A porous silicon microcavity biosensor and a preparation method thereof, and a gibberellin fluorescence image detection method

Abstract

The application discloses a porous silicon microcavity biosensor and a preparation method thereof, and a gibberellin fluorescence image detection method. The preparation method of the porous silicon microcavity biosensor comprises the following steps: (1) a p-type boron-doped single crystal silicon is prepared into a porous silicon microcavity by using an electrochemical etching method; and (2) a functionalization treatment is performed on the porous silicon microcavity, so that the central wavelength is blue-shifted to 625 nm, and a treated porous silicon microcavity, namely the porous silicon microcavity biosensor, is obtained. The porous silicon microcavity biosensor and the preparation method thereof, and the gibberellin fluorescence image detection method have the advantages of low cost and high sensitivity by using the porous silicon microcavity and modifying the same for the fluorescence image detection of gibberellin.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a porous silicon microcavity biosensor and its preparation method, and a method for detecting gibberellin fluorescence images. Background Technology

[0002] Porous silicon is used in biosensing due to its advantages such as large specific surface area, good bioactivity, compatibility and ease of fabrication into various types of photonic devices, and can be fabricated into high-performance optical biosensors.

[0003] Porous silicon optical biosensors primarily employ two detection mechanisms: one based on refractive index changes, and the other based on fluorescence changes. Fluorescence-based detection in porous silicon offers advantages such as high sensitivity and rapid, convenient operation. Fluorescence-based markers typically fall into two categories: the first is organic dye molecules, which, due to their strong fluorescence, can be used as fluorescent markers for biomolecules; the second is semiconductor quantum dots, which, due to their unique optical properties (including good photostability, high quantum yield, wide excitation spectral range, and tunable emission spectrum), are also excellent nanoluminescent materials and can be used as markers for highly sensitive biosensors.

[0004] Digital imaging can be used to detect fluorescent bioreactions in porous silicon. Antibodies labeled with quantum dots are used to specifically bind to antigens of the analyte immobilized on the inner wall of a porous silicon microcavity. The images are then captured using a digital microscope, and the average grayscale value of the image is calculated using digital processing software.

[0005] Gibberellin is a commonly used plant growth regulator in crops. Excessive use can pose health risks to humans, making its detection crucial for food safety. However, fluorescence imaging detection methods for gibberellin suffer from drawbacks such as high cost and low sensitivity.

[0006] In view of this, the present invention proposes a novel porous silicon microcavity biosensor and a method for detecting gibberellin fluorescence images, which can achieve low-cost and highly sensitive biological detection. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a porous silicon microcavity biosensor, which, after improvement, can effectively enhance the sensitivity of gibberellin detection.

[0008] To achieve the above objectives, the technical solution adopted is as follows:

[0009] A method for fabricating a porous silicon microcavity biosensor includes the following steps:

[0010] (1) A porous silicon microcavity was prepared by electrochemical etching of boron-doped p-type single crystal silicon.

[0011] (2) The porous silicon microcavity is functionalized to blue shift its center wavelength to 625nm, thus obtaining the processed porous silicon microcavity, namely the porous silicon microcavity biosensor.

[0012] Furthermore, in step (1), when preparing the porous silicon microcavity by electrochemical etching, the current densities for the high-porosity layer, low-porosity layer, and intermediate layer are set to 90 mA / cm², respectively. 2 40mA / cm 2 90mA / cm 2 The corrosion times were 1.7s, 2.0s, and 3.4s, respectively.

[0013] Furthermore, in step (1), when preparing porous silicon microcavities by electrochemical etching, the electro-etching solution is a mixture of hydrofluoric acid and anhydrous ethanol in a volume ratio of 1:1±0.2.

[0014] Furthermore, the functionalization process includes the following steps:

[0015] After immersing the porous silicon microcavity in hydrogen peroxide solution, it is dried, rinsed with water, and dried again to obtain an oxidized porous silicon microcavity.

[0016] The porous silicon microcavity after oxidation treatment is immersed in silanization solution, then rinsed with water and dried to obtain the porous silicon microcavity after silanization treatment.

[0017] After the silanization treatment, the porous silicon microcavity was immersed in glutaraldehyde solution, rinsed repeatedly with phosphate buffer and water, and dried in nitrogen to obtain the glutaraldehyde-treated porous silicon microcavity.

[0018] Furthermore, the concentration of the hydrogen peroxide solution is 35-45%, and the immersion time is 2.5-3.5 hours;

[0019] The silanization solution contains APTES, water, and methanol in a volume ratio of 1:8-12:8-12, and the immersion time is 50-70 min.

[0020] The glutaraldehyde solution has a concentration of 2.0-3.0% Vt%, and the immersion time is 50-70 min.

[0021] Furthermore, the concentration of the hydrogen peroxide solution is 40%, and the immersion time is 3 hours;

[0022] The silanization solution contains APTES, water, and methanol in a volume ratio of 1:10:10, and the immersion time is 60 min.

[0023] The glutaraldehyde solution was 2.5% Vt, and the immersion time was 60 min.

[0024] Another objective of this invention is to provide a porous silicon microcavity biosensor, which is prepared using the above-described preparation method.

[0025] Another objective of this invention is to provide a method for detecting the fluorescence image of gibberellin, based on the aforementioned porous silicon microcavity, for detecting the concentration of biomolecules, which has the advantages of low cost and high sensitivity.

[0026] To achieve the above objectives, the technical solution adopted is as follows:

[0027] A fluorescence image detection method for gibberellin is disclosed, which uses the aforementioned porous silicon microcavity biosensor to detect gibberellin via fluorescence image detection.

[0028] Furthermore, in the detection process, the semiconductor quantum dots are carboxyl water-soluble CdSe / ZnS quantum dots, which are coupled with gibberellin antibody, and the quantum dots are excited using 375nm excitation light.

[0029] Furthermore, during the detection process, a fluorescence image of the porous silicon surface is obtained using a digital microscope. The concentration of the target biomolecule is calculated by calculating the average gray value change of the image before and after the biological reaction and using the amount of average gray value change.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention experimentally demonstrates that, when the center wavelength of the porous silicon Bragg reflector and the wavelength of the defect states in the porous silicon microcavity structure coincide with the fluorescence wavelength of the quantum dots, the fluorescence intensity enhancement of the quantum dots using the microcavity structure is approximately three times that of the Bragg structure. Based on these experimental conclusions, a porous silicon biosensor was successfully fabricated using fluorescence imaging to detect the analyte.

[0032] This invention then designs a porous silicon microcavity structure with a center wavelength of 633 nm. The porous silicon microcavity is prepared using an electrochemical etching method and functionalized to blue-shift its center wavelength to 625 nm. Antibodies to the analyte are labeled with carboxyl-based water-soluble CdSe / ZnS quantum dots, which specifically react with the antigen of the analyte immobilized within the porous silicon pores. A 375 nm ultraviolet semiconductor laser is used as the excitation source. The quantum dots in the reactants are excited within the porous silicon pores of the microcavity structure, generating fluorescence. The porous silicon microcavity structure further enhances the fluorescence generated by the quantum dots. The concentration of the target biomolecule is detected by analyzing the grayscale values ​​of the fluorescence images and calculating the average grayscale value change of the digital images before and after the biological reaction. Attached Figure Description

[0033] Figure 1 The reflection spectra of the functionalized porous silicon microcavity structure (left) and the porous silicon Bragg mirror (right);

[0034] Figure 2 The fluorescence spectra of two porous silicon samples with different structures after being combined with CdSe / ZnS quantum dots;

[0035] Figure 3 The fluorescence spectra of quantum dots excited by excitation light of three wavelengths are shown.

[0036] Figure 4 The reflectance spectrum of a porous silicon microcavity after oxidation, silanization, and glutaraldehyde treatment;

[0037] Figure 5 Comparison of reflectance spectra of porous silicon microcavity samples with added gibberellin antigen;

[0038] Figure 6 The fluorescence spectra are shown before and after conjugation of quantum dots with gibberellin antibody at a concentration of 1 mg / L;

[0039] Figure 7 This is a flowchart illustrating the detection of GAs antigens using a porous silicon optical biosensor.

[0040] Figure 8 This is a detection optical path diagram of the porous silicon microcavity sensor of the present invention;

[0041] Figure 9 A trend graph showing the changes in average gray value for seven concentrations of gibberellin antigen solutions;

[0042] Figure 10 This is a linear relationship graph showing the relationship between four low concentrations of gibberellin antigen and their corresponding changes in average gray value. Detailed Implementation

[0043] To further illustrate the porous silicon microcavity biosensor and its preparation method, as well as the gibberellin fluorescence image detection method of the present invention, and to achieve the intended objectives of the invention, the following detailed description, in conjunction with preferred embodiments, details the specific implementation, structure, features, and effects of the porous silicon microcavity biosensor and its preparation method, as well as the gibberellin fluorescence image detection method proposed according to the present invention. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable manner.

[0044] The following will provide a detailed description of the porous silicon microcavity biosensor and its fabrication method, as well as the method for detecting gibberellin fluorescence images, based on specific embodiments of the present invention:

[0045] This invention first experimentally demonstrates that porous silicon microcavity structures enhance the fluorescence of quantum dots more strongly than porous silicon Bragg mirrors. A porous silicon microcavity photonic crystal sensor is fabricated and combined with digital imaging methods for the detection of gibberellin. Semiconductor quantum dots are used as fluorescent markers to label antibodies against the analyte, resulting in a specific reaction with the antigen of the analyte immobilized on the inner wall of the porous silicon. The porous silicon microcavity structure further enhances the fluorescence generated by the semiconductor quantum dots. Fluorescence images of the porous silicon surface are obtained using a digital microscope. By calculating the change in average grayscale value of the images before and after the biological reaction, the target biomolecule can be detected with high sensitivity.

[0046] The technical solution adopted in this invention is as follows:

[0047] A method for fabricating a porous silicon microcavity biosensor includes the following steps:

[0048] (1) A porous silicon microcavity was prepared by electrochemical etching of boron-doped p-type single crystal silicon.

[0049] (2) The porous silicon microcavity is functionalized to blue-shift its center wavelength to 625nm, and then semiconductor quantum dots are fixed on the pore wall to obtain the processed porous silicon microcavity, namely the porous silicon microcavity biosensor.

[0050] Furthermore, in step (1), when preparing the porous silicon microcavity by electrochemical etching, the current densities for the high-porosity layer, low-porosity layer, and intermediate layer are set to 90 mA / cm², respectively. 2 40mA / cm 2 90mA / cm 2 The corrosion times were 1.7s, 2.0s, and 3.4s, respectively.

[0051] Furthermore, in step (1), when preparing porous silicon microcavities by electrochemical etching, the electro-etching solution is a mixture of hydrofluoric acid and anhydrous ethanol in a volume ratio of 1:1±0.2.

[0052] Furthermore, the functionalization process includes the following steps:

[0053] After immersing the porous silicon microcavity in hydrogen peroxide solution, it is dried, rinsed with water, and dried again to obtain an oxidized porous silicon microcavity.

[0054] The porous silicon microcavity after oxidation treatment is immersed in silanization solution, then rinsed with water and dried to obtain the porous silicon microcavity after silanization treatment.

[0055] After the silanization treatment, the porous silicon microcavity was immersed in glutaraldehyde solution, rinsed repeatedly with phosphate buffer and water, and dried in nitrogen to obtain the glutaraldehyde-treated porous silicon microcavity.

[0056] Furthermore, the concentration of the hydrogen peroxide solution is 35-45%, and the immersion time is 2.5-3.5 hours;

[0057] The silanization solution contains APTES, water, and methanol in a volume ratio of 1:8-12:8-12, and the immersion time is 50-70 min.

[0058] The glutaraldehyde solution has a concentration of 2.0-3.0% Vt%, and the immersion time is 50-70 min.

[0059] Furthermore, the concentration of the hydrogen peroxide solution is 40%, and the immersion time is 3 hours;

[0060] The silanization solution contains APTES, water, and methanol in a volume ratio of 1:10:10, and the immersion time is 60 min.

[0061] The glutaraldehyde solution was 2.5% Vt, and the immersion time was 60 min.

[0062] A fluorescence image detection method for gibberellin is disclosed, which uses the aforementioned porous silicon microcavity biosensor to detect gibberellin via fluorescence image detection.

[0063] Furthermore, in the detection process, the semiconductor quantum dots are carboxyl water-soluble CdSe / ZnS quantum dots, which are coupled with gibberellin antibody, and the quantum dots are excited using 375nm excitation light.

[0064] Furthermore, during the detection process, a fluorescence image of the porous silicon surface is obtained using a digital microscope. The concentration of the target biomolecule is calculated by calculating the average gray value change of the image before and after the biological reaction and using the amount of average gray value change.

[0065] In the technical solution of this invention, the fluorescence signal is further enhanced by the porous silicon microcavity because the porous silicon microcavity structure has no fluorescence before the specific biological reaction occurs. After the specific reaction occurs, the quantum dots in the reactants are excited in the porous silicon pores of the microcavity structure to generate fluorescence. The porous silicon microcavity structure can reflect the fluorescence falling on its high reflectivity band, causing the downward-emitted fluorescence to be reflected upward-emitted fluorescence, thus enhancing the fluorescence through the microcavity structure. Therefore, this invention has the characteristics of low detection cost, simple detection device, fast detection speed, and high detection sensitivity, requiring only a digital microscope and general optical devices to complete the detection.

[0066] Example 1.

[0067] The specific operating steps are as follows:

[0068] (1) Using p-type boron-doped single crystal silicon (crystal orientation) <100> Porous silicon microcavities (with resistivity 0.03-0.06 Ω·cm and thickness 400±10 μm) were fabricated using a single-cell anodic electrochemical etching method. The electrolytic etching solution was prepared by a ratio of hydrofluoric acid (40% by mass) to anhydrous ethanol (C2H5OH, concentration ≥99%) of 1:1±0.2 (volume ratio).

[0069] When fabricating porous silicon microcavities using electrochemical etching, the current densities for the high-porosity layers, low-porosity layers, and intermediate layers are 90 mA / cm². 2 40mA / cm 2 90mA / cm 2 The corrosion times were 1.7s, 2.0s, and 3.4s, respectively.

[0070] (2) Functionalization of porous silicon microcavities resulted in a blue shift of the center (defect state) wavelength of the treated porous silicon microcavities to 625 nm.

[0071] The steps of functionalization are as follows:

[0072] ① Immerse the newly prepared porous silicon microcavity sample completely in hydrogen peroxide solution (concentration: 35-45%) for 2.5-3.5 hours, place it in a vacuum drying oven and set it to 60°C. After waiting for 3 hours, take it out, rinse it several times with deionized water (DI), and then dry it in the air.

[0073] ② Prepare a 5% aminopropyltriethoxysilane (volume ratio 1:8-12:8-12 = APTES:DI:methanol) solution for silanization treatment. Immerse the oxidized porous silicon microcavity sample in the silanization solution for 50-70 min. After taking it out, rinse it repeatedly with deionized water and then place it in a vacuum drying oven at 100℃ for 10 min.

[0074] ③ Immerse the porous silicon microcavity sample in a 2.0-3.0% glutaraldehyde solution (using a mixture of DI and 50% glutaraldehyde solution) for 50-70 minutes. After removing it, rinse it repeatedly with phosphate buffer (PBS) and DI to remove residual glutaraldehyde solvent from the porous silicon microcavity sample, and then dry it in nitrogen.

[0075] The semiconductor quantum dots are carboxyl-based water-soluble CdSe / ZnS quantum dots, and the quantum dots are excited using 375nm excitation light.

[0076] (3) Using the above-mentioned porous silicon microcavity biosensor, gibberellin was detected by fluorescence image detection method.

[0077] During the detection process, a fluorescent image of the porous silicon surface was obtained using a digital microscope. The concentration of the target biomolecule was calculated by calculating the change in the average gray value of the image before and after the biological reaction.

[0078] Example 2.

[0079] The specific operating steps are as follows:

[0080] S10: Uses p-type boron-doped single-crystal silicon (crystal orientation) <100> Porous silicon Bragg mirrors (with resistivity of 0.03-0.06 Ω·cm and thickness of 400±10 μm) were fabricated using a single-cell anodic electrochemical etching method. The electrolytic etching solution was prepared by a 1:1 (volume ratio) ratio of hydrofluoric acid (40% by mass) to anhydrous ethanol (C2H5OH, concentration ≥99%). During the electrochemical etching of the porous silicon Bragg mirrors, the current densities for setting the high and low porosity layers were 90 mA / cm², respectively. 2 and 40mA / cm 2 The etching times were 1.7 s and 2.0 s, respectively. The porous silicon microcavity was constructed by adding a high-porosity defect layer between two perfectly symmetrical porous silicon Bragg mirrors. The current density and time parameters for the high-porosity and low-porosity layers were set to be the same as those for the Bragg mirrors, and the etching current density of the defect layer was 90 mA / cm². 2 The corrosion time was controlled at 3.4 seconds.

[0081] Assuming a high refractive index of 1.61 and a low refractive index of 1.23, the corresponding thicknesses are 98 nm for the high refractive index layer and 128 nm for the low refractive index layer. The center wavelength of the porous silicon Bragg mirror is 633 nm. In the porous silicon microcavity structure, the high refractive index layer has a thickness of 104 nm and a corresponding refractive index of 1.52, the low refractive index layer has a thickness of 131 nm and a corresponding refractive index of 1.21, and the intermediate defect layer has a thickness of 262 nm and a corresponding refractive index of 1.21. The wavelength of the defect state in the porous silicon microcavity structure is calculated to be 633 nm using the transfer matrix method.

[0082] S20: The Bragg structure and microcavity structure samples were functionalized, respectively. The center wavelengths of the porous silicon samples with these two structures were blue-shifted to 625 nm. Quantum dots were then fixed onto the pore walls of the functionalized porous silicon, and the fluorescence intensity of the two porous silicon devices was detected using fluorescence intensity detection. The fluorescence intensity of the two (porous silicon) samples combined with quantum dots was measured using a fluorescence spectrometer. The excitation wavelength used for the samples was 375 nm, the voltage was 700 V, and the slit width was 2.5 nm. Figure 1 The reflection spectra of the functionalized Bragg structure and the microcavity structure are presented, demonstrating the successful preparation and functionalization of porous silicon samples with both structures.

[0083] Test results as follows Figure 2 As shown, Figure 2 Fluorescence spectra of porous silicon samples with two different structures after being combined with quantum dots are presented. Figure 2 It is known that the fluorescence performance of the microcavity structure is stronger, up to 3 times stronger.

[0084] A semiconductor laser with a wavelength of 375 nm is used to generate excitation light, which is then used to excite the quantum dots in the reactants. The semiconductor quantum dots are excited by the 375 nm wavelength, reaching their strongest fluorescence peak. In the gibberellin detection process, using a semiconductor laser allows the quantum dots to be excited to their strongest fluorescence peak, while also reducing the size and weight of the detection device. This enables more cost-effective, highly sensitive, rapid, and miniaturized food safety detection.

[0085] The semiconductor quantum dots are carboxyl-based water-soluble CdSe / ZnS quantum dots. This invention uses excitation light at three wavelengths: 375 nm, 488 nm, and 532 nm. When the quantum dots are excited by a wavelength of 375 nm, they reach their strongest fluorescence peak, which is located at 625 nm. Figure 3 The fluorescence spectra of quantum dots excited by excitation light of three wavelengths show that the excitation effect at 375 nm is the best.

[0086] The steps for functionalization are as follows:

[0087] S21: Immerse the newly prepared porous silicon microcavity sample completely in hydrogen peroxide solution (concentration: 40%) for 3 hours, place it in a vacuum drying oven and adjust it to 60°C. After waiting for 3 hours, take it out, rinse it several times with deionized water (DI), and then dry it in the air.

[0088] S22: Prepare a 5% aminopropyltriethoxysilane (volume ratio 1:10:10 = APTES:DI:methanol) solution for silanization treatment. Immerse the oxidized porous silicon microcavity sample in the silanization solution for 1 hour. After taking it out, rinse it repeatedly with deionized water and then place it in a vacuum drying oven at 100°C for 10 minutes.

[0089] S23: Immerse the porous silicon microcavity sample in a 2.5% glutaraldehyde solution (volume ratio 19:1 = DI: 50% glutaraldehyde solution) for 1 hour. After removal, rinse repeatedly with phosphate buffer (PBS) and DI to remove residual glutaraldehyde solvent from the porous silicon microcavity sample, and dry in nitrogen.

[0090] Oxidation and silanization treatments can make the surface of newly prepared porous silicon samples more stable, while glutaraldehyde treatment can help biomolecules adhere to the inner wall of the porous silicon. Figure 4The reflectance spectra of the porous silicon microcavity after oxidation, silanization, and glutaraldehyde treatment demonstrate that each step of the functionalization process was successful.

[0091] S30: Dilute the gibberellin antigen solution with PBS to seven concentrations (0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL).

[0092] Using a micropipette, 45 μL of the desired concentration of gibberellin antigen solution was dropped onto the porous silica sample. The sample was then placed in a 37°C incubator for 2 hours to allow the gibberellin antigen to fully penetrate the sample. After removal, the sample was repeatedly rinsed with the silica sample and diethyl ether (DI) to remove excess gibberellin antigen solution from the pore walls. The sample was then dried under nitrogen. Next, the porous silica sample was immersed in 3M ethanolamine hydrochloride solution (4% concentration) and placed in a 37°C incubator for 1 hour. It was then rinsed repeatedly with PBS to prevent unreacted aldehyde groups, ensuring that the gibberellin antibody binds only to the gibberellin antigen. Finally, the sample was repeatedly rinsed with PBS and DI and dried under nitrogen.

[0093] Figure 5 A comparison of the reflectance spectra of porous silicon microcavity samples before and after the addition of gibberellin antigen shows that the reflectance spectrum of PSMCs with added gibberellin antigen exhibits a red shift, proving that the gibberellin antigen was successfully immobilized on the inner wall of the PSMC.

[0094] S40: Using a micropipette, add 50 μL of 8 μM QDs (carboxyl water-soluble CdSe / ZnS quantum dots) to a 1.5 mL microcentrifuge tube, then add 340 μL of PBS. After stirring for 10 min, add 30 μL each of 0.01 M EDC (1-ethyl-3-dimethylaminopropyl) and 0.01 M sulfo-NHS (N-hydroxythiosuccinimide). Perform a quantum dot activation and shaking reaction for 10 min to allow for coupling with gibberellin antibody, resulting in a final quantum dot concentration of 1 μM. Then, add 200 μL of 1 mg / L gibberellin antibody and shake in the dark for 10 h to ensure complete coupling between the quantum dots and the gibberellin antibody. After removal, centrifuge at 10000 rpm for 10 min to remove the supernatant and precipitate.

[0095] Figure 6 The fluorescence spectra of QDs before and after conjugation with gibberellin antibody at a concentration of 1 mg / L are shown. The fluorescence emission peak red-shifts from 625 nm to 627 nm, indicating that QDs were successfully conjugated with gibberellin antibody.

[0096] S50: Using a micropipette, 40 μL of quantum dot-conjugated gibberellin antibody solution was added dropwise to a porous silicon surface immobilized with seven concentrations of gibberellin antigen. The surface was then placed in a 37°C incubator for 2 hours to allow specific binding of the gibberellin antigen and antibody. Afterward, the surface was removed and repeatedly rinsed with PBS and DI to remove unreacted gibberellin antibody and quantum dots, and then dried in nitrogen. The flowchart for gibberellin antigen detection using a porous silicon optical biosensor is shown below. Figure 7 As shown.

[0097] S60: An ultraviolet semiconductor laser (λ = 375 nm, 30 mW) is used as the excitation source. The collimation and beam expansion system is formed by two lenses, L1 and L2. After beam expansion, a portion of the laser light reflected by the beam splitter is detected by detector D to eliminate the influence of excitation light intensity fluctuations on the fluorescence grayscale value. The other portion of the transmitted light illuminates the surface of the porous silicon sample positioned at the center of the goniometer G (resolution 1′), exciting the quantum dots to produce fluorescence. The fluorescence passes through a fiber filter into a digital microscope and is imaged on a computer, as shown below. Figure 8 As shown.

[0098] S70: Use a digital microscope to obtain digital images of seven different concentrations of gibberellin antigen and quantum dot-conjugated gibberellin antibody before and after the specific reaction, and calculate the average gray value of the digital images of the seven different concentrations of gibberellin antigen and quantum dot-conjugated gibberellin antibody before and after the specific reaction.

[0099] S80: By analyzing the gray values ​​of the fluorescence image, the average gray value before and after the immune reaction is calculated to obtain the linear relationship between the change in average gray value and the concentration of the analyte, and the detection limit is determined.

[0100] The trend graphs of the changes in average gray value of seven concentrations of gibberellin antigen solutions are shown below. Figure 9 As shown, gibberellin antigen at concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, and 0.5 ng / mL was selected, and the detection limit of this method was calculated.

[0101] Figure 10 It can be seen that the change in the average gray value of the PSM digital image has a good linear relationship with the four different concentrations of gibberellin antigen solution. Using the 3σ rule to calculate the experimental results, the detection limit of gibberellin is approximately 21.84 pg / mL.

[0102] This invention first experimentally demonstrates that porous silicon microcavity structures enhance the fluorescence of quantum dots more strongly than porous silicon Bragg mirrors. A porous silicon microcavity photonic crystal sensor is fabricated and combined with digital imaging methods for the detection of gibberellin. Semiconductor quantum dots are used as fluorescent markers to label antibodies against the analyte, resulting in a specific reaction with the antigen of the analyte immobilized on the inner wall of the porous silicon. The porous silicon microcavity structure further enhances the fluorescence generated by the semiconductor quantum dots. Fluorescence images of the porous silicon surface are obtained using a digital microscope. By calculating the change in average grayscale value of the images before and after the biological reaction, the target biomolecule can be detected with high sensitivity.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting gibberellin using fluorescence images, characterized in that, A porous silicon microcavity biosensor was used to detect gibberellin using fluorescence image detection. In the detection process, carboxyl water-soluble CdSe / ZnS quantum dots are coupled with gibberellin antibody, and the quantum dots are excited using 375nm excitation light; During the detection process, a fluorescent image of the porous silicon surface is obtained using a digital microscope. The concentration of the target biomolecule is calculated by calculating the average gray value change of the image before and after the biological reaction and using the average gray value change. The method for fabricating the porous silicon microcavity biosensor includes the following steps: (1) A porous silicon microcavity was prepared by electrochemical etching of p-type boron-doped single crystal silicon. When preparing porous silicon microcavities using the electrochemical etching method, the current densities for the high-porosity layer, low-porosity layer, and intermediate layer are 90 mA / cm², respectively. 2 40mA / cm 2 90mA / cm 2 The corrosion times were 1.7s, 2.0s, and 3.4s, respectively. (2) The porous silicon microcavity is functionalized to blue shift its center wavelength to 625nm, thus obtaining the processed porous silicon microcavity, namely the porous silicon microcavity biosensor.

2. The fluorescence image detection method according to claim 1, characterized in that, In step (1), when preparing porous silicon microcavities by electrochemical etching, the electro-etching solution is a mixture of hydrofluoric acid and anhydrous ethanol in a volume ratio of 1:1±0.

2.

3. The fluorescence image detection method according to claim 1, characterized in that, The functionalization process is as follows: After immersing the porous silicon microcavity in hydrogen peroxide solution, it is dried, rinsed with water, and dried again to obtain an oxidized porous silicon microcavity. The porous silicon microcavity after oxidation treatment is immersed in silanization solution, then rinsed with water and dried to obtain the porous silicon microcavity after silanization treatment. After the silanization treatment, the porous silicon microcavity was immersed in glutaraldehyde solution, rinsed repeatedly with phosphate buffer and water, and dried in nitrogen to obtain the glutaraldehyde-treated porous silicon microcavity.

4. The fluorescence image detection method according to claim 3, characterized in that, The concentration of the hydrogen peroxide solution is 35-45%, and the immersion time is 2.5-3.5 hours. The silanization solution contains APTES, water, and methanol in a volume ratio of 1:8-12:8-12, and the immersion time is 50-70 min. The glutaraldehyde solution has a concentration of 2.0-3.0% Vt%, and the immersion time is 50-70 min.

5. The fluorescence image detection method according to claim 4, characterized in that, The concentration of the hydrogen peroxide solution is 40%, and the immersion time is 3 hours. The silanization solution contains APTES, water, and methanol in a volume ratio of 1:10:10, and the immersion time is 60 min. The glutaraldehyde solution was 2.5% Vt, and the immersion time was 60 min.

Images