0089] 2. Prepare hydrogel patch: Mix hydrogel monomer with LAMP reaction reagent, add into sealed chamber, crosslink and gel at room temperature. Specifically, 1× isothermal amplification buffer, 6 mM MgSO 4, 1.4 mM dNTPs, 640 U / mL Bst 2.0 DNA polymerase, 1×SYBR Green, 20% hydrogel monomer, LAMP primer mixture (1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.4 μM LB), 300 U / mL reverse transcriptase, and the system was supplemented to 25 μL with sterile deionized water.

[0090] 3. The formed hydrogel patch is attached to the surface of the solid inorganic sample and the biological sample respectively, so that the target to be tested is fixed in situ at the interface layer between the hydrogel patch and the solid sample. The entire reaction system is sealed with a sealing chamber and a cover.

[0091] 4. The entire solid sample to be tested is heated at a constant temperature of 65°C to perform an in situ interface LAMP amplification reaction.

[0092] 5. Use the SYBR Green excitation light source of an ordinary fluorescence microscope to illuminate the hydrogel in situ detection system, showing the fluorescent bright spots formed by the hydrogel patch on the solid sample, take images, and compare the in situ imaging results of the hydrogel patch on the solid inorganic sample and the biological sample.

[0093] like Figure 3 As shown, the hydrogel patch can achieve nucleic acid amplification on the inorganic glass surface in just 10 minutes, but no fluorescent spots can be observed in biological leaf samples under the same conditions.

[0094] The present invention improves the in-situ imaging process based on the amplification characteristics of the solid biological sample surface. When the heating time is extended to 20 minutes, nucleic acid fluorescence bright spots can be amplified on flat biological leaves, but nucleic acids on rough biological leaf samples cannot be amplified. This may be due to the thickness of the hydrogel patch, which is affected by the curved and rough sample.

[0095] The present invention compares the in situ imaging of hydrogel patches with thicknesses of 200 μm and 400 μm on rough leaf surfaces. The nucleic acid interface amplification of the 400 μm hydrogel patch is significantly improved, showing clear fluorescent bright spots.

[0096] By comparison, it was found that there were differences in the amplification of in situ imaging technology on different solid sample surfaces. Inorganic glass was heated in situ for 10 min with a 200 μm thick hydrogel patch, flat biological samples were heated in situ for 20 min with a 200 μm thick hydrogel patch, and rough biological samples were heated in situ for 20 min with a 400 μm thick hydrogel patch.

[0097] Example 2

[0098] This embodiment uses a flexible hydrogel patch in situ imaging device to quantify the target on the surface of a solid sample. Specifically, a commercial glass slide is used as a solid sample, and the new coronavirus RNA molecule is used as a representative of the target. Different concentrations of the new coronavirus RNA are applied to the glass slide and dried for detection.

[0099] 1. The sealed chamber was subjected to plasma treatment for 2 min in a vacuum environment to remove the completely cross-linked hydrogel sheet. Then the LAMP reaction system and hydrogel monomer mixture were added to gel at room temperature to synthesize a flexible hydrogel sheet with biological functions.

[0100] The components of the hydrogel sheet in situ reaction system include: 1× isothermal amplification buffer, 6 mM MgSO 4 , 1.4 mM dNTPs, 640 U / mL Bst 2.0 DNA polymerase, 1× SYBR Green, 20% hydrogel monomer, 1× LAMP primer mixture (1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.4 μM LB), 300 U / mL reverse transcriptase, and the system was supplemented to 25 μL with sterile deionized water.

[0101] 2. Attach the flexible hydrogel sheet to the area to be tested on the surface of the solid sample, and confine the target object in situ in the middle interface layer between the solid sample and the hydrogel sheet.

[0102] 3. The entire solid sample to be tested is heated at a constant temperature to perform an in-situ interface LAMP amplification reaction;

[0103] 4. After amplification, observe the in-situ imaging signal under the excitation light source of SYBR Green, showing the fluorescent bright spots formed by nucleic acid molecules on the solid sample. If the solid sample's own fluorescence interference is too strong or the curved surface is large, tear off the hydrogel sheet for observation, count the in-situ imaging fluorescent spots, and obtain the contamination concentration and original position of the nucleic acid on the surface of the solid sample.

[0104] The present invention uses different gradients of template concentration to evaluate the quantitative performance of hydrogel in situ imaging. Figure 4 As shown in a, templates of different concentrations can produce clear fluorescent spots. As the concentration of template molecules increases, the number of fluorescent spots also increases. Figure 4 As shown in b, the number of amplification points and template concentration show a positive linear relationship (R 2 =9962), indicating that the hydrogel in situ detection method has high accuracy.

[0105] Example 3

[0106] This embodiment compares the positions of the target object on the surface of the solid sample before and after in situ imaging. Taking Escherichia coli as a representative, the specific positions of the bacteria before and after in situ amplification in the hydrogel are compared.

[0107] 1. Add 1×SYBR Green to the bacteria before amplification and stain in the dark for more than 30 min to allow the dye to fully stain the bacterial DNA. Fix the stained bacteria on a slide.

[0108] 2. Mix the hydrogel monomer with the LAMP reaction reagent and cross-link to form a gel at room temperature to prepare a hydrogel sheet with a thickness of 200 μm. The composition of the E. coli in situ reaction system includes: 1× isothermal amplification buffer, 6 mM MgSO 4 , 1.4 mM dNTPs, 640 U / mL Bst 2.0 DNA polymerase, 1× SYBR Green, 20% hydrogel monomer and 1× LAMP primer mixture (1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.4 μM LF and LB), 1.0 mg / mL BSA, 0.1 mg / mL lysozyme, and the system was supplemented to 25 μL with sterile deionized water.

[0109] 3. The hydrogel sheet and the sealed chamber formed by room temperature cross-linking are attached to the stained bacterial slide, and the cover is used to seal the entire reaction system for subsequent in situ amplification of the hydrogel sheet.

[0110] 4. Heat at 65°C for 10 min.

[0111] 5. Image the same position of the bacteria stained before amplification and the solid sample after in situ amplification under the SYBR Green excitation light source of an ordinary fluorescence microscope, take photos, and compare and observe the imaging positions.

[0112] 6. Result analysis:

[0113] The position of bacteria before and after in situ amplification of the hydrogel was photographed using a microscope objective lens at 10× magnification. Figure 5 As shown in the figure, 1×SYBR Green was used to stain bacteria in the dark reaction before amplification, which showed very weak fluorescence. Even under high exposure conditions, it was impossible to observe clear targets, and the background fluorescence interference was serious, and it was impossible to distinguish bacterial fluorescence from other fluorescent impurities. However, after the bacteria stained on the sample surface were amplified in situ, under the same shooting conditions and positions (there were corresponding weak fluorescent spots at the same positions before amplification), obvious fluorescent amplification spots could be seen, the signal-to-noise ratio was high, the background fluorescence interference was weak, and the fluorescent impurities and detection targets could be distinguished, indicating excellent in situ imaging capabilities.

[0114] The above description of the hydrogel interface detection method provided by the present invention can confine and amplify pathogenic microorganisms or nucleic acids on the surface of solid samples in situ, and the position of the target object remains highly consistent before and after the reaction, and the spatial position of the target object on the solid sample can be accurately imaged.

[0115] Example 4

[0116] This example explores the in-situ interface imaging of nucleic acids on the surface of rough solid samples using flexible hydrogel patches. A grapefruit leaf with a relatively large curved surface was used as a rough sample for hydrogel in-situ interface LAMP amplification, and the detection object was represented by the nucleic acid molecules of the new coronavirus.

[0117] 1. The hydrogel monomer was mixed with the LAMP reaction reagent. The composition of the reaction system was the same as that of Example 2. The hydrogel sheet with a thickness of 400 μm was prepared by crosslinking and gelation at room temperature.

[0118] 2. A 400 μm thick hydrogel sheet is attached to the curved blade, so that the target to be tested is fixed in situ at the interface layer between the hydrogel sheet and the solid sample. The entire reaction system is sealed with a sealing chamber and a cover sheet.

[0119] 3. The isothermal amplification reaction conditions are heating at 65°C for 20 min.

[0120] 4. Use the SYBR Green excitation light source of the laser confocal fluorescence microscope to illuminate the hydrogel in situ detection system on the rough solid sample, select the appropriate z-axis height range for shooting, scan and shoot layer by layer and integrate them to obtain a three-dimensional image of a single fluorescent amplification point on the solid sample, and observe the microscopic morphology of a single amplification point at the interface layer between the hydrogel and the solid sample.

[0121] 5. Result analysis:

[0122] like Figure 6 As shown in a, the hydrogel sheet has flexible and conformable properties, and the leaf vein pattern can be clearly presented on the hydrogel. Figure 6 As shown in b, the single-molecule fluorescence amplification point is obviously located at the interface where the hydrogel layer and the solid sample contact, and the three-dimensional structure presents a morphology similar to that of a hemisphere. This indicates that the template molecules on the lower solid sample are fixed in situ under the confinement of the upper hydrogel. As the heating time increases, the template molecules begin to amplify, the concentration of the nucleic acid clusters continues to gather and increase, and gradually diffuses into the surrounding hydrogel matrix, eventually forming a hemispherical state that diffuses from the bottom to the surrounding. In addition, there are inconsistent micro-heights at the bottom of the nucleic acid amplification cluster on the rough solid sample surface. The three-dimensional structure of the amplification point can clearly present its specific position. There is a certain height at different amplification point positions on the curved blade and the hydrogel interface layer, directly imaging the spatial distribution and position of the target on the curved blade.

[0123] The above results all illustrate that the hydrogel sheet provided by the present invention has good flexibility and conformability, and can confine the amplification target to the interface between the solid sample surface and the hydrogel layer for in situ amplification and detection.

[0124] Example 5

[0125] This example explores the in situ imaging of hydrogels on the surface of solid samples contaminated by aerosol droplets of nucleic acids and viruses. Specifically, a glass slide is used as a representative, and a 6 μL droplet containing nucleic acid molecules (E. coli DNA, SARS-CoV-2 RNA, and SARS-CoV-2 pseudovirus particles containing a protein shell) is added to simulate the aerosol droplets in the sneezing process, and the aerosol droplets are used after drying.

[0126] 1. Mix the hydrogel monomer with the LAMP reaction reagent, cross-link and gel at room temperature to prepare a hydrogel sheet with a thickness of 200 μm.

[0127] 2. The formed hydrogel sheet and sealed chamber are attached to the surface of the solid sample contaminated by aerosol droplets; the nucleic acid to be tested is fixed in situ at the interface layer between the hydrogel sheet and the solid sample, and the cover sheet is used to seal the entire reaction system for isothermal amplification, which is heated at 65°C for 10 min.

[0128] 3. Use the SYBR Green excitation light source of an ordinary fluorescence microscope to illuminate the hydrogel in situ detection system, showing the fluorescent bright spots formed by nucleic acids and viruses on the solid sample, and take photos.

[0129] 4. Result analysis:

[0130] Small molecules such as nucleic acids and viruses cannot be seen with the naked eye without signal amplification, and the distribution of aerosol droplet contamination on solid samples cannot be clearly determined. Currently, there is no report on the distribution of nucleic acid contamination in aerosol droplets. This embodiment provides a method for in-situ spatial imaging of nucleic acid and virus aerosol droplet contamination, such as Figure 7 As shown in the figure, after the aerosol droplets of E. coli DNA, SARS-CoV-2 RNA, and SARS-CoV-2 pseudovirus particles on the solid sample dried, they all showed the "coffee ring" phenomenon, which was more obvious at higher concentrations, similar to the "coffee ring" of nanomaterials. Hydrogel interfacial amplification amplifies the target signal in situ, and the fluorescent signal of the amplified product reflects the spatial position distribution of the target on the solid sample, which simply and intuitively shows the contamination distribution pattern of nucleic acids or virus small molecules on the surface of the solid sample by aerosol droplet contamination.

[0131] Example 6

[0132] This example explores the in-situ imaging of pathogenic microorganisms on the surface of various inorganic solid samples by in-situ interface of flexible hydrogel patches. Specifically, glass, polypropylene plastics, ceramics and stainless steel metals commonly used in life are used as representatives, and droplets containing new coronavirus, Escherichia coli or Listeria monocytogenes are sprayed with a spray bottle to simulate the contamination of pathogenic bacteria during irrigation, rain and sneezing, and then dried for use.

[0133] 1. The hydrogel monomers were mixed with LAMP reaction reagents. The compositions of the in-situ reaction systems of various pathogenic microorganisms were referred to Examples 2 and 3. The hydrogel sheets with a thickness of 200 μm were prepared by crosslinking and gelation at room temperature.

[0134] 2. The formed hydrogel sheet and the in-situ sealed chamber are attached to the area to be tested on the surface of various inorganic solid samples, so that the new coronavirus, Escherichia coli or Listeria monocytogenes to be tested are fixed in situ at the interface layer between the hydrogel sheet and the inorganic solid sample. The cover sheet is used to seal the entire reaction system for isothermal amplification and heated at 65°C for 10 min.

[0135] 3. Use the SYBR Green excitation light source of an ordinary fluorescence microscope to illuminate the hydrogel in situ detection system, showing the fluorescent bright spots formed by Escherichia coli or Listeria monocytogenes on the inorganic solid sample, and take photos.

[0136] 4. Result analysis:

[0137] like Figure 8 As shown in the figure, on the surfaces of inorganic solid samples of various materials such as glass, polypropylene plastic, ceramics and stainless steel, the hydrogel patch can be used to image the new coronavirus, Escherichia coli or Listeria monocytogenes in situ. This shows that the hydrogel in situ imaging method has high versatility on the surfaces of inorganic solid samples of various materials, and can realize the in situ rapid detection of pathogenic microorganisms on the surfaces of container materials that are commonly used or contacted in the fields of food, environment, medical treatment and agriculture.

[0138] Example 7

[0139] This embodiment explores the in-situ imaging of Escherichia coli or Listeria monocytogenes on the surface of biological solid samples by in-situ interface of flexible hydrogel patches. Specifically, various biological solid samples are used as representatives for hydrogel in-situ imaging, including the detection of Escherichia coli on the surface of tea leaves and fruits such as apples, cherry tomatoes and dates, as well as Listeria monocytogenes on the surface of raw lettuce that is often eaten directly.

[0140] 1. The hydrogel monomers were mixed with LAMP reaction reagents. The composition of the in-situ reaction system of various pathogenic microorganisms was referred to Example 2 and Example 3. The hydrogel sheets with a thickness of 400 μm were prepared by crosslinking and gelation at room temperature.

[0141] 2. The formed hydrogel sheet is attached to the area to be tested on the surface of various biological solid samples, and the Escherichia coli or Listeria monocytogenes to be tested is fixed in situ at the interface layer between the hydrogel sheet and the biological solid sample. The entire reaction system is sealed with a sealing chamber and a cover sheet for isothermal amplification and heated at 65°C for 20 min.

[0142] 3. Use the SYBR Green excitation light source of a common fluorescence microscope to illuminate the hydrogel in-situ detection system, showing the fluorescent bright spots formed by Escherichia coli or Listeria monocytogenes on the biological solid sample, and take photos. Convert the bright field field to take photos of the biological sample at the same position to observe the contamination distribution of the target object on the biological solid sample.

[0143] 4. Result analysis:

[0144] like Fig. 9 As shown in a and b, Escherichia coli on the surface of tea leaves and fruits such as apples, cherry tomatoes and dates can be amplified and imaged in situ, indicating that flexible hydrogels have excellent properties for in situ detection on the surface of biological edible solid samples.

[0145] like Fig. 9 As shown in Figure c, Listeria monocytogenes can also be imaged in situ on the surface of lettuce. When the fluorescence image of the amplification point and the bright field image of the lettuce are combined, the amplification product is mainly located in the leaf surface of the lettuce, rather than the veins and stems. This indicates that pathogenic microorganisms tend to contaminate the leaf surface area of ​​the lettuce, clarifying the distribution of contamination on biological solid samples.

[0146] The above embodiments are only explanations of the present invention, but the implementation of the present invention is not limited by the above embodiments. For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. An in situ spatial imaging method for single molecules or single cells on the surface of solid samples, characterized in that: The following steps are involved: (1) adding a hydrogel monomer to a nucleic acid amplification reaction system without an amplification template to obtain a mixed solution, wherein the reaction system includes primers for specifically amplifying a target nucleic acid DNA fragment and a fluorescent dye, and adding the mixed solution to a gel-forming mold after plasma treatment to form a hydrogel sheet with a thickness of 200 to 400 μm as a flexible conformable device; The hydrogel monomers include four-arm ethylene glycol acrylate and mercapto-polyethylene glycol-mercapto, the molecular weight of the four-arm ethylene glycol acrylate is 10000 MW, the molecular weight of the mercapto-polyethylene glycol-mercapto is 3400 MW, the addition amount of the four-arm ethylene glycol acrylate in the reaction system is 0.064 mg / μL, and the addition amount of the mercapto-polyethylene glycol-mercapto is 0.044 mg / μL; The nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction; (2) During the test, the flexible hydrogel sheet is directly attached to the surface of the solid sample to be tested, and the hydrogel sheet is pressed so that the hydrogel sheet is in full contact with the sample surface, and then the sample attached to the hydrogel sheet is placed under nucleic acid amplification conditions for nucleic acid amplification reaction; the solid sample is any one of glass, plastic, ceramic, metal, leaves, vegetables, and fruits; (3) Fluorescence imaging technology is used to analyze the fluorescence signal on the hydrogel sheet after the nucleic acid amplification reaction, calculate the amount of nucleic acid or microbial contamination on the surface of the solid sample, and determine the spatial distribution location of the contamination.

2. The in situ spatial imaging method for single molecules or single cells on the surface of solid samples according to claim 1, characterized in that: The plasma treatment is to place the gel-forming mold in a vacuum environment and perform plasma treatment for 2 to 5 minutes.

3. The in situ spatial imaging method for single molecules or single cells on the surface of solid samples according to claim 1, characterized in that: When the detection target is DNA, the reaction system I consists of: 1× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTPs, 640 U / mL Bst 2.0 DNA polymerase, fluorescent dye, 0.064 mg / μL four-arm ethylene glycol acrylate, 0.044 mg / μL thiol-polyethylene glycol-thiol and 1×LAMP primer mixture, the LAMP primer mixture is 1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.4 μM LF and LB; when the detection target is bacteria, 1.0 mg / mL BSA and 0.1 mg / mL lysozyme are added to reaction system I; when the detection target is virus or RNA, 300 U / mL reverse transcriptase is added to reaction system I.

4. The in situ spatial imaging method for single molecules or single cells on the surface of solid samples according to claim 1, characterized in that: In step (2), the entire solid sample with the hydrogel sheet attached is placed under constant temperature conditions for incubation, and the reaction conditions are heating at 63-68° C. for 10-20 min.

5. The in situ spatial imaging method for single molecules or single cells on the surface of solid samples according to claim 4, characterized in that: For flat solid samples, a hydrogel sheet with a thickness of 200-400 μm was used and heated at 65°C for 10 min. For non-flat solid samples, a hydrogel sheet with a thickness of 400 μm was used and heated at 65°C for 20 min.

6. The in situ spatial imaging method for single molecules or single cells on the surface of solid samples according to claim 1, characterized in that: For solid samples with strong autofluorescence interference or large curved surfaces, in step (3), the hydrogel sheet is torn off to observe the in situ imaging fluorescence points to determine the spatial contamination distribution of nucleic acids or microorganisms on the surface of the solid sample.

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