A full-printing photonic crystal biochip and a preparation method and application thereof

Abstract

This invention belongs to the fields of materials science and biology, and relates to a fully printed photonic crystal biochip, its fabrication method, and its applications. The chip includes a substrate and a photonic crystal microarray disposed on at least one surface of the substrate. The photonic crystal microarray includes at least two photonic crystal units, a capture antibody disposed on the surface of the photonic crystal unit, and a soluble spot disposed around the periphery of the photonic crystal unit. The soluble spot includes a detection antibody, and the capture antibody and the detection antibody are used to bind to an antigen. This invention enables real-time detection of trace samples with high sensitivity and a wide detection range, and can be used for clinical testing.

Description

Technical Field

[0001] This invention belongs to the fields of materials science and biology, and in particular relates to a miniaturized, integrated, fully printed photonic crystal biochip, its preparation method and application, such as its application in detecting biomarkers in a drop of blood. Background Technology

[0002] Blood analysis is an important traditional method for disease detection. Depending on the needs of the analysis, medical staff need to collect venous or capillary blood once or multiple times. In order to obtain more stable test results, the amount of blood collected is usually greater than the actual amount needed, with each collection being 1-3 mL, resulting in a large waste of blood. At the same time, the use of lancets to prick the skin during blood collection can cause pain and skin trauma. People who are more sensitive to pain, such as infants and young children, may react violently, making blood collection difficult. There are many shortcomings in using large amounts of blood for health checks, blood donor physical examinations, and clinical examinations, making it difficult to carry out screening for infectious diseases, such as rapid home testing for COVID-19.

[0003] Protein microarrays are devices that integrate multiple active protein molecules on a small area. Using trace amounts of physiological or biological sampling, they can simultaneously detect and study the interactions between different biomolecules and the expression of gene functions, obtain changes in the proteome under various conditions, and enable disease protein diagnosis and gene function research. With the increasing maturity and refinement of protein microarray technology, it has been widely applied in clinical medicine, enabling the detection of multiple antigens and antibodies. Examples include detecting multiple tumor antigens with a single drop of blood; pre-transfusion screening for hepatitis B, hepatitis C, HIV, and syphilis in blood recipients; screening organ transplant recipients for matching and blood typing; detection of infertility antibodies for prenatal and postnatal care; and detection of antibodies against autoimmune diseases.

[0004] However, the current protein biochip preparation process is quite complex, requiring the manual addition of different materials one by one, which is difficult to operate, has a long production cycle, and the manual operation is subject to unavoidable errors, resulting in poor repeatability between different batches of products, making it difficult to achieve mass production. At the same time, the detection sensitivity of existing protein biochips is low, with a linear detection range of several hundred ug / mL, which is of little significance for actual clinical testing.

[0005] Therefore, it is particularly important to develop a fast, high-throughput, miniaturized, and low-cost detection platform or method. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention provides a fully printed photonic crystal biochip, the chip comprising a substrate and a photonic crystal microarray disposed on at least one side surface of the substrate; the photonic crystal microarray comprises at least two photonic crystal units, a capture antibody disposed on the surface of the photonic crystal unit, and a soluble spot disposed around the periphery of the photonic crystal unit, the soluble spot comprising a detection antibody, the capture antibody and the detection antibody being used to bind to an antigen.

[0007] According to an embodiment of the present invention, when the detection antibody and the capture antibody bind to the antigen, they form a double-antibody sandwich detection system or a competitive detection system.

[0008] Preferably, the photonic crystal microarray is a dot pattern, a line pattern, or a surface pattern.

[0009] Preferably, the substrate is a hydrophobic substrate, for example, the contact angle of the substrate surface is greater than or equal to 80°.

[0010] According to an embodiment of the present invention, the photonic crystal in the photonic crystal unit is a photonic crystal with or without a linking group.

[0011] Preferably, the linking group includes at least one selected from carboxyl, hydroxyl, mercapto, and amino groups.

[0012] Preferably, the photonic crystal is at least one of opal photonic crystal, inverse opal photonic crystal, and two-dimensional photonic crystal.

[0013] According to an embodiment of the present invention, the capture antibody and the detection antibody are independently selected from antibodies, streptavidin, biotin, or base fragments capable of specifically binding to antigens.

[0014] Preferably, the detection antibody is attached to a receptor substance, and the receptor substance will generate fluorescence gain when it is close to the photonic crystal, thus amplifying the fluorescence signal of the receptor substance.

[0015] Preferably, the receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

[0016] A method for fabricating the above-mentioned fully printed photonic crystal biochip, the method comprising the following steps:

[0017] 1) Prepare photonic crystal ink, capture antibody solution, and detection antibody solution;

[0018] 2) A photonic crystal array comprising photonic crystal units is formed by printing photonic crystal ink on the substrate surface. A capture antibody solution is printed on the photonic crystal units to set the capture antibody on the surface of the photonic crystal units. A detection antibody solution is printed around the photonic crystal units to form soluble spots.

[0019] According to an embodiment of the present invention, the photonic crystal ink includes a photonic crystal, a humectant, and a wetting agent.

[0020] According to an embodiment of the present invention, the photonic crystal exists in the ink in the form of an emulsion, for example, the photonic crystal exists in the form of latex spheres.

[0021] Preferably, the photonic crystal latex spheres can be selected from at least one of photonic crystal-poly(methyl methacrylate-acrylic-styrene) latex spheres, photonic crystal-silica microspheres, and photonic crystal-polystyrene microspheres.

[0022] Preferably, in the photonic crystal ink, the mass ratio of the photonic crystal to the humectant and wetting agent is (80-90):(10-20):(0.1-1).

[0023] One claim is the application of the above-described fully printed photonic crystal biochip in biomarker detection.

[0024] A method for detecting biomarkers, the method comprising the following steps: contacting the sample to be tested with the above-described fully printed photonic crystal biochip, and measuring the fluorescence value.

[0025] According to an embodiment of the present invention, the method includes the following steps:

[0026] a) Contact the fully printed photonic crystal biochip with standard biomarker samples of different concentrations, measure the fluorescence value, and construct a standard curve for the biomarker;

[0027] b) The sample to be tested is brought into contact with the fully printed photonic crystal biochip, the fluorescence value is measured, and the content of the biomarker is calculated according to the standard curve.

[0028] Preferably, the volume of the sample to be tested is 10 μL to 50 μL.

[0029] Preferably, the method can be applied to the detection of a single drop of blood.

[0030] Preferably, the drop of blood can be a drop of venous blood or a drop of blood from a fingertip.

[0031] Beneficial effects

[0032] 1. This invention designs a universal photonic crystal biodetector chip. By printing a photonic crystal onto a biochip, capturing antibodies are printed on the photonic crystal, and detection antibodies labeled with fluorescent substances are printed around the photonic crystal. After the detection antibody binds to the antigen, due to the hydrophilic-hydrophobic patterned structure, the antibody-antigen conjugate will approach and accumulate towards the crystal detection unit and specifically bind to the capturing antibody. The photonic crystal produces a fluorescence enhancement effect on the fluorescent substances, enabling point-of-care testing (POCT) of trace samples. It has high detection sensitivity and a wide detection range, and can be used for clinical testing.

[0033] 2. This invention achieves full printing of photonic crystals, detection antibodies, and capture antibodies through full printing technology. The operation is simple and straightforward, and the entire chip preparation cycle can be controlled within 2 hours. Compared with the existing technology that involves manually preparing and then dropping the materials onto the substrate, this invention is not only more efficient and lower in cost, but also has better repeatability between different batches of products, enabling large-scale production and application, and facilitating clinical diagnostic use.

[0034] 3. The photonic crystal biochip of the present invention can realize the joint detection of multiple biomarkers and the detection of multiple samples on the same chip. At the same time, only a small amount of sample, such as a drop of blood (about 10μl to 50μl), is required for detection. Compared with the prior art, which requires multiple blood collections and each blood collection volume is 1ml to 3mL, it can reduce the number of blood collections and the amount of blood collected, thereby reducing the difficulty of detection caused by blood collection and facilitating the screening of infectious diseases. Attached Figure Description

[0035] Figure 1 This is a physical image of the photonic crystal biochip used in this invention.

[0036] Figure 2 This is a photograph of a photonic crystal biochip used in this invention to detect a drop of blood.

[0037] Figure 3 The standard curve is for the photonic crystal biochip (using CRP as an example) prepared in Example 2.

[0038] Figure 4 The standard curve is for the multifunctional photonic crystal biochip (CRP) prepared in Example 3. Detailed Implementation

[0039] [Fully Printed Photonic Crystal Biochip]

[0040] As described above, the present invention provides a fully printed photonic crystal biochip, which includes a substrate and a photonic crystal microarray disposed on at least one side surface of the substrate; the photonic crystal microarray includes at least two photonic crystal units, a capture antibody disposed on the surface of the photonic crystal unit, and a soluble spot disposed around the periphery of the photonic crystal unit, the soluble spot including a detection antibody, the capture antibody and the detection antibody being used to bind to an antigen.

[0041] According to an embodiment of the present invention, when the detection antibody and the capture antibody bind to the antigen, they form a double-antibody sandwich detection system or a competitive detection system.

[0042] According to an embodiment of the present invention, the soluble spots on the periphery of different photonic crystal units do not contact each other.

[0043] According to an embodiment of the present invention, the photonic crystal unit and the corresponding soluble spot do not contact each other. Preferably, the distance between the photonic crystal unit and the soluble spot is 0.1 mm to 1 mm. Further, the distance between the photonic crystal unit and the soluble spot is 0.3 mm to 1 mm, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1 mm.

[0044] According to the implementation method of the present invention, the substrate is a hydrophobic substrate. Specifically, the contact angle of the substrate surface is greater than or equal to 80°; further, the contact angle of the substrate surface is greater than or equal to 100°; exemplaryly, the contact angle of the substrate surface is 80° to 150°, for example, 80°, 85°, 88°, 90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145° or 150°.

[0045] According to the implementation method of the present invention, the material of the hydrophobic substrate is selected from hydrophobic plastics (such as PET), glass, or silicon wafers.

[0046] According to an embodiment of the present invention, the photonic crystal microarray is, for example, a dot pattern, a line pattern, or a surface pattern. Specifically, the photonic crystal microarray is a plurality of dot patterns, for example, forming a basic pattern as shown in the figure. Figure 1 The dot matrix pattern.

[0047] According to an embodiment of the present invention, the photonic crystal in the photonic crystal unit is a photonic crystal with or without a linking group.

[0048] Specifically, the linking group includes at least one of carboxyl, hydroxyl, mercapto, and amino groups, for example, the linking group is a carboxyl group.

[0049] According to an embodiment of the present invention, the photonic crystal is at least one of opal photonic crystal, inverse opal photonic crystal, and two-dimensional photonic crystal.

[0050] According to an embodiment of the present invention, the capture antibody and the detection antibody are independently selected from antibodies, streptavidin, biotin, or base fragments capable of specifically binding to antigens.

[0051] According to an embodiment of the present invention, the detection antibody is attached to a receptor substance, and the receptor substance generates fluorescence gain when it is close to the photonic crystal, thereby amplifying the fluorescence signal of the receptor substance.

[0052] According to an embodiment of the present invention, the receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

[0053] Preferably, the semiconductor nanocrystals are selected from at least one of silicon dioxide nanocrystals, titanium dioxide nanocrystals, zirconium oxide nanocrystals, zinc oxide nanocrystals, cadmium sulfide nanocrystals, cadmium telluride nanocrystals, gold nanodots, and silver nanodots.

[0054] Preferably, the fluorescent dye labeling molecule is selected from at least one of Rhodamine B, Rhodamine 6G, and fluorescein, for example, Rhodamine B.

[0055] [Preparation Method of Fully Printed Photonic Crystal Biochips]

[0056] This invention also provides a method for fabricating a fully printed photonic crystal biochip, the method comprising the following steps:

[0057] 1) Prepare photonic crystal ink, capture antibody solution, and detection antibody solution;

[0058] 2) A photonic crystal array comprising photonic crystal units is formed by printing photonic crystal ink on the substrate surface. A capture antibody solution is printed on the photonic crystal units to set the capture antibody on the surface of the photonic crystal units. A detection antibody solution is printed around the photonic crystal units to form soluble spots.

[0059] According to an embodiment of the present invention, the photonic crystal ink includes a photonic crystal, a humectant, and a wetting agent.

[0060] Preferably, the photonic crystal has the definition described above.

[0061] According to an embodiment of the present invention, the photonic crystal exists in the ink in the form of an emulsion.

[0062] According to an embodiment of the present invention, the photonic crystal exists in the ink in the form of latex spheres.

[0063] According to an embodiment of the present invention, the photonic crystal latex ball can be selected from at least one of photonic crystal-poly(methyl methacrylate-acrylic acid-styrene) latex balls, photonic crystal-silica microspheres, photonic crystal-polystyrene microspheres, etc., for example, photonic crystal-poly(methyl methacrylate-acrylic acid-styrene) latex balls.

[0064] Preferably, the latex balls are monodisperse latex balls.

[0065] According to an embodiment of the present invention, in the photonic crystal ink, the mass ratio of the photonic crystal to the humectant and wetting agent is (80-90):(10-20):(0.1-1), preferably the mass ratio of the photonic crystal to the humectant and wetting agent is (82-86):(12-17):(0.3-0.7), and further, the mass ratio of the photonic crystal to the humectant and wetting agent is (84-85):(15-16):(0.4-0.5).

[0066] According to an embodiment of the present invention, the moisturizer is selected from at least one of glycerin, ethylene glycol, and sorbitol, for example, ethylene glycol.

[0067] According to an embodiment of the present invention, the wetting agent is a commonly used surfactant, preferably at least one of alkyl glucoside (APG), fatty acid glycerides, fatty acid sorbitan (Span), polysorbate (Tween), Triton X-100, BYK-3455 (polyether modified polydimethylsiloxane), for example BYK-3455.

[0068] According to an embodiment of the present invention, the capture antibody solution comprises a capture antibody and an activator, wherein the capture antibody has the definition described above.

[0069] According to an embodiment of the present invention, the activator is selected from at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS), and dimethylacetamide (DMAC), for example, an EDC / NHS mixed solution.

[0070] According to an embodiment of the present invention, the detection antibody solution comprises a detection antibody labeled with a fluorescent dye labeling molecule and a humectant, wherein the detection antibody has the definition described above.

[0071] Preferably, the detection antibody solution further includes a solvent used to dissolve the detection antibody and the humectant.

[0072] According to an embodiment of the present invention, the humectant is selected from at least one of trehalose, polyethylene glycol, and sucrose, for example, trehalose or polyethylene glycol.

[0073] Preferably, the volume ratio of the detection antibody to the solvent is 1:1-3, for example, 1:1.

[0074] According to an embodiment of the present invention, the printing temperature in step 2) is 15 to 30°C; exemplaryly, the printing temperature is 20 to 27°C, for example, 15°C, 20°C, 22°C, 25°C, 27°C, 19°C or 30°C.

[0075] According to an embodiment of the present invention, in step 2), the printing is performed under conditions of 25-40% humidity, exemplarily, the humidity is 30-35%, for example 25%, 28%, 30%, 35%, 36%, 38% or 40%.

[0076] According to an embodiment of the present invention, in step 2), the ink output frequency of the printing is 1800 to 2500 Hz. For example, the ink output frequency is 2000 to 2200 Hz, such as 1800 Hz, 1900 Hz, 2000 Hz, 2100 Hz, 2200 Hz, 2300 Hz, 2400 Hz or 2500 Hz.

[0077] As an example, photonic crystal ink is printed on a hydrophobic PET plastic substrate, and an antibody solution is captured and printed in situ onto the surface of the photonic crystal. The detection antibody solution is then printed around the photonic crystal to form soluble spots, thus obtaining a photonic crystal biochip.

[0078] Applications of fully printed photonic crystal biochips

[0079] The present invention also provides an application of the above-mentioned fully printed photonic crystal biochip in biomarker detection.

[0080] According to an embodiment of the present invention, the biomarker includes protein and / or nucleic acid fragments, such as those used to detect four inflammatory markers (CRP, SAA, PCT, IL-6), six cardiac markers (AST, CK, CKMB, LDH, CTNI), and COVID-19.

[0081] According to an embodiment of the present invention, the detection is a point-of-care test, also known as POCT (point-of-care testing); or a one-drop blood test.

[0082] [Methods for Detection Using Fully Printed Photonic Crystal Biochips]

[0083] The present invention also provides a method for detecting biomarkers using the above-mentioned fully printed photonic crystal biochip, the method comprising the following steps: contacting the sample to be tested with the fully printed photonic crystal biochip and measuring the fluorescence value.

[0084] According to an embodiment of the present invention, the method includes the following steps:

[0085] a) Contact the fully printed photonic crystal biochip with standard biomarker samples of different concentrations, measure the fluorescence value, and construct a standard curve for the biomarker;

[0086] b) The sample to be tested is brought into contact with the fully printed photonic crystal biochip, the fluorescence value is measured, and the content of the biomarker is calculated according to the standard curve.

[0087] According to an embodiment of the present invention, the volume of the sample to be tested is 10 μL to 50 μL, preferably 10 μL to 20 μL, and further, 10 μL to 15 μL. The method of the present invention can be applied to the detection of a single drop of blood.

[0088] According to an embodiment of the present invention, the drop of blood may be a drop of venous blood or a drop of blood from a fingertip.

[0089] According to an embodiment of the present invention, in step a), the number of biomarker standard curves can be one or more, depending on the number of types of biomarkers to be detected.

[0090] According to an embodiment of the present invention, the contact time between the sample and the fully printed photonic crystal biochip is 5 min to 15 min, preferably 5 min to 10 min, for example, the contact time is 5 min, 8 min, 10 min, 12 min or 15 min.

[0091] According to an embodiment of the present invention, the sample to be tested can be any sample containing biomarkers, including but not limited to whole blood, serum, cerebrospinal fluid, saliva, plasma, tissue fluid, and cerebrospinal fluid.

[0092] According to an embodiment of the present invention, before step b), the step of diluting the sample to be tested is further included.

[0093] Preferably, the diluent is a PBS solution.

[0094] The detection process of the photonic crystal biochip of this invention is as follows: When the substance to be detected (droplet) is dropped onto the detection chip, the soluble spot (the spot containing the detection antibody that is dried around the photonic crystal) will dissolve rapidly and bind with the biomarker in the droplet. Because the substrate is hydrophobic and the photonic crystal array is hydrophilic, a hydrophilic array pattern is formed on the surface of the hydrophobic substrate. During the drying process, the droplet accumulates towards the photonic crystal. The retraction of the three-phase interface causes the detection antibody and biomarker to accumulate towards the capture antibody fixed on the surface of the photonic crystal, and then they are captured and reported by fluorescence, thus realizing the rapid detection of biomarkers.

[0095] The structure, preparation method, and application of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0096] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0097] Preparation Example 1

[0098] The specific steps for fabricating a photonic crystal biochip are as follows:

[0099] Photonic crystal ink, capture antibody solution, and detection antibody solution are prepared and stored in different reservoirs in the printing system.

[0100] S1, design photonic crystal microarrays on the substrate surface, such as dot, line, or surface patterns, and input the graphics into the control system of the printing device.

[0101] S2. The control system controls the printhead to print the photonic crystal ink onto a PET (polyethylene terephthalate) plastic substrate (contact angle > 80°) according to the designed pattern (the photonic crystal ink consists of 10% by mass of photonic crystal emulsion, ethylene glycol and surfactant BYK-3455, mixed in a mass ratio of 919:80:1).

[0102] S3. After the crystal ink on the substrate surface dries, the capture antibody solution is printed in situ onto the surface of the photonic crystal microarray pattern and incubated for 30 minutes.

[0103] The capture antibody is an antibody that detects the target substance. For example, when the target substance is the inflammatory factor CRP, the target substance is antibody 1 for CRP. The capture antibody solution includes: a capture antibody and an activator with a mixing ratio of 1 mg: 100 μL, wherein the antibody concentration is 1 mg / mL, the activator is a mixed solution of 6 mg / mL EDC and 10 mg / mL NHS, and the solvent is PBS buffer solution.

[0104] S4. After the capture antibody solution on the surface of the photonic crystal microarray pattern dries, the detection antibody solution is printed around the photonic crystal to form a soluble dot matrix. After drying, it is stored in a refrigerator at 4°C to obtain the photonic crystal biochip.

[0105] The detection antibody is a secondary antibody that detects the target substance, and the secondary antibody is labeled with fluorescence. For example, if the target substance to be detected is the inflammatory factor CRP, the detection antibody is antibody 2 of CRP labeled with fluorescence. In the detection antibody solution, the mixing ratio of detection antibody and trehalose is 1 mg:100 μL, the antibody concentration is 1 mg / mL, the trehalose concentration is 1 mg / mL, and the solvent is PBS buffer solution.

[0106] The printing environment for steps S2-S4 is a room temperature of 25℃, a humidity of 30%, and a printing ink output frequency of 2000Hz.

[0107] Preparation Example 2

[0108] The specific steps for fabricating a multifunctional photonic crystal biochip with multiple detection capabilities are as follows:

[0109] In this preparation example, three types of photonic crystal inks with red, green and blue light emission wavelengths were used instead of the single-wavelength photonic crystal ink in Preparation Example 1. The photonic crystals in different lattices of the photonic crystal microarray have the same or different light emission wavelengths. The detection antibodies and capture antibodies corresponding to the photonic crystals with different wavelengths are different, and the target analytes to be detected are also different. Apart from this, the other methods and steps are the same as in Preparation Example 1.

[0110] The multifunctional photonic crystal biochip prepared in this example can simultaneously detect multiple target substances.

[0111] Example 1

[0112] Photonic crystal biochips are used to detect biomarkers (taking CRP (C-reactive protein) as an example). In this embodiment, a double antibody sandwich system is used as an example.

[0113] CRP antigen and paired capture antibody (Ab1) and detection antibody (Ab2) were selected. The detection antibody (Ab2) was labeled with a red fluorescent tag of Rhodamine B (RhB), i.e., Ab2-RhB. Photonic crystals were matched to polystyrene microspheres with a particle size of 260 nm according to the fluorescence emission wavelength of Ab2-RhB. The specific steps are as follows:

[0114] (1) A photonic crystal solution was obtained by mixing polystyrene microsphere emulsion with a mass fraction of 10%, ethylene glycol and surfactant BYK-3455 in a mass ratio of 919:80:1.

[0115] (2) Prepare a mixed solution by mixing 6 mg / mL EDC and 10 mg / mL NHS at a volume ratio of 1:1. Prepare the solution immediately before use.

[0116] (3) Take 100 μL of the mixed solution from (2) above and add it to 1 mg of Ab1. Vortex to disperse it evenly and store the capture antibody solution at 4°C.

[0117] (4) Prepare 1 mg / mL Ab2-RhB by mixing 1 mL of 1 mg / mL Ab2-RhB with 100 μL of 1 mg / mL trehalose solution and storing the antibody solution at 4°C.

[0118] (5) Using the method in Preparation Example 1, the solution in steps (1)-(4) was inkjet printed onto a PET plastic substrate to prepare a photonic crystal biochip.

[0119] (6) Dilute the standard samples to the following concentrations (here, FBS fetal bovine serum is used for dilution): 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL; drop them onto the photonic crystal biochip. The soluble Ab2-RhB will quickly dissolve into the liquid environment of the droplet and bind to the analyte CRP. Due to the hydrophilic-hydrophobic patterned structure design, as the water evaporates, the Ab2-RhB bound to the analyte CRP in the liquid will accumulate around the hydrophilic photonic crystal and be captured by Ab1 fixed on the photonic crystal. RhB matches the emission wavelength of the photonic crystal, and light is emitted in a total emission, producing a fluorescence gain effect, and thus producing fluorescence. The fluorescence signal intensity corresponding to different concentration samples is different. Collect the fluorescence signals of different concentration gradients and establish a standard curve for the analyte CRP.

[0120] (7) Set up a negative control. Add pure FBS diluted solution to the detection chip and collect fluorescence signals repeatedly.

[0121] (8) Based on the standard curve established by concentration-fluorescence signal in step (6), all data are calibrated by subtracting the non-specific adsorption fluorescence intensity value from (7) as the baseline.

[0122] Example 2

[0123] Photonic crystal biochips are used to detect biomarkers (taking CRP as an example), with a competitive assay system as an example. CRP antigen, a CRP antigen-labeled rhodamine B red fluorescent tag (Antigen-RhB), and a CRP antibody (Ab-1) are selected. The photonic crystal is matched to the fluorescence emission wavelength of Antigen-RhB to form polystyrene microspheres with a particle size of 260 nm. The specific steps are as follows:

[0124] (1) A photonic crystal solution was obtained by mixing polystyrene microsphere emulsion with a mass fraction of 10%, ethylene glycol and surfactant BYK-3455 in a mass ratio of 919:80:1.

[0125] (2) Prepare a mixed solution by mixing 6 mg / mL EDC and 10 mg / mL NHS at a volume ratio of 1:1. Prepare the solution immediately before use.

[0126] (3) Take 100 μL of the mixed solution from (2) above and add it to 1 mg of Ab-1. Vortex to disperse it evenly and store the detection antibody solution at 4°C.

[0127] (4) Prepare 1 mg / mL Antigen-RhB by mixing 1 mL of 1 mg / mL Antigen-RhB with 100 μL of 1 mg / mL trehalose and storing the capture antibody solution at 4 °C.

[0128] (5) The above-prepared solution was used to prepare a photonic crystal biochip by inkjet printing using the method of Preparation Example 1.

[0129] (6) Dilute the standard samples to the following concentrations: 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL (here diluted with FBS), and drop them onto different detection units of the photonic crystal biochip. The soluble Antigen-RhB will quickly dissolve into the liquid environment of the droplets and bind to the analyte CRP. Due to the hydrophilic-hydrophobic patterned structure design, as the water evaporates, the Antigen-RhB bound to the analyte CRP in the liquid will accumulate around the hydrophilic photonic crystal and be captured by Ab-1 fixed on the photonic crystal. RhB matches the emission wavelength of the photonic crystal, and light is emitted in a total emission, producing a fluorescence gain effect, and thus reporting fluorescence.

[0130] The reaction time was 10 min. After the reaction, the unreacted biochip was rinsed 3 to 5 times with PBS buffer solution. Fluorescence signals corresponding to standard samples with different concentration gradients were collected repeatedly to establish an inverse standard curve for the analyte CRP.

[0131] (7) Set up a negative control by adding pure FBS diluted solution onto the detection chip and repeatedly collecting the corresponding fluorescence intensity values.

[0132] (8) Based on the standard curve established in (6), take (7) as the full value, subtract the fluorescence intensity value of the standard curve established in (6) from all data, and calibrate the CRP standard curve. See [link to relevant documentation]. Figure 3 As shown in the figure, the linear relationship between the actual sample concentration and fluorescence intensity value in this embodiment is shown. As can be seen from the figure, the detection limit of this embodiment can be as low as 0.01 ng / mL, and the detection range is from 0.01 ng / mL to 100 ng / mL, which is a wide detection range and can be used for clinical testing.

[0133] Example 3

[0134] Multiple biomarkers can be detected using a photonic crystal biochip. Taking CRP / SAA / PCT as an example, this embodiment uses a double antibody sandwich method.

[0135] CRP antigen, SAA antigen, and PCT antigen were selected. The detection antibody for CRP antigen was CRP-labeled with a red fluorescent tag of Rhodamine B (Ab12-RhB), and the capture antibody for CRP was Ab-1. The detection antibody for SAA antigen was SAA-labeled with a green fluorescent tag of FITC (Ab22-FITC), and the capture antibody for SAA was Ab-2. The detection antibody for PCT antigen was PCT-labeled with a blue fluorescent tag of DAPI (Ab32-DAPI), and the capture antibody for PCT was Ab-3.

[0136] The photonic crystals include: Ab12-RhB with a fluorescence emission wavelength matched to polystyrene microspheres with a particle size of 260 nm (hereinafter referred to as the first photonic crystal); Ab22-FITC with a fluorescence emission wavelength matched to photonic crystal polymer microspheres with a particle size of 220 nm (hereinafter referred to as the second photonic crystal); and Ab32-DAPI with a fluorescence emission wavelength matched to photonic crystal polymer microspheres with a particle size of 180 nm (hereinafter referred to as the third photonic crystal).

[0137] The specific steps are as follows:

[0138] (1) A first photonic crystal solution is obtained by mixing 10% polystyrene microsphere emulsion with a particle size of 260 nm, ethylene glycol, and surfactant BYK-3455 in a mass ratio of 919:80:1; a second photonic crystal solution is obtained by mixing 10% methyl methacrylate-acrylic acid-styrene polylatex microsphere emulsion with a particle size of 220 nm, ethylene glycol, and surfactant BYK-3455 in a mass ratio of 919:80:1; and a third photonic crystal solution is obtained by mixing 10% methyl methacrylate-acrylic acid-styrene polylatex microsphere emulsion with a particle size of 180 nm, ethylene glycol, and surfactant BYK-3455 in a mass ratio of 919:80:1.

[0139] (2) Prepare a mixed solution by mixing 6 mg / mL EDC and 10 mg / mL NHS at a volume ratio of 1:1. Prepare the solution immediately before use.

[0140] (3) Take three 100 μL portions of the above (2) mixed solution and add them to 1 mg of Ab-1, Ab-2 and Ab-3 in sequence. Vortex to disperse them evenly and store the capture antibody solution at 4°C.

[0141] (4) Prepare Ab12-RhB, Ab22-FITC and Ab32-DAPI solutions with a concentration of 1 mg / mL. Mix 1 mL of each solution with 100 μL of 1 mg / mL trehalose and store the antibody solution at 4 °C.

[0142] (5) Prepare solutions of (1)-(4) and use the method in Preparation Example 2 to prepare a multifunctional photonic crystal biochip for joint detection by inkjet printing.

[0143] (6) Dilute the standard samples to the following concentrations: 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL (using FBS for dilution). Add these concentrations to different detection units of the multifunctional photonic crystal biochip. The soluble Ab12-RhB, Ab22-FITC, and Ab32-DAPI will rapidly dissolve in the liquid environment of the droplets, binding to the corresponding analytes CRP, SAA, or PCT. Due to the hydrophilic-hydrophobic patterned structure design, as the water evaporates, the bound analytes in the liquid will accumulate around the hydrophilic photonic crystal, where they will be captured by Ab-1, Ab-2, or Ab-3 fixed on the photonic crystal, thus reporting fluorescence.

[0144] The reaction time was 10 min. After the reaction was complete, the crystal biochip was rinsed with PBS buffer. Fluorescence signals at different concentration gradients were repeatedly collected to establish standard curves for the detectants CRP, SAA, and PCT.

[0145] (7) Set up a negative control. Add pure FBS diluted solution to the detection chip and collect three different fluorescence intensity values ​​repeatedly.

[0146] (8) Based on the standard curve established in (6), subtract the fluorescence intensity value of the standard curve established in (6) from all data using (7) as the full value, and calibrate the CRP standard curve. See [link to relevant documentation]. Figure 4 As shown, the actual sample concentration and fluorescence intensity value in this embodiment exhibit a good linear relationship.

[0147] Example 4

[0148] This example demonstrates the detection of a single biomarker in a drop of blood from a real clinical patient sample. Detectable blood samples include serum, plasma, and whole blood; this example uses whole blood.

[0149] In Example 2, a photonic crystal biodetector chip for a specific biomarker was prepared. Whole blood of the person to be tested was diluted with PBS (1:9) to obtain a diluent. 10 μL of the diluent was added to one unit of the photonic crystal biochip. After 10 min, the unreacted solution was rinsed with PBS buffer solution. Fluorescence signals were collected from the unit on the biochip, and the corresponding concentration value was calculated by referring to the standard curve.

[0150] Example 5

[0151] A single drop of blood can be used to detect multiple biomarkers in real clinical patient samples. Samples that can be tested include serum, plasma, and whole blood; whole blood is used as an example here.

[0152] Using the photonic crystal biodetector chip for specific biomarkers prepared in Example 3, whole blood of the person to be tested was diluted with PBS (1:9). 10 μL of the diluted solution was added to several multifunctional detection units of the photonic crystal biodetector chip. After 10 min, the unreacted solution was rinsed with PBS buffer solution. Fluorescence signals of different colors of multiple biomarkers (e.g., red, green, blue) were collected. The concentration values ​​of the corresponding substances to be detected were calculated based on the corresponding standard curve.

[0153] As above, simultaneous rapid testing of multiple patient samples can be performed, using the same method.

[0154] Example 6

[0155] Detection of a single biomarker in a drop of blood from a real clinical patient sample.

[0156] Sampling fingertip blood: Apply iodine to the fingertip area of ​​the left ring finger, prick the fingertip with a disposable fingertip blood sampling needle, squeeze out the first drop of blood, and wipe it away with a cotton swab; squeeze out the second drop of blood and apply it to the reference. The specific biomarker photonic crystal biodetector chip was prepared using the methods of Preparation Example 1 and Preparation Example 2 (the biomarker is selected according to the detection requirements). After 10 minutes, the unreacted solution was rinsed with PBS buffer solution, and the fluorescence signal of the unit on the biochip was collected. The corresponding concentration value was calculated by referring to the standard curve.

[0157] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fully printed photonic crystal biochip, characterized in that, The chip includes a hydrophobic substrate with a contact angle greater than or equal to 80° and a photonic crystal microarray disposed on at least one side of the substrate; the photonic crystal microarray is hydrophilic and includes at least two photonic crystal units, a capture antibody disposed on the surface of the photonic crystal unit, and a soluble spot disposed around the periphery of the photonic crystal unit. The photonic crystal unit and the corresponding soluble spot do not contact each other. The soluble spot includes a detection antibody. The capture antibody and the detection antibody are used to bind to the antigen. The detection antibody is attached to a receptor substance, and the receptor substance will generate fluorescence amplification when it is close to the photonic crystal. The receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

2. The fully printed photonic crystal biochip according to claim 1, characterized in that, When the detection antibody and the capture antibody bind to the antigen, they form a double-antibody sandwich detection system or a competitive detection system.

3. The fully printed photonic crystal biochip according to claim 1, characterized in that, The photonic crystal microarray can be a dot pattern, a line pattern, or a surface pattern.

4. The fully printed photonic crystal biochip according to claim 1, characterized in that, The distance between the photonic crystal unit and the soluble spot is 0.1 mm to 1 mm.

5. The fully printed photonic crystal biochip according to claim 1, characterized in that, The photonic crystal in the photonic crystal unit may or may not contain linking groups.

6. The fully printed photonic crystal biochip according to claim 5, characterized in that, The linking group includes at least one of carboxyl, hydroxyl, mercapto, and amino groups.

7. The fully printed photonic crystal biochip according to claim 1, characterized in that, The photonic crystal is at least one of opal photonic crystal, inverse opal photonic crystal, and two-dimensional photonic crystal.

8. The fully printed photonic crystal biochip according to claim 1, characterized in that, The capture antibody and detection antibody are independently selected from antibodies, streptavidin, biotin, or base fragments that can specifically bind to the antigen.

9. A method for fabricating a fully printed photonic crystal biochip according to any one of claims 1 to 8, characterized in that, The method includes the following steps: 1) Prepare photonic crystal ink, capture antibody solution, and detection antibody solution; 2) A photonic crystal array comprising photonic crystal units is formed by printing photonic crystal ink on the substrate surface. A capture antibody solution is printed on the photonic crystal units to set the capture antibody on the surface of the photonic crystal units. A detection antibody solution is printed around the photonic crystal units to form soluble spots.

10. The preparation method according to claim 9, characterized in that, The photonic crystal ink includes a photonic crystal, a humectant, and a wetting agent.

11. The preparation method according to claim 10, characterized in that, In the ink, the photonic crystal exists in the form of an emulsion.

12. The preparation method according to claim 11, characterized in that, The photonic crystal exists in the form of a photonic crystal latex ball.

13. The preparation method according to claim 12, characterized in that, The photonic crystal latex spheres are selected from at least one of photonic crystal-poly(methyl methacrylate-acrylic acid-styrene) latex spheres, photonic crystal-silica microspheres, and photonic crystal-polystyrene microspheres.

14. The preparation method according to claim 10, characterized in that, In the photonic crystal ink, the mass ratio of the photonic crystal to the humectant and wetting agent is (80~90):(10~20):(0.1~1).

15. The application of the fully printed photonic crystal biochip according to any one of claims 1 to 8 in the detection of biomarkers.

16. A method for detecting biomarkers, characterized in that, The method includes the following steps: bringing the sample to be tested into contact with the fully printed photonic crystal biochip according to any one of claims 1 to 8, and measuring the fluorescence value.

17. The method according to claim 16, characterized in that, The method includes the following steps: a) Contact the fully printed photonic crystal biochip with standard biomarker samples of different concentrations, measure the fluorescence value, and construct a standard curve for the biomarker; b) The sample to be tested is brought into contact with the fully printed photonic crystal biochip, the fluorescence value is measured, and the content of the biomarker is calculated according to the standard curve.

18. The method according to claim 17, characterized in that, The volume of the sample to be tested is 10. L~50 L.

19. The method according to claim 17, characterized in that, The method is applicable to the detection of a single drop of blood.

20. The method according to claim 19, characterized in that, The drop of blood mentioned refers to a drop of venous blood or a drop of blood from a fingertip.

Images