Method for manufacturing biosensor

Abstract

The present invention provides a method for manufacturing a biosensor, which is used to solve the problem that the existing method for manufacturing glass-based biosensors uses a strong acid solution or a strong alkaline solution, or performs oxygen plasma treatment. The manufacturing method includes: treating a silicon-containing substrate with an ethanol solution so that at least one surface of the silicon-containing substrate has a negative charge; forming at least one active polymer layer with a positive charge on the surface of the silicon-containing substrate, the active polymer layer having a binding surface and an active surface, the active polymer layer is bound to the at least one surface of the silicon-containing substrate; and allowing a plurality of capture biomolecules to bind to the active surface. The present invention also relates to a biosensor obtained by the manufacturing method, the biosensor includes a silicon-containing substrate; at least one active polymer layer, each having a binding surface and an active surface opposite to each other, the active polymer layer is respectively bound to the at least one surface of the silicon-containing substrate; and a plurality of capture biomolecules are bound to the active surface.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a biosensor, and more particularly to a method for manufacturing a biosensor that reduces waste liquid generation. The present invention also relates to a biosensor manufactured by the aforementioned manufacturing method. Background Art

[0002] In order to detect whether a suspected patient is infected with a virus, quantitative real-time polymerase chain reactive (RT-qPCR) can generally be used in combination with a specific primer pair to detect the virus. Although quantitative real-time polymerase chain reaction (RT-qPCR) has high specificity and high sensitivity, it requires complex sample pre-treatment procedures, expensive laboratory instruments and equipment, and workers also need to undergo complete training to perform this method. It is still inconvenient for real-time detection of specific viruses (point-of-care, POC).

[0003] In order to conduct real-time detection, existing optical biosensors have been developed that can be used with smartphones to detect molecules or cancer biomarkers in body fluids. If a sample contains an antigen of a specific virus, and the antigen is able to specifically bind to an antibody provided on the surface of the existing optical biosensor, the optical signal generated by the specific binding of the antibody and the antigen can be detected by the smartphone, thereby confirming that the sample has been infected by the specific virus.

[0004] However, in the manufacturing method of the existing optical biosensor, a strong acid solution or a strong alkali solution (such as sodium hydroxide solution) is used to form a negative charge on the surface of a silicon-containing substrate, and then a positively charged active polymer layer is adsorbed on the surface of the silicon-containing substrate, and the negatively charged antibody can be adsorbed on the surface of the active polymer layer, thereby obtaining the existing optical biosensor. However, the strong acid solution or strong alkali solution used in the manufacturing method of the existing optical biosensor not only increases the operating risk of the workers, but also the large amount of waste liquid containing the strong acid solution or the strong alkali solution generated will also pollute the environment.

[0005] In addition, the existing optical biosensor manufacturing method can also form a negative charge on the surface of the silicon-containing substrate through oxygen plasma, but workers need to use special equipment such as an oxygen plasma cleaner to perform oxygen plasma treatment under special conditions such as high temperature and high pressure, which will greatly increase the manufacturing cost of the existing optical biosensor.

[0006] In view of this, there is still a need to improve the existing biosensor manufacturing method. Summary of the invention

[0007] To solve the above problems, an object of the present invention is to provide a method for manufacturing a biosensor without using a strong acid solution or a strong base solution.

[0008] Another object of the present invention is to provide a method for manufacturing a biosensor, which can reduce the manufacturing cost of the biosensor.

[0009] Another object of the present invention is to provide a biosensor manufactured by the aforementioned biosensor manufacturing method.

[0010] The directions or their approximate terms described throughout the present invention, such as "front", "rear", "left", "right", "up (top)", "down (bottom)", "inside", "outside", "side", etc., mainly refer to the directions of the accompanying drawings, and each direction or its approximate terms are only used to assist in the explanation and understanding of the embodiments of the present invention, and are not used to limit the present invention.

[0011] The components and members described throughout the present invention use the quantifiers "a" or "an" only for convenience of use and to provide a general meaning of the scope of the present invention; in the present invention, they should be interpreted as including one or at least one, and a single concept also includes plural cases, unless it is obvious that it means otherwise.

[0012] The similar terms such as "combination", "assembly" or "assembly" described throughout the present invention mainly include the connection that can be separated without damaging the components, or the connection that makes the components inseparable, which can be selected by technicians in this field according to the materials of the components to be connected or the assembly requirements.

[0013] A method for manufacturing a biosensor comprises: providing a silicon-containing substrate having at least one surface; treating the silicon-containing substrate with an ethanol solution so that the at least one surface of the silicon-containing substrate carries a negative charge; forming at least one active polymer layer carrying a positive charge on the at least one surface of the silicon-containing substrate, the at least one active polymer layer having a binding surface and an active surface, the at least one active polymer layer being bound to the silicon-containing substrate via the binding surface; and binding a plurality of capture biomolecules to the active surface of the at least one active polymer layer.

[0014] Accordingly, in the manufacturing method of the biosensor of the present invention, by using the ethanol solution, the at least one surface of the silicon-containing substrate can be made to have a negative charge without using a strong acid solution or a strong alkali solution. This can not only improve the safety of the workers' working environment, but also reduce the treatment cost of the waste liquid containing the strong acid solution and the strong alkali solution, and further prevent the discharge of the waste liquid containing the strong acid solution and the strong alkali solution from causing adverse effects on environmental organisms or buildings, which is the effect of the present invention.

[0015] Furthermore, in the manufacturing method of the biosensor of the present invention, by using the ethanol solution, at least one surface of the silicon-containing substrate can be made to have a negative charge without using special equipment such as an oxygen plasma cleaner, and the high temperature and high pressure environment required for oxygen plasma treatment can also be avoided, which helps to reduce the manufacturing cost of the biosensor.

[0016] In the method for manufacturing the biosensor of the present invention, the silicon-containing substrate can be treated with an ethanol aqueous solution with a concentration of 60% to 99.8%. Thus, by selecting an ethanol aqueous solution with a suitable ethanol concentration, the at least one surface of the silicon-containing substrate can be provided with a sufficient amount of negative charge, so that the at least one surface of the silicon-containing substrate can be stably combined with the at least one active polymer layer with a positive charge.

[0017] In the method for manufacturing the biosensor of the present invention, the plurality of captured biomolecules are respectively negatively charged, so that the plurality of captured biomolecules are respectively electrostatically bound to the active surface of the at least one active polymer layer. Thus, compared with using a cross-linker to bind the plurality of captured biomolecules to the at least one active polymer layer, the cumbersome steps can be greatly reduced.

[0018] In the method for manufacturing a biosensor of the present invention, the plurality of captured biomolecules bind to a covered area of ​​the active surface of the at least one active polymer layer, and the active surface of the at least one active polymer layer further includes an exposed area. Preferably, the manufacturing method further includes making a blocking layer cover the exposed area of ​​the active surface of the at least one active polymer layer. Thus, by covering the exposed area of ​​the active surface with the blocking layer, impurities in a sample can be prevented from non-specifically binding to the active surface, thereby achieving the effect of improving the detection specificity of the biosensor.

[0019] The manufacturing method of the biosensor of the present invention, wherein each of the plurality of captured biomolecules is respectively bound to the active surface of the at least one active polymer layer through a plurality of precious metal nanoparticles; for example, the plurality of precious metal nanoparticles are respectively negatively charged, so that the plurality of precious metal nanoparticles are respectively electrostatically bound to the active surface of the at least one active polymer layer, and the plurality of captured biomolecules are respectively covalently bound to the plurality of precious metal nanoparticles. Thus, through the presence of the precious metal nanoparticles, a larger steric hindrance can be formed, making the plurality of captured biomolecules more easily exposed, thereby achieving the effect of improving the detection sensitivity of the biosensor.

[0020] In the method for manufacturing a biosensor of the present invention, the plurality of noble metal nanoparticles are combined with a covering area of ​​the active surface of the at least one active polymer layer, and the active surface of the at least one active polymer layer further comprises an exposed area. Preferably, the manufacturing method further comprises making a blocking layer cover the exposed area of ​​the active surface of the at least one active polymer layer. Thus, by covering the exposed area of ​​the active surface with the blocking layer, impurities in a sample can be prevented from non-specifically binding to the active surface, thereby achieving the effect of improving the detection specificity of the biosensor.

[0021] In the method for manufacturing the biosensor of the present invention, the active surface of the at least one active polymer layer has a functional group, and the functional group is selected from the group consisting of amine groups and ammonium groups. Thus, through the aforementioned functional group, the active surface of the at least one active polymer layer has a strong positive charge, so that it can quickly bind to the captured biomolecule.

[0022] In the method for manufacturing the biosensor of the present invention, the at least one active polymer layer is formed by a polymer, and the polymer is selected from the group consisting of polyethyleneimine (such as linear polyethyleneimine or branched polyethyleneimine), polyallylamine hydrochloride, polyβ-amino ester (such as linear polyβ-amino ester or branched polyβ-amino ester), polydiallyl dimethyl ammonium chloride and polyacrylamide. Thus, by forming the at least one active polymer layer by the aforementioned polymer, the active surface of the at least one active polymer layer has a strong positive charge, so that it can quickly bind to the captured biomolecule.

[0023] According to the aforementioned method for manufacturing a biosensor, the biosensor of the present invention can be manufactured, and the biosensor comprises a silicon-containing substrate having at least one surface; at least one active polymer layer having a binding surface and an active surface, respectively, and the at least one active polymer layer is bound to the at least one surface of the silicon-containing substrate with the binding surface; and a plurality of capture biomolecules bound to the active surface of the at least one active polymer layer.

[0024] Accordingly, the biosensor of the present invention is manufactured by the aforementioned biosensor manufacturing method, and the selected substrate is the silicon-containing substrate (e.g., a SiO2-based substrate). In other words, the biosensor is not a plastic product and can be recycled and reused after melting. Moreover, in the process of manufacturing the biosensor, it is not necessary to use the strong acid solution or the strong alkali solution, so that the biosensor is an environmentally friendly good, which is the effect of the present invention.

[0025] In the biosensor of the present invention, the active surface of the at least one active polymer layer comprises a covering area and an exposed area, and the plurality of captured biomolecules bind to the covering area of ​​the active surface of the at least one active polymer layer, and preferably, a blocking layer covers the exposed area. Thus, by covering the exposed area of ​​the active surface with the blocking layer, impurities in a sample can be prevented from non-specifically binding to the active surface, thereby achieving the effect of improving the detection specificity of the biosensor.

[0026] In the biosensor of the present invention, the plurality of captured biomolecules are respectively bound to the active surface of the at least one active polymer layer through a plurality of noble metal nanoparticles. Thus, the presence of the noble metal nanoparticles can form a larger steric hindrance, making it easier for the plurality of captured biomolecules to be exposed, thereby achieving the effect of improving the detection sensitivity of the biosensor.

[0027] In the biosensor of the present invention, the active surface of the at least one active polymer layer comprises a covering area and an exposed area, and the plurality of noble metal nanoparticles are bound to the covering area of ​​the active surface of the at least one active polymer layer, and preferably, a blocking layer covers the exposed area. Thus, by covering the exposed area of ​​the active surface with the blocking layer, impurities in a sample can be prevented from non-specifically binding to the active surface, thereby achieving the effect of improving the detection specificity of the biosensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : A side cross-sectional view of a biosensor manufactured by the method for manufacturing a biosensor according to the first embodiment of the present invention;

[0029] Figure 2 : Figure 1 A local enlarged view of region A of the biosensor;

[0030] Figure 3 :exist Figure 2 Schematic diagram of a biosensor after a blocking layer is formed;

[0031] Figure 4 : Spectra at wavelengths between 300 and 500 nm of the glass test pieces of group A1 in test (A) that were not pretreated with an ethanol aqueous solution, and the glass test pieces of groups A2 to A4 that were pretreated with the ethanol aqueous solution and then treated with 0.1 wt%, 1.0 wt% and 2.5 wt% branched polyethyleneimine aqueous solutions, respectively;

[0032] Figure 5 : In the test (B), the calibration curve of the grayscale of the mixed solution of immunoglobulin M specific to the SARS-CoV-2 novel coronavirus (●) and the calibration curve of the concentration of immunoglobulin G specific to the SARS-CoV-2 novel coronavirus (▲) against the grayscale of the mixed solution;

[0033] Figure 6 : In test (C), the calibration curve of the concentration of immunoglobulin M specific for the SARS-CoV-2 novel coronavirus versus the absorbance value of the mixed solution;

[0034] Figure 7 : In the test (D), the absorbance values ​​of immunoglobulin M and immunoglobulin G specific to SARS-CoV-2 in blood samples from healthy individuals (Group D1), blood samples from patients with other diseases (Group D2), blood samples from patients infected with SARS-CoV-2 and in the early stages of infection (Group D3), and blood samples from patients infected with SARS-CoV-2 and in the middle and late stages of infection (Group D4);

[0035] Figure 8 : A schematic diagram of a biosensor manufactured by the method for manufacturing a biosensor according to the second embodiment of the present invention;

[0036] Fig. 9 :exist Figure 8 Schematic diagram of a biosensor after a blocking layer is formed on the biosensor;

[0037] Fig.10 : A flow chart of the use status of the biosensor manufactured by the method for manufacturing the biosensor according to the second embodiment of the present invention;

[0038] Fig.11 : Spectra of the glass test pieces of groups E1 and E2 at wavelengths between 400 and 650 nm before and after the plurality of noble metal nanoparticles are bonded to the active surface of the active polymer layer in test (E);

[0039] Fig.12: Fluorescence image of the glass test piece of group F1 before the plurality of captured biomolecules are respectively bound to the plurality of noble metal nanoparticles in experiment (F);

[0040] Fig.13 : Fluorescence image of the F2 group of glass test pieces after the plurality of captured biomolecules are respectively bound to the plurality of noble metal nanoparticles in experiment (F);

[0041] Fig.14 : In test (G), the surface roughness bar graphs of the untreated glass test piece (Group G1), the glass test piece combined with the active polymer layer (Group G2), the glass test piece combined with the plurality of noble metal nanoparticles (Group G3), the glass test piece combined with the plurality of captured biomolecules (Group G4), and the glass test piece combined with the blocking layer (Group G5);

[0042] Fig.15 : Spectra of urine samples containing different concentrations of FXYD3 protein at wavelengths between 350 and 550 nm in experiment (H);

[0043] Fig.16 : In the test (I), the FXYD3 protein content in urine samples from healthy individuals (Group I1), urine samples from individuals with low-stage lower urinary tract epithelial cell carcinoma (Group I2), urine samples from individuals with low-stage upper urinary tract epithelial cell carcinoma (Group I3), urine samples from individuals with high-stage lower urinary tract epithelial cell carcinoma (Group I4), and urine samples from individuals with high-stage upper urinary tract epithelial cell carcinoma (Group I5).

[0044] Description of Reference Numerals

[0045] [The present invention]

[0046] 1: Silicon-containing substrate

[0047] 11: Surface

[0048] 2: Active polymer layer

[0049] 21: Bonding Surface

[0050] 22: Active Surface

[0051] 22a: Coverage area

[0052] 22b: Exposed area

[0053] 3: Capturing biomolecules

[0054] 4: Blocking layer

[0055] 5: Precious metal nanoparticles

[0056] B1: Plastic bottle

[0057] B2: Plastic bottle

[0058] B3: Glass bottle

[0059] C: Bottle cap

[0060] S: Biosensor. DETAILED DESCRIPTION

[0061] In order to make the above and other purposes, features and advantages of the present invention more clearly understood, preferred embodiments of the present invention are listed below and described in detail with reference to the accompanying drawings as follows:

[0062] Please refer to Figure 1 , Figure 2 As shown, the manufacturing method of the first embodiment of the present invention can first provide a silicon-containing substrate 1, then form an active polymer layer 2 on the silicon-containing substrate 1, and then allow a plurality of capture biomolecules 3 to bind to the active polymer layer 2.

[0063] In detail, the silicon-containing substrate 1 can be a SiO2-based substrate, etc., and can be in various three-dimensional shapes such as a sheet or a bottle. This can be adjusted by technicians in the technical field of the present invention according to their needs and is not limited here.

[0064] The silicon-containing substrate 1 can be treated with an ethanol solution so that at least one surface 11 of the silicon-containing substrate 1 has a negative charge. For example, the ethanol solution can be a 60% to 99.8% ethanol aqueous solution (ethanol concentration is 60% to 99.8%). It is worth noting that if the ethanol concentration of the ethanol aqueous solution is less than 60%, it will not be able to effectively clean the impurities or grease attached to at least one surface 11 of the silicon-containing substrate 1, resulting in the inability to form a sufficient amount of negative charge on at least one surface 11 of the silicon-containing substrate 1, or causing the negative charge on at least one surface 11 of the silicon-containing substrate 1 to be unevenly distributed, so that the active polymer layer 2 cannot be stably bonded to at least one surface 11 of the silicon-containing substrate. The worker can use the following method: Figure 1 The glass bottle (volume is about 2 mL) shown is used as the silicon-containing substrate 1. The ethanol aqueous solution (volume is 1 mL) is added to the glass bottle and shaken at room temperature (temperature of 22-28° C.) for about 10 minutes, so that a plurality of hydroxide ions (OH-) with negative charge can be formed on an inner surface of the glass bottle. In other words, the glass bottle is the silicon-containing substrate 1, and the inner surface of the glass bottle is the surface 11 of the silicon-containing substrate 1.

[0065] In addition, the worker can also use a glass chip as the silicon-containing substrate 1. After immersing the glass chip in the ethanol aqueous solution, a plurality of hydroxide ions with negative charges can be formed on two opposite surfaces of the glass chip. In other words, the glass chip is the silicon-containing substrate 1, and the two opposite surfaces of the ionization bottle are the surfaces 11 of the silicon-containing substrate 1.

[0066] Furthermore, before being treated with the ethanol solution, the silicon-containing substrate 1 may also be subjected to a pre-treatment to remove dust, grease or impurities attached to the surface 11 of the silicon-containing substrate 1. For example, the worker may use a tris (hydroxymethyl)aminomethane (Tris) buffer solution containing 0.1% polysorbate 20 (Tween 20) to clean the surface 11 of the silicon-containing substrate 1, or may use acetone or deionized water to clean the surface 11 of the silicon-containing substrate 1.

[0067] Please refer to Figure 1 , Figure 2As shown, after obtaining the surface 11 with negative charge, the worker can form the active polymer layer 2 with positive charge on the surface 11, and the active polymer layer 2 has a binding surface 21 and an active surface 22 opposite to each other, and the active polymer layer 2 can be electrostatically bonded to the surface 11 of the silicon-containing substrate 1 through the binding surface 21. It is worth noting that the active polymer layer 2 preferably has a functional group with positive charge, and the functional group can be selected from the group consisting of amine group (-NH2) and ammonium root (-NH4+), so that the active polymer layer 2 can be bonded to the plurality of capture biomolecules 3 through the functional group. For example, the active polymer layer 2 can be formed by a polymer, and the polymer is selected from the group consisting of polyethyleneimine (PEI), poly(allylamine hydrochloride), PAH, poly(β-amino ester), PAE, polydiallyldimethylammonium chloride (PDDA) and polyacrylamide, wherein the polyethyleneimine can be linear polyethyleneimine (linear PEI) or branched polyethyleneimine (branched PEI), and the poly(β-amino ester) can be linear poly(β-amino ester) (linear PAE) or branched poly(β-amino ester) (branched PAE).

[0068] In this embodiment, the worker can prepare a 0.1 wt% aqueous solution of branched polyethyleneimine (branched PEI, Co#408727, purchased from Sigma-Aldrich) and Figure 1 0.1 mL of branched polyethyleneimine aqueous solution is added to the glass bottle shown, and reacted at room temperature for 2 hours, so that the branched polyethyleneimine can form an active polymer layer 2 with positive charges formed by amine groups, and electrostatically bonded to the inner surface of the glass bottle (i.e., the surface 11 of the silicon-containing substrate 1) with the bonding surface 21. The worker can also use deionized water to wash away the branched polyethyleneimine that is not bonded to the inner surface of the glass bottle, and can heat the glass bottle to 80°C, and keep the temperature for more than 15 minutes, and then slowly cool it down to room temperature to strengthen the bonding force between the active polymer layer 2 and the silicon-containing substrate 1.

[0069] Next, please refer to Figure 1 , Figure 2As shown, the worker can make the multiple capture biomolecules 3 bind to the active surface 22 of the active polymer layer 2 (i.e., the surface of the active polymer layer 2 that is not bound to the silicon-containing substrate 1), so that the active polymer layer 2 can be located between the multiple capture biomolecules 3 and the silicon-containing substrate 1. The worker can select the capture biomolecule 3 according to a target biomolecule that the biosensor S is to specifically detect. For example, the capture biomolecule 3 can be an antibody, antigen, enzyme, substrate, aptamer, etc. that is specific to the target biomolecule, so that the biosensor S can specifically detect the corresponding target biomolecules such as antigens, antibodies, receptors, enzymes, nucleic acids and cells.

[0070] The worker can make the capture biomolecule 3 electrostatically bond with the active surface 22 of the active polymer layer 2 with a positive charge, for example, by selecting a capture biomolecule 3 that itself has a negative charge, or by adjusting the pH value of the solution containing the capture biomolecule 3 so that the pH value of the solution containing the capture biomolecule 3 is higher than the isoelectric point of the capture biomolecule 3 so that the capture biomolecule 3 has a negative charge, or by modifying the capture biomolecule 3 with a thiol group (-SH) so that the capture biomolecule 3 has a negative charge and can electrostatically bond to the active surface 22 of the active polymer layer 2.

[0071] In this embodiment, a negatively charged SARS-CoV-2 novel coronavirus nucleocapsid protein is selected as the capture biomolecule 3. The SARS-CoV-2 novel coronavirus nucleocapsid protein can specifically bind to immunoglobulin M (IgM) specific to the SARS-CoV-2 novel coronavirus, and can also specifically bind to immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus. In other words, the biodetector containing the SARS-CoV-2 novel coronavirus nucleocapsid protein can be used to detect whether a sample contains immunoglobulin M (IgM) specific to the SARS-CoV-2 novel coronavirus and / or immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus. The worker can dissolve the SARS-CoV-2 novel coronavirus nucleocapsid protein in phosphate buffered saline (PBS) to form a SARS-CoV-2 novel coronavirus nucleocapsid protein solution with a concentration of 100 ng / mL, and then add 0.1 mL of the SARS-CoV-2 novel coronavirus nucleocapsid protein solution into the glass bottle and react at room temperature for 1 hour, so that the negatively charged SARS-CoV-2 novel coronavirus nucleocapsid protein can electrostatically bind to the active surface 22 of the active polymer layer 2 located on the inner surface of the glass bottle.

[0072] Furthermore, the captured biomolecules 3 may not form a captured biomolecule layer that completely covers the active surface 22. In other words, there are still some areas of the active surface 22 that are not bound to the captured biomolecules 3. Therefore, the active surface 22 can be divided into a covered area 22a that is bound to the captured biomolecules 3 and an exposed area 22b that is not bound to the captured biomolecules 3. Since the specimen from the organism usually contains a large amount of impurities (for example, serum albumin, bilirubin, lipids and hemoglobin, etc.), in order to prevent the aforementioned impurities from non-specifically binding to the exposed area 22b of the active polymer layer 2 and affecting the interpretation of the biosensor S, the worker may preferably add a blocking solution such as a bovine serum albumin (BSA) solution or a casein solution into the glass bottle so that the blocking solution can cover the exposed area 22b of the active surface 22 of the active polymer layer 2 with a blocking layer 4. In this embodiment, the blocking solution is a bovine serum albumin aqueous solution (containing 2 wt% bovine serum albumin). 1 mL of the bovine serum albumin aqueous solution is added to the glass bottle and reacted at room temperature for 1 hour to form the blocking layer 4 on the exposed area 22b of the active surface 22 of the active polymer layer 2 located on the inner surface of the glass bottle. The blocking layer 4 is then washed with tris(hydroxymethyl)aminomethane buffer to obtain the following. Figure 3 The biosensor S shown.

[0073] The method of using the biosensor S manufactured according to the manufacturing method of the first embodiment is as follows:

[0074] Preparation of probe solution: The probe solution contains 250 ng / mL of anti-human immunoglobulin M (IgM) secondary antibody labeled with horseradish peroxidase (HRP) and / or 250 ng / mL of anti-human immunoglobulin G (IgG) secondary antibody labeled with horseradish peroxidase (HRP), dissolved in tri(hydroxymethyl)aminomethane (Tris) buffer containing 0.001% polysorbate 20 (Tween 20).

[0075] Preparation of chromogen solution: The chromogen solution contains 0.5 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) and 0.5% hydrogen peroxide (H2O2) dissolved in 0.1 M sodium acetate (NaOAc) aqueous solution with a pH of 5.5.

[0076] Preparation of terminating reagent: The terminating reagent is a 1 M hydrochloric acid aqueous solution.

[0077] Sample sampling: In order to confirm whether a suspected patient is infected with the new coronavirus SARS-CoV-2 using the biosensor S, the sample used can be a whole blood sample (such as a venous whole blood sample or a fingertip whole blood sample), a serum sample, a plasma sample, a urine sample, a saliva sample, etc. from the suspected patient. In this embodiment, in order to facilitate the real-time detection of the new coronavirus SARS-CoV-2, the blood sample collected from the fingertip is selected.

[0078] The operation process of the biosensor S is as follows:

[0079] 1 mL of the probe solution is added to the glass bottle, and then 5 μL of the blood sample is added. After uniform mixing, the mixture is allowed to stand at room temperature for 15 minutes, so that the secondary antibody containing anti-human immunoglobulin M (IgM) labeled with horseradish peroxidase (HRP) and / or the secondary antibody containing anti-human immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) in the probe solution can specifically bind to human immunoglobulin M (IgM) specific to the new coronavirus SARS-CoV-2 and / or human immunoglobulin G (IgG) specific to the new coronavirus SARS-CoV-2 in the sample, and then specifically bind to the capture biomolecule 3 (nucleocapsid protein of SARS-CoV-2) on the inner surface of the glass bottle.

[0080] After cleaning the glass bottle, add 0.5 mL of chromogen solution into the glass bottle and let it stand for 2 minutes to observe the color change of the mixed solution in the glass bottle. At this time, under the action of horseradish peroxidase (HRP), the mixed solution in the glass bottle will gradually turn dark blue.

[0081] Finally, the stop reagent is added to the dark blue mixed solution. At this time, the dark blue mixed solution will turn into yellow under the action of the stop reagent. In this way, the worker can observe the color change of the mixed solution with the naked eye, or measure the change of absorbance at a specific wavelength (such as 450 nm) with a spectrometer.

[0082] When the biosensor S manufactured by the manufacturing method according to the first embodiment is used, if the suspected patient is indeed infected with the SARS-CoV-2 novel coronavirus, the capture biomolecule 3 (SARS-CoV-2 nucleocapsid protein) can capture the human immunoglobulin M (IgM) and / or the human immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus in the specimen of the suspected patient, and further bind to the secondary antibody against human immunoglobulin M (IgM) labeled with horseradish peroxidase (HRP) and / or the secondary antibody against human immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) in the probe solution, so that when the chromogen solution is added, the chromogen solution will be affected by the horseradish peroxidase (HRP) and change color. In other words, if the color of the mixed solution in the glass bottle eventually turns blue, it shows that the suspected patient has been infected with the SARS-CoV-2 novel coronavirus.

[0083] In order to test the detection specificity of the biosensor S prepared according to the manufacturing method of the first embodiment for the novel coronavirus SARS-CoV-2, the following test was performed:

[0084] (A) Adjustment of the concentration of branched polyethyleneimine aqueous solution

[0085] As shown in Table 1, the test was conducted by first soaking a glass test piece in a 95% ethanol aqueous solution, then preparing a 0.1 wt% branched polyethyleneimine aqueous solution, soaking the glass test piece in the branched polyethyleneimine aqueous solution, reacting at room temperature for 1 hour, and then washing the glass test piece. The glass test piece was then soaked in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) aqueous solution to allow the 2,4,6-trinitrobenzenesulfonic acid to react with the amine groups on the surface of the glass test piece to form a chromophore, which has a maximum absorbance at a wavelength of 340 nm. Finally, the absorbance of the glass test piece (Group A2) at a wavelength between 300 and 500 nm was analyzed.

[0086] In this experiment, a branched polyethyleneimine aqueous solution with a concentration of 1.0 wt % and a branched polyethyleneimine aqueous solution with a concentration of 2.5 wt % were used to replace the aforementioned branched polyethyleneimine aqueous solution with a concentration of 0.1 wt %, so as to obtain glass test pieces of groups A3 and A4, respectively.

[0087] In addition, in this test, another glass test piece was taken which was not previously treated by immersion in the ethanol aqueous solution, and was directly immersed in the branched polyethyleneimine aqueous solution with a concentration of 0.1 wt % as the glass test piece of group A1.

[0088] Table 1. Treatment conditions of each group of glass specimens in this test

[0089]

[0090] Please refer to Figure 4 As shown, for the glass test piece of group A1 which was not pre-treated with the ethanol aqueous solution, no negatively charged hydroxide ions were formed on the surface of the glass test piece, so that the active polymer layer 2 formed by the branched polyethyleneimine could not be bound to the surface of the glass test piece, and thus no characteristic peak was observed at a wavelength of 340 nm. However, the glass test pieces of groups A2 to A4 which were pre-treated with the ethanol aqueous solution first all had a peak at a wavelength of 340 nm, indicating that the branched polyethyleneimine aqueous solution with a concentration of 0.1 to 2.5 wt % could form positively charged amine groups on the surface of the glass test piece, that is, the active polymer layer 2 had been formed on the surface of the glass test piece.

[0091] (B) Test results of calibration curve (I)

[0092] In this test, a standard of human immunoglobulin M (IgM) specific to the SARS-CoV-2 novel coronavirus and a standard of human immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus were taken and diluted to a concentration of 10 -1 , 10 0 , 10 1 , 10 2 and 10 3 ng / mL, and then the above method was used for detection, and the color of the mixed solution was photographed with a smart phone (iPhone 7 plus), and the grayscale of each mixed solution was calculated according to the formula (1) below, and then a linear regression analysis was performed. Among them, R, G, and B in formula (1) refer to the red value, green value, and blue value, respectively.

[0093] Formula (I).

[0094] Please refer to Figure 5 As shown, whether using human immunoglobulin M (IgM) specific to the new coronavirus SARS-CoV-2 or human immunoglobulin G (IgG) specific to the new coronavirus SARS-CoV-2, a linear calibration curve can be drawn, indicating that the biosensor S manufactured by the manufacturing method of the first embodiment has good linearity for detecting blood samples.

[0095] (C) Test results of calibration curve (II)

[0096] In this test, a standard of human immunoglobulin M (IgM) specific to the SARS-CoV-2 novel coronavirus and a standard of human immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus were taken and diluted to a concentration of 10 1.0 , 10 1.3 , 10 2.0 , 10 2.5 , 10 3.0 , 10 3.5 , 10 4.0 and 10 4.5 pg / mL, and then detected by the above method, using a spectrometer (SpectraMax M2) to measure the absorbance at a wavelength of 450 nm, and performing linear regression analysis.

[0097] Please refer to Figure 6 The regression equation of the calibration curve is shown in the following formula (II), and the coefficient of determination (R 2 ) is 0.98838.

[0098] Formula (2).

[0099] (D) Test results of blood samples

[0100] In this study, blood samples from healthy individuals were collected as Group D1 (a total of 10 cases), blood samples from patients with other diseases (such as influenza A / B, pneumonia, tuberculosis, lung cancer, liver cancer, etc., who had symptoms of infection suspected of being infected with the new coronavirus SARS-CoV-2, such as fever, cough, sore throat, runny nose) as Group D2 (a total of 109 cases), and blood samples from patients confirmed to be infected with the new coronavirus SARS-CoV-2 as Groups D3 and D4 (a total of 29 cases). Among them, the blood samples of groups D3 and D4 were simultaneously confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (RT-qPCR) to confirm the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels and virus levels in the blood samples. They were then divided into group D3 (8 cases in total) in the early stage of infection and group D4 (21 cases in total) in the middle and late stages of infection. They were then tested using the aforementioned method and the absorbance at a wavelength of 450 nm was measured using a spectrometer (SpectraMax M2).

[0101] Please refer to Figure 7 As shown, it is difficult to detect the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus in blood samples from healthy individuals (Group D1) or blood samples from patients with other diseases (Group D2). However, in blood samples from patients confirmed to be infected with the SARS-CoV-2 novel coronavirus (Groups D3 and D4), immunoglobulin M (IgM) and immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus can be detected. Among them, the blood samples from patients in the early stage of infection (Group D3) have a higher level of immunoglobulin M (IgM) specific to the SARS-CoV-2 novel coronavirus, and the blood samples from patients in the middle and late stages of infection (Group D4) have a higher level of immunoglobulin G (IgG) specific to the SARS-CoV-2 novel coronavirus.

[0102] Based on the same technical concept, please refer to Figure 8 As shown, the manufacturing method of the second embodiment of the present invention can also first provide the silicon-containing substrate 1, then form the active polymer layer 2 on the silicon-containing substrate 1, and then allow a plurality of capture biomolecules 3 to bind to the active polymer layer 2.

[0103] In this embodiment, the glass sheet is used as the silicon-containing substrate 1, and the glass sheet is immersed in a 60% to 99.8% ethanol aqueous solution (ethanol concentration is 60 to 99.8%) for 1 hour at room temperature, so that hydroxide ions are formed on both opposite surfaces of the glass sheet. In other words, in this embodiment, the two opposite surfaces of the glass sheet that form hydroxide ions with negative charges correspond to the surface 11 of the silicon-containing substrate 1. Then, the glass sheet is immersed in the branched polyethyleneimine aqueous solution (0.1 wt%) for 2 hours at room temperature, so that the branched polyethyleneimine forms two active polymer layers 2 with positive charges formed by amino groups, and the two opposite surfaces of the glass sheet (i.e., the two surfaces 11 of the silicon-containing substrate 1) are electrostatically bonded to the bonding surface 21, respectively.

[0104] It is worth noting that in the manufacturing method of the second embodiment, each capture biomolecule 3 is not directly bound to the active surface 22 of the active polymer layer 2 , but is indirectly bound to the active surface 22 of the active polymer layer 2 via a noble metal nanoparticle 5 .

[0105] In detail, the worker may select the noble metal nanoparticles 5 with negative charge so that the noble metal nanoparticles 5 can be electrostatically bonded to the active surface 22 of the active polymer layer 2 with positive charge, and because the noble metal nanoparticles 5 can form a covalent bond with the capture biomolecules 3, the multiple capture biomolecules 3 can be bonded to the active surface 22 of the active polymer layer 2 through the noble metal nanoparticles 5. For example, the noble metal nanoparticles 5 can be selected from the group consisting of gold nanoparticles, platinum nanoparticles, silver nanoparticles and palladium nanoparticles.

[0106] Furthermore, the worker may first allow the noble metal nanoparticles 5 to electrostatically bind to the active surface 22 of the active polymer layer 2, and then allow the capture biomolecules 3 to covalently bind to the noble metal nanoparticles 5; or the worker may allow the noble metal nanoparticles 5 to covalently bind to the capture biomolecules 3 to form a complex, and then allow the noble metal nanoparticles 5 in the complex to electrostatically bind to the active surface 22 of the active polymer layer 2, so that the capture biomolecules 3 can indirectly bind to the active surface 22 of the active polymer layer 2 via the noble metal nanoparticles 5. This is something that a technician in the technical field of the present invention can adjust on his own according to needs and is not limited here.

[0107] In this embodiment, a sodium citrate (Na3C6H5O7) aqueous solution (10 mL, 38.8M) is added to a boiling chloroauric acid (H[AuCl4]) aqueous solution (temperature is about 100°C). After the color of the mixed solution changes from light yellow to wine red, the mixed solution is slowly cooled to room temperature to form a plurality of gold nanoparticles with a size between 10 and 50 nm. Finally, the worker can wash the plurality of gold cores and re-suspend the plurality of gold nanoparticles in deionized water for standby use to obtain a gold nanoparticle suspension.

[0108] Next, at room temperature, the glass sheet is immersed in 0.5 mL of a gold nanoparticle suspension so that the multiple gold nanoparticles can be electrostatically bonded to the active surface 22 of the active polymer layer 2. After being washed with deionized water, the glass sheet is immersed in 0.5 mL of an antibody solution (containing 500 ng / mL thiolated rabbit anti-FXYD3 polyclonal antibodies dissolved in phosphate buffer) and reacted at room temperature for 2 hours so that the rabbit anti-FXYD3 polyclonal antibodies can form covalent bonds with the multiple gold nanoparticles and bond to the active surface 22 of the active polymer layer 2 through the multiple gold nanoparticles, thereby obtaining a Figure 8 The biosensor S shown.

[0109] In addition, the worker can also treat the glass sheet with the blocking solution to cover the blocking layer 4 on the exposed area 22b of the active surface 22 (i.e., the area not bonded with the noble metal nanoparticles 5). In this embodiment, the glass sheet is immersed in 1 mL of a bovine serum albumin aqueous solution (containing 2 wt% bovine serum albumin) for 1 hour at room temperature, so that the blocking layer 4 can be formed on the exposed areas 22b of the active surfaces 22 of the two active polymer layers 2 located on the two opposite surfaces of the glass sheet, and then washed with tris(hydroxymethyl)aminomethane buffer to obtain the following. Fig. 9 The biosensor S shown.

[0110] The method of using the biosensor S manufactured according to the manufacturing method of the second embodiment is as follows:

[0111] Preparation of probe solution: The worker first added 10 μL of thiolated rabbit anti-FXYD3 polyclonal antibody (10 ng / μL) and 10 μL of horseradish peroxidase (HRP, 20 mg / mL) to the aforementioned gold nanoparticle suspension, and reacted at room temperature in the dark for 2 hours to allow the thiolated rabbit anti-FXYD3 polyclonal antibody and the horseradish peroxidase (HRP) to bind to the gold nanoparticles. The mixture was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed. Finally, 200 μL of bovine serum albumin aqueous solution (containing 2 wt% bovine serum albumin) was added. After reacting for 30 minutes, the mixture was centrifuged at 12,000 rpm for 10 minutes again, and the supernatant was removed. Finally, the mixture was washed with 200 μL of tris(hydroxymethyl)aminomethane buffer (containing 0.1% polysorbate 20). After removing the supernatant, the resulting probe particles were resuspended in 200 μL of 1% tris(hydroxymethyl)aminomethane buffer. μL of phosphate buffer to obtain the probe solution.

[0112] Preparation of cleaning solution: tri(hydroxymethyl)aminomethane (Tris) buffer containing 0.001% polysorbate 20 (Tween 20).

[0113] Preparation of chromogen solution: The chromogen solution contains 0.5 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) and 0.5% hydrogen peroxide (H2O2) dissolved in 0.1 M sodium acetate aqueous solution with a pH of 5.5.

[0114] Preparation of the stop reagent: The stop reagent is a 1 M hydrochloric acid aqueous solution.

[0115] Sample sampling: In order to confirm whether a suspected patient suffers from urothelial carcinoma (UC) using the biosensor S, the sample used can be a whole blood sample, a serum sample, a plasma sample, a urine sample, etc. from the suspected patient. In this embodiment, in order to facilitate the real-time detection of urothelial carcinoma, a clean-catch urine sample is selected. After obtaining the urine sample, it is necessary to centrifuge at 5,000 rpm for 10 minutes at a temperature of 4°C. The obtained precipitate is then treated with a protein extraction solution (PROPREP protein extraction solution, purchased from iNtRON Biotechnology, Inc., Cat. No. 17081) for 15 minutes to lyse the cells, and then immediately centrifuged at 13,000 rpm for 10 minutes at a temperature of 4°C. The obtained supernatant can be stored at -80°C for future use.

[0116] Please refer to Fig.10 As shown, the operation process of the biosensor S is as follows:

[0117] Will be like Fig. 9 The biosensor S shown is adhered to the inner side of a bottle cap C, so that when the bottle cap C is combined with a bottle body, the captured biomolecules 3 can face the bottle body.

[0118] Next, 0.15 mL of the probe solution was added to a plastic bottle B1, and then 0.05 mL of the urine specimen was added, and the plastic bottle B1 was bonded with the bottle cap C. After being inverted, the bottle was left to stand at room temperature for 15 minutes, so that the probe particles in the probe solution could specifically bind to the FXYD3 protein in the specimen, and then specifically bind to the capture biomolecule 3 (rabbit anti-FXYD3 polyclonal antibody) on the inner surface of the glass bottle.

[0119] After opening the bottle cap C, the bottle cap C is combined with another plastic bottle body B2 (the plastic bottle body B2 is filled with 1 mL of cleaning solution) and turned upside down again to clean the biosensor S adhered to the inner side of the bottle cap C.

[0120] Then, the bottle cap C is combined with a glass bottle body B3 (the glass bottle body B3 is filled with 0.2 mL of the pigment solution), and is inverted again. The bottle cap C is left standing for 2 minutes to observe the color change of the mixed solution in the glass bottle body B3. At this time, under the action of horseradish peroxidase (HRP), the mixed solution in the glass bottle body B3 gradually turns dark blue.

[0121] Finally, the stop reagent is added to the dark blue mixed solution. At this time, the dark blue mixed solution will turn into yellow under the action of the stop reagent. In this way, the worker can observe the color change of the mixed solution with the naked eye, or measure the change of absorbance value at a specific wavelength (such as 450 nm) with a spectrometer.

[0122] When the biosensor S manufactured by the manufacturing method of the second embodiment is used, if the suspected patient indeed suffers from urothelial cell carcinoma, the capture biomolecule 3 (rabbit anti-FXYD3 polyclonal antibody) can capture the FXYD3 protein in the specimen of the suspected patient and further react with horseradish peroxidase (HRP). Therefore, when the chromogen solution is added, the chromogen solution will be affected by the horseradish peroxidase (HRP) and change color. In other words, if the color of the mixed solution in the glass bottle eventually changes to blue, it shows that the specimen of the suspected patient indeed contains FXYD3 protein, which means that the suspected patient indeed suffers from urothelial cell carcinoma.

[0123] In order to test the detection specificity of the biosensor S made according to the manufacturing method of the second embodiment for the biomarker (FXYD3 protein) of urothelial cell carcinoma, the following test was performed:

[0124] (E) Incorporation of noble metal nanoparticles

[0125] This test is as shown in Table 2, the active polymer layer 2 (formed by branched polyethyleneimine) is formed on the silicon-containing substrate 1 (glass sheet) to obtain the E1 group of glass test pieces, and after the active polymer layer 2 (formed by branched polyethyleneimine) is formed on the silicon-containing substrate 1 (glass sheet), the noble metal nanoparticles 5 (the aforementioned gold nanoparticles) are electrostatically bonded to the active surface 22 of the active polymer layer 2 to obtain the E2 group of glass test pieces.

[0126] Table 2. Treatment conditions of each group of glass specimens in this test

[0127]

[0128] Next, due to the localized surface plasmon resonance (LSPR) characteristics of the gold nanoparticles, which can produce a characteristic absorption peak at 520 nm (as stated in the journal article "Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay" published by Huang et al. in 2020), the absorbance values ​​of each group of glass specimens at wavelengths between 400 and 650 nm were analyzed.

[0129] Please refer to Fig.11 As shown, the glass test piece of group E2 has a peak at a wavelength of 520 nm, indicating that the noble metal nanoparticles 5 (the gold nanoparticles) have indeed been electrostatically bonded to the active surface 22 of the active polymer layer 2 .

[0130] (F) Capturing the binding of biomolecules

[0131] As shown in Table 3, this experiment is to form the active polymer layer 2 (formed by branched polyethyleneimine) on the silicon-containing substrate 1 (glass sheet), and then the noble metal nanoparticles 5 (the aforementioned gold nanoparticles) are electrostatically bonded to the active surface 22 of the active polymer layer 2, and then the blocking layer 4 is formed with the blocking solution (aqueous solution of bovine serum albumin) to obtain the F1 group of glass test pieces, and after the noble metal nanoparticles 5 (the aforementioned gold nanoparticles) are electrostatically bonded to the active surface 22 of the active polymer layer 2, the capture biomolecules 3 (rabbit anti-FXYD3 polyclonal antibodies) are covalently bonded to the noble metal nanoparticles 5, and then the blocking layer 4 is formed with the blocking solution (aqueous solution of bovine serum albumin) to obtain the F2 group of glass test pieces.

[0132] Table 3. Treatment conditions of each group of glass test pieces in this test

[0133]

[0134] Next, each set of glass test pieces is treated with a goat anti-rabbit iFluor488 secondary antibody labeled with a green fluorescent label, so that the captured biomolecule 3 is labeled with the green fluorescent label, and finally a fluorescent image of each set of glass test pieces is captured.

[0135] Please refer to Fig.12 , Fig.13As shown, almost no green fluorescence signal can be observed on the glass test piece of group F1, while uniform green fluorescence signal can be observed on the glass test piece of group F2, indicating that the capture biomolecule 3 (rabbit anti-FXYD3 polyclonal antibody) has been evenly distributed on the glass test piece through the precious metal nanoparticles 5, and no obvious secondary antibody green fluorescence is shown in the F1 group, confirming that the biosensor has good anti-interference ability to reduce non-specific molecular binding.

[0136] (G) Changes in surface roughness

[0137] As shown in Table 4, the present experiment takes an untreated silicon-containing substrate 1 (glass sheet) as the first group of glass test pieces, forms the active polymer layer 2 (formed by branched polyethyleneimine) on the silicon-containing substrate 1 (glass sheet) to form the second group of glass test pieces, and after the active polymer layer 2 (formed by branched polyethyleneimine) is formed on the silicon-containing substrate 1 (glass sheet), the noble metal nanoparticles 5 (the aforementioned gold nanoparticles) are electrostatically bonded to the active surface 22 of the active polymer layer 2 to obtain the third group of glass test pieces. The glass test piece, after the noble metal nanoparticles 5 (the aforementioned gold nanoparticles) are electrostatically bonded to the active surface 22 of the active polymer layer 2, the capture biomolecules 3 (rabbit anti-FXYD3 polyclonal antibodies) are covalently bonded to the noble metal nanoparticles 5 to obtain the G4 group of glass test pieces, and after the capture biomolecules 3 (rabbit anti-FXYD3 polyclonal antibodies) are covalently bonded to the noble metal nanoparticles 5, the blocking solution (bovine serum albumin aqueous solution) is used to form the blocking layer 4 to obtain the G5 group of glass test pieces.

[0138] Table 4. Treatment conditions of each group of glass specimens in this test

[0139]

[0140] Next, the surface roughness of each set of glass specimens was analyzed using atomic force microscopy (AFM).

[0141] Please refer to Fig.14As shown, the center line average roughness (R) of the glass test piece of group G1 is 0.149 nm, the center line average roughness of the glass test piece of group G2 is 0.252 nm, the center line average roughness of the glass test piece of group G3 is 2.662 nm, the center line average roughness of the glass test piece of group G4 is 2.786 nm, and the center line average roughness of the glass test piece of group G5 is 2.539 nm, indicating that when a large number of precious metal nanoparticles 5 and captured biomolecules 3 are located on the surface of the glass test piece, the surface roughness of the glass test piece will be greatly improved, and the formation of the blocking layer 4 will reduce the roughness of the surface of the glass test piece.

[0142] (H) Test results of calibration curve

[0143] In this study, a standard of FXYD3 protein (i.e., a biomarker for urothelial carcinoma) was taken and added to a urine sample from a healthy individual that did not contain FXYD3 protein, so that the concentration of the standard was 1, 2.5, 10, 100, 500, and 1,000 pg / mL, respectively. The standard was then detected using the aforementioned method, with the absorbance at a wavelength of 450 nm measured by a spectrometer, and a linear regression analysis was performed.

[0144] Please refer to Fig.15 As shown, as the FXYD3 protein concentration in the urine sample increases, the absorbance value measured at a wavelength of 450 nm also increases, and the regression equation of the calibration curve obtained by linear regression analysis is shown in the following formula (III), and the determination coefficient (R2) of the regression equation is 0.9960.

[0145] Formula (III).

[0146] (I) Urine test results

[0147] The experiment collected patients who were diagnosed with urothelial carcinoma by clinical cystoscopy and biopsy before standard clinical practice. Among them, urine samples from healthy individuals were used as group I1 (a total of 4 cases), urine samples from individuals with low-grade urothelial carcinoma (low-grade UC) were used as groups I2 and I3 (a total of 6 cases, including 4 cases of lower urinary tract bladder cancer in group I2 and 2 cases of upper urothelial carcinoma in group I3), and urine samples from individuals with high-grade urothelial carcinoma (high-grade UC) were used as groups I4 and I5 (a total of 30 cases, including 19 cases of lower urinary tract bladder cancer in group I4 and 11 cases of upper urothelial carcinoma in group I5).

[0148] Next, the above-mentioned method was used for detection, and the absorbance value at a wavelength of 450 nm was measured by a spectrometer, and the concentration of FXYD3 protein in each urine sample was calculated. In addition, the absorbance value at a wavelength of 450 nm measured by enzyme-linked immunosorbent assay (ELISA) was used as a control, and the concentration of FXYD3 protein in each urine sample was calculated.

[0149] Please refer to Fig.16 As shown, whether the urine samples were from healthy individuals (Group I1) or from individuals with stage T4 urothelial carcinoma (Groups I2 to I7), the concentrations of FXYD3 protein measured were similar when the urine samples were tested by the aforementioned method or by enzyme-linked immunosorbent assay (ELISA).

[0150] According to the above test data, it can be known that the biosensor S prepared by the manufacturing method of the present invention does have good sensitivity, so only 5 to 50 μL of blood sample (or urine sample) is needed to detect the target biological molecules (immunoglobulin M (IgM) specific to the new coronavirus SARS-CoV-2, immunoglobulin G (IgG) or FXYD3 protein specific to the new coronavirus SARS-CoV-2). Compared with the traditional quantitative real-time polymerase chain reaction (RT-qPCR), the detection method using the biosensor S prepared by the manufacturing method of the present invention is not only simple to operate and has a shorter reaction time, but also does not require the operator to be exposed to the risk of infection; and compared with the traditional enzyme-linked immunosorbent assay (ELISA), the detection method using the biosensor S prepared by the manufacturing method of the present invention has a lower detection cost, and can significantly shorten the reaction time to less than 15 minutes, and the detection result can be directly judged only by the naked eye.

[0151] In summary, in the method for manufacturing the biosensor of the present invention, by using the ethanol solution, at least one surface of the silicon-containing substrate can be made to have a negative charge without using a strong acid or a strong base, which can not only improve the safety of the working environment of the workers, but also reduce the treatment cost of strong acid and strong base waste liquids, and further prevent the discharge of strong acid and strong base waste liquids from causing adverse effects on environmental organisms or buildings, which is the effect of the present invention.

[0152] Furthermore, in the manufacturing method of the biosensor of the present invention, by using the ethanol solution, at least one surface of the silicon-containing substrate can be made to have a negative charge without using special equipment such as an oxygen plasma cleaner, and the high temperature and high pressure environment required for oxygen plasma treatment can also be avoided, which helps to reduce the manufacturing cost of the biosensor.

[0153] In addition, the biosensor of the present invention is manufactured by the aforementioned biosensor manufacturing method, and the selected substrate is the silicon-containing substrate (e.g., a glass substrate, a silicon dioxide substrate, a quartz substrate or a siloxane substrate). In other words, the biosensor is not a plastic product and can be recycled and reused after melting. Moreover, in the process of manufacturing the biosensor, no strong acid solution or strong alkali solution is used, so that the biosensor is an environmentally friendly good, which is the effect of the present invention.

Claims

1. A method for manufacturing a biosensor, characterized in that: It consists of the following steps: providing a silicon-containing substrate having at least one surface; Treating the silicon-containing substrate with an ethanol solution, shaking at a temperature of 22-28° C. for 10 minutes, so that a plurality of hydroxide ions with negative charges are formed on the at least one surface of the silicon-containing substrate, wherein the ethanol solution is an ethanol aqueous solution with an ethanol concentration of 60-99.8%; Forming at least one active polymer layer with positive charge on the at least one surface of the silicon-containing substrate, the at least one active polymer layer having a binding surface and an active surface opposite to each other, the at least one active polymer layer being bound to the silicon-containing substrate via the binding surface; and A plurality of capture biomolecules are bound to the active surface of the at least one active polymer layer.

2. The method for manufacturing a biosensor according to claim 1, wherein: The plurality of capture biomolecules are respectively negatively charged, so that the plurality of capture biomolecules are respectively electrostatically bound to the active surface of the at least one active polymer layer.

3. The method for manufacturing a biosensor according to claim 2, wherein: The plurality of capture biomolecules are bound to a covered area of ​​the active surface of the at least one active polymer layer, and the active surface of the at least one active polymer layer further comprises an exposed area.

4. The method for manufacturing a biosensor according to claim 1, wherein: The plurality of captured biomolecules are respectively combined with the active surface of the at least one active polymer layer through a plurality of noble metal nanoparticles.

5. The method for manufacturing a biosensor according to claim 4, wherein: The plurality of noble metal nanoparticles are respectively negatively charged, so that the plurality of noble metal nanoparticles are respectively electrostatically bonded to the active surface of the at least one active polymer layer.

6. The method for manufacturing a biosensor according to claim 5, wherein: The plurality of capture biomolecules are covalently bound to the plurality of noble metal nanoparticles respectively.

7. The method for manufacturing a biosensor according to claim 6, wherein: The plurality of noble metal nanoparticles are combined with a covering area of ​​the active surface of the at least one active polymer layer, and the active surface of the at least one active polymer layer further comprises an exposed area.

8. The method for manufacturing a biosensor according to claim 1, wherein: The active surface of the at least one active polymer layer has a functional group, and the functional group is selected from the group consisting of amine groups and ammonium groups.

9. The method for manufacturing a biosensor according to claim 8, wherein: The at least one active polymer layer is formed by a polymer selected from the group consisting of polyethyleneimine, polyallylamine hydrochloride, polyβ-amino ester, polydiallyl dimethyl ammonium chloride and polyacrylamide.

10. The method for manufacturing a biosensor according to claim 9, wherein: The polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

11. The method for manufacturing a biosensor according to claim 9, wherein: The poly β-amino ester is a linear poly β-amino ester or a branched poly β-amino ester.

12. A method for manufacturing a biosensor, characterized in that: The method comprises the following steps: providing a silicon-containing substrate having at least one surface; Treating the silicon-containing substrate with an ethanol solution, shaking at a temperature of 22-28° C. for 10 minutes, so that a plurality of hydroxide ions with negative charges are formed on the at least one surface of the silicon-containing substrate, wherein the ethanol solution is an ethanol aqueous solution with an ethanol concentration of 60-99.8%; Forming at least one active polymer layer with positive charge on the at least one surface of the silicon-containing substrate, the at least one active polymer layer having a binding surface and an active surface opposite to each other, the at least one active polymer layer being bound to the silicon-containing substrate via the binding surface; Allowing a plurality of capture biomolecules each having a negative charge to electrostatically bind to a covered area of ​​the active surface of the at least one active polymer layer, and the active surface of the at least one active polymer layer further comprises an exposed area; and A blocking layer is provided to cover the exposed area of ​​the active surface of the at least one active polymer layer.

13. The method for manufacturing a biosensor according to claim 12, wherein: The active surface of the at least one active polymer layer has a functional group, and the functional group is selected from the group consisting of amine groups and ammonium groups.

14. The method for manufacturing a biosensor according to claim 13, wherein: The at least one active polymer layer is formed by a polymer selected from the group consisting of polyethyleneimine, polyallylamine hydrochloride, polyβ-amino ester, polydiallyl dimethyl ammonium chloride and polyacrylamide.

15. The method for manufacturing a biosensor according to claim 14, wherein: The polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

16. The method for manufacturing a biosensor according to claim 14, wherein: The poly β-amino ester is a linear poly β-amino ester or a branched poly β-amino ester.

17. A method for manufacturing a biosensor, characterized in that: It consists of the following steps: providing a silicon-containing substrate having at least one surface; Treating the silicon-containing substrate with an ethanol solution, shaking at a temperature of 22-28° C. for 10 minutes, so that a plurality of hydroxide ions with negative charges are formed on the at least one surface of the silicon-containing substrate, wherein the ethanol solution is an ethanol aqueous solution with an ethanol concentration of 60-99.8%; Forming at least one active polymer layer with positive charge on the at least one surface of the silicon-containing substrate, the at least one active polymer layer having a binding surface and an active surface opposite to each other, the at least one active polymer layer being bound to the silicon-containing substrate via the binding surface; Allowing a plurality of captured biomolecules to bind to a covered area of ​​the active surface of the at least one active polymer layer through a plurality of noble metal nanoparticles, respectively, and the active surface of the at least one active polymer layer further comprises an exposed area; and A blocking layer is provided to cover the exposed area of ​​the active surface of the at least one active polymer layer.

18. The method for manufacturing a biosensor according to claim 17, wherein: The active surface of the at least one active polymer layer has a functional group, and the functional group is selected from the group consisting of amine groups and ammonium groups.

19. The method for manufacturing a biosensor according to claim 18, wherein: The at least one active polymer layer is formed by a polymer selected from the group consisting of polyethyleneimine, polyallylamine hydrochloride, polyβ-amino ester, polydiallyl dimethyl ammonium chloride and polyacrylamide.

20. The method for manufacturing a biosensor according to claim 19, wherein: The polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

21. The method for manufacturing a biosensor according to claim 19, wherein: The poly β-amino ester is a linear poly β-amino ester or a branched poly β-amino ester.

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