AuNP / BP composite biosensor and pathogen detection method

Abstract

The invention discloses an AuNP / BP composite material biosensor and a pathogen detection method. The biosensor comprises an AuNP / BP composite material and a biological recognition element, wherein the composite material is of a multi-layer structure, and each layer of structure is composed of a black phosphorus substrate with a target thickness and surface-modified gold nanoparticles. The biological recognition element is fixed on the surfaces of the gold nanoparticles and is used for specifically capturing target pathogens and antigens. The sensor can be used for rapidly, sensitively and specifically detecting infectious pathogens. Based on the terahertz wave technology, in combination with the effects of specific capture and electromagnetic induction enhancement of AuNP / BP on target pathogens, the pathogens are identified and detected through spectral information, and intelligent identification of the pathogens becomes possible. And an important basis is provided for diagnosis and subsequent treatment of infectious diseases.

Description

Technical Field

[0001] This solution relates to the field of optical technology, and in particular to AuNP / BP composite biosensors and pathogen detection methods. Background Technology

[0002] Commonly used methods for pathogen detection include isolation and culture, immunoassay, and nucleic acid detection. Isolation and culture is a relatively direct diagnostic method, but its results are easily affected by various factors such as sampling and culture conditions, and it has limitations such as long processing time (usually 48-96 hours) and low sensitivity. Compared to isolation and culture, immunoassay (such as enzyme-linked immunosorbent assay, ELISA) and nucleic acid detection (such as real-time quantitative polymerase chain reaction, qPCR) significantly improve the sensitivity and specificity of detection and are widely used in pathogen detection. However, ELISA and PCR are labor-intensive, time-consuming (≥2 hours), and costly. Summary of the Invention

[0003] This solution aims to at least solve the technical problems existing in the prior art. To this end, the first aspect of the present invention proposes an AuNP / BP composite material biosensor, the biosensor comprising an AuNP / BP composite material and a biometric element. The composite material has a multilayer structure, each layer consisting of a black phosphorus substrate of target thickness and gold nanoparticles modified on the surface; the thickness of the black phosphorus is 5-30 nm, and the particle size of the gold nanoparticles is 5-50 nm.

[0004] The biometric element is fixed on the surface of the gold nanoparticles for the specific capture of target pathogens and antigens.

[0005] Optionally, the biometric element is an antibody or aptamer, which is fixed to the surface of the composite material by the adsorption of the gold nanoparticles.

[0006] Optionally, the target pathogen includes bacteria, viruses, fungi, and parasites.

[0007] A second aspect of this invention provides a pathogen detection method based on an AuNP / BP composite material biosensor, the method comprising: A solution of a specific antibody or aptamer for the target pathogen is dropped onto the surface of an AuNP / BP composite biosensor of the target thickness and incubated at room temperature for 30-60 minutes to allow the gold nanoparticles of the biosensor to efficiently adsorb the antibody or aptamer solution; the AuNP / BP composite biosensor is the biosensor described in the first aspect. BSA solution was added to the surface of the biosensor and sealed for 30 minutes to reduce non-specific adsorption. The material was then rinsed with PBS buffer to obtain the AuNP / BP detection material. The AuNP / BP detection material was immersed in a solution containing the pathogen to be detected and incubated for 30 minutes to obtain an immune complex; The unbound pathogens on the immune complex were washed away with the PBS buffer to obtain the sample to be tested. The sample to be tested is scanned using a terahertz time-domain spectrometer to obtain target absorbance data, and the target absorbance data is used to identify whether the sample to be tested contains the target pathogen.

[0008] Optionally, after identifying whether the target pathogen is present in the sample to be tested based on the target absorbance data, the method further includes: If it is determined that the sample to be tested does not contain the first pathogen, then an AuNP / BP composite biosensor using a specific antibody or aptamer for the second pathogen as a biorecognition element is selected to detect the sample according to the method described in the second aspect; when the test result is positive, it is determined that the sample to be tested contains the second pathogen; when the test result is still negative, the method described in the second aspect is repeated sequentially with AuNP / BP composite biosensors targeting specific antibodies or aptamers for other candidate pathogens until the pathogen category in the sample to be tested is identified.

[0009] Optionally, before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the method further includes: A solution of a specific antibody or aptamer for the target pathogen was dropped onto the surface of the AuNP / BP composite biosensor, and absorbance data was obtained by scanning multiple frequency points using a terahertz time-domain spectrometer to obtain the first absorbance value at the multiple frequency points. Based on the mapping relationship between the frequency points and the first absorbance value, a blank group curve is obtained; A solution of a specific antibody or aptamer for the target pathogen and a solution containing the target pathogen were sequentially dropped onto the surface of the AuNP / BP composite biosensor, and absorbance data were obtained by scanning multiple frequency points using a terahertz time-domain spectrometer to obtain a second absorbance value at the multiple frequency points. Based on the mapping relationship between the frequency points and the second absorbance value, a standard curve of the target pathogen is obtained.

[0010] Optionally, identifying whether the target pathogen is present in the sample to be detected based on the target absorbance data includes: Obtain the absorbance value corresponding to each frequency point from the target absorbance data; Based on the mapping relationship between the frequency points and the absorbance values, the curve to be detected is obtained; The test curve is compared with the standard curve of the target pathogen, and the similarity between the test curve and the standard curve is used to determine whether the test sample contains the target pathogen.

[0011] Optionally, before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the method further includes: Several candidate composite materials composed of black phosphorus substrates of different thicknesses and surface-modified gold nanoparticles were prepared. Specific antibodies or aptamers of the target pathogen are immobilized on the plurality of candidate composite materials, respectively; The thickness of the candidate composite material that produces the maximum absorbance response is selected as the target thickness using terahertz detection.

[0012] Optionally, the step of scanning the sample to be detected using a terahertz time-domain spectrometer to obtain target absorbance data includes: The sample to be tested is placed in a terahertz time-domain spectrometer to remove moisture from the sample, and nitrogen gas is continuously introduced into the spectrometer. Using a transmission scanning mode, the spectrum at each frequency point is rapidly and repeatedly scanned 300 times, and the absorbance data is collected each time. The absorbance data is then averaged to obtain the target absorbance data.

[0013] Optionally, before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the method further includes: The AuNP / BP composite biosensor and terahertz time-domain spectrometer were used to detect target pathogen samples of different concentrations, and the absorbance-concentration logarithm plot of the AuNP / BP composite biosensor was obtained. The detection limit of the AuNP / BP composite biosensor is calculated based on the absorbance-concentration logarithmic graph and the preset detection limit formula.

[0014] The embodiments of the present invention have the following beneficial effects: This invention provides a biosensor based on AuNP / BP composite material, comprising an AuNP / BP composite material and a biorecognition element. The composite material has a multilayer structure, each layer consisting of a black phosphorus substrate of a target thickness and surface-modified gold nanoparticles. The black phosphorus substrate has a thickness of 5-30 nm, and the gold nanoparticles have a particle size of 5-50 nm. The biorecognition element is fixed to the surface of the gold nanoparticles for specifically capturing target pathogens and antigens. This sensor can rapidly, sensitively, and specifically detect infectious pathogens. Based on terahertz wave technology, combined with the specific capture of target pathogens and the electromagnetic induction enhancement effect of AuNP / BP, pathogens can be identified and detected through spectral information, enabling intelligent pathogen identification. This provides important evidence for the diagnosis and subsequent treatment of infectious diseases, and is of great significance for treating patients, controlling the spread of epidemics, and reducing casualties. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of an AuNP / BP composite material biosensor provided in an embodiment of the present invention; Figure 2 This is a flowchart illustrating the steps of a pathogen detection method based on an AuNP / BP composite material biosensor provided in an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the relationship between AuNP / BP thickness and absorbance provided in an embodiment of the present invention; Figure 4 This is a schematic diagram illustrating the relationship between sample concentration and absorbance provided in an embodiment of the present invention; Figure 5 This is a comparison chart of absorbance values ​​for different samples provided in an embodiment of the present invention; Figure 6 This is an embodiment of the present invention providing AuNP / BP of different thicknesses. E. coli Absorbance comparison chart; Figure 7 This is an embodiment of the invention providing different concentrations of... E. coli Absorbance comparison chart of solutions; Figure 8 This is an embodiment of the present invention providing AuNP / BP of different thicknesses. Leishmania Absorption rate comparison chart. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present solution, and not all embodiments. Based on the embodiments of the present solution, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present solution.

[0017] Hereinafter, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of embodiments of this disclosure, unless otherwise stated, "a plurality of" means two or more. Furthermore, the use of "based on" or "according to" implies openness and inclusiveness, because processes, steps, calculations, or other actions "based on" or "according to" one or more of the stated conditions or values ​​may in practice be based on additional conditions or beyond the stated values.

[0018] Commonly used methods for pathogen detection include isolation and culture, immunoassay, and nucleic acid detection. Isolation and culture is a relatively direct diagnostic method, but its results are easily affected by various factors such as sampling and culture conditions, and it has limitations such as long processing time (usually 48-96 hours) and low sensitivity. Compared to isolation and culture, immunoassay (such as enzyme-linked immunosorbent assay, ELISA) and nucleic acid detection (such as real-time quantitative polymerase chain reaction, qPCR) significantly improve the sensitivity and specificity of detection and are widely used in pathogen detection. However, even with these improved methods, ELISA and PCR still involve labor-intensive steps, time (≥2 hours), and high costs.

[0019] Terahertz detection technology offers a novel and rapid method for diagnosing biomedical diseases. In the electromagnetic spectrum, terahertz waves (THz) encompass wavelengths between 0.1 and 10 THz. Terahertz waves (THz) are electromagnetic waves with frequencies ranging from 0.1 to 10 THz and wavelengths from 3000 to 30 μm. They possess characteristics such as high resolution, high transmittance, low photon energy, high safety, and molecular "fingerprinting," enabling the revelation of conformational information of biomolecules based on intermolecular or intramolecular vibrational modes, leading to their widespread application in the biomedical field. This method has already been used for pathogen detection, such as bacterial component characterization, spore identification, and colony detection. However, due to the mismatch between the terahertz radiation wavelength and the size of the virus being detected, the sensitivity of free-space terahertz waves is limited when detecting trace amounts of analytes. Furthermore, interference from impurities in complex biological samples reduces the accuracy of pathogen detection. Researchers have combined resonant metasurfaces with terahertz technology to enhance the coupling effect between the analyte and the terahertz wave, thereby addressing this bottleneck. Existing metamaterials are not yet widely used for two reasons. First, fabricating metamaterials requires complex and often expensive processes. Second, the connection between metamaterials and biological interfaces can create gaps at the interface, significantly reducing sensing performance. Therefore, researchers are seeking sensitive, metamaterial-free, and flexible sensors that can adapt to terahertz frequencies and have simple fabrication methods.

[0020] In recent years, two-dimensional materials such as graphene and transition metal dichalcogenides (TMDs) have been widely used in terahertz biomedical detection to improve detection sensitivity. As a new member of the emerging family of two-dimensional materials, black phosphorus (BP) exhibits excellent performance in the terahertz frequency range due to its high carrier mobility, tunable bandgap, and plasmon resonance. Therefore, black phosphorus is an ideal material for enhancing terahertz-based response signals. Gold nanoparticles possess good biocompatibility and a large specific surface area, enabling rapid adsorption of large amounts of antibodies or aptamers. They also exhibit surface plasmon resonance effects, significantly enhancing terahertz detection signals. Currently, gold nanoparticles are used in the field of terahertz detection of cells and disease biomarkers, significantly improving the sensitivity of terahertz detection of biological samples. Therefore, combining terahertz detection technology with black phosphorus and gold nanoparticles holds promise for achieving rapid, specific, and highly sensitive detection of pathogens.

[0021] This invention provides an electromagnetically enhanced AuNP / BP terahertz biosensor that enhances the terahertz detection signal through electromagnetic induction, enabling rapid, specific, and highly sensitive detection of pathogens in fields such as food safety, medical health, and animal husbandry.

[0022] Figure 1 This is a schematic diagram of an AuNP / BP composite material biosensor provided in an embodiment of the present invention. Figure 1As shown, the biosensor includes an AuNP / BP composite material and a biometric element; The composite material has a multilayer structure, each layer consisting of a black phosphorus substrate 1 of the target thickness and surface-modified gold nanoparticles 2; the thickness of the black phosphorus 1 is 5-30 nm, and the particle size of the gold nanoparticles 2 is 5-50 nm. The biorecognition element 3 is fixed on the surface of the gold nanoparticles for specifically capturing target pathogens and antigens 4.

[0023] The AuNP / BP composite biosensor is placed on substrate 5, which is typically a silicon wafer.

[0024] BP, or black phosphorus, is a novel two-dimensional nanomaterial. Its mechanism of action is chemical enhancement. The two-dimensional layered structure provides a vast surface area, allowing for the loading of numerous biological probe molecules and increasing the opportunities for contact with target molecules. It exhibits excellent biocompatibility, demonstrating good interactions with many biomolecules.

[0025] Black phosphorus exhibits high carrier mobility. When target molecules bind to the surface of black phosphorus, charge transfer occurs, altering the conductivity of the black phosphorus. This change in conductivity directly affects its response to terahertz waves, thereby amplifying the detection signal.

[0026] AuNP refers to gold nanoparticles, a typical plasmonic material. Its mechanism of action is electromagnetic enhancement. When terahertz waves irradiate AuNPs, they excite localized surface plasmon resonances on their surface. This effect generates a strong, localized, enhanced electromagnetic field around the nanoparticles. If the biomolecules being measured (such as DNA, proteins, or viruses) are located within this electromagnetic field region, the intensity of the electromagnetic field they experience will be greatly increased, thus significantly amplifying their absorption or scattering of terahertz waves.

[0027] The BP with a thickness of about 5-30 nm and its embedded 5-50 nm AuNP were prepared and optimized by electrochemical intercalation and exfoliation combined with in-situ electrochemical deposition (EI&ED) method, which can generate the maximum terahertz response signal for the target pathogen.

[0028] AuNPs with a particle size of 5-50 nm exhibit excellent surface plasmon resonance effect and good biocompatibility. They can efficiently adsorb specific antibodies or aptamers through an embedded structure and form high-density plasmon "hot spots".

[0029] Biometric element 3, fixed to the surface of AuNP, is used to specifically capture target pathogens and antigens 4.

[0030] The mosaic structure of this composite material generates a synergistic enhancement effect through the electromagnetic induction effect of BP and AuNP, which significantly amplifies the terahertz response signal (such as absorbance) after the target pathogen binds, improves the sensitivity of terahertz detection of pathogens, and enhances interface stability.

[0031] As an optional embodiment, the biometric element 3 is an antibody or aptamer, which is fixed to the surface of the composite material by the adsorption of the gold nanoparticles 2.

[0032] Aptamers and antibodies are both biomolecules with specific recognition capabilities.

[0033] Nucleic acid aptamers are oligonucleotide fragments (DNA or RNA) composed of nucleic acids that bind with high specificity and selectivity to a variety of target substances such as pathogens, biological macromolecules and small molecules.

[0034] Antibodies are proteins produced by the immune system. They have extremely high specificity and affinity, and can recognize target antigens and pathogens with high specificity.

[0035] Gold nanoparticles 2 firmly immobilize antibodies or aptamers with specific recognition functions on the surface of high-performance AuNP / BP composite materials through various physicochemical adsorption processes, laying the foundation for subsequent high-sensitivity biological detection.

[0036] As an optional embodiment, the target pathogen 4 includes bacteria, viruses, fungi, and parasites.

[0037] For example, the target pathogen may include: Escherichia coli, Candida albicans, Staphylococcus aureus, novel coronavirus, herpes zoster virus, Leishmania, etc.

[0038] In summary, the AuNP / BP composite material-based biosensor provided in this invention comprises an AuNP / BP composite material and a biorecognition element. The composite material has a multilayer structure, with each layer consisting of a black phosphorus substrate of a target thickness and surface-modified gold nanoparticles. The black phosphorus substrate has a thickness of 5-30 nm, and the gold nanoparticles have a particle size of 5-50 nm. The biorecognition element is fixed to the surface of the gold nanoparticles for specifically capturing target pathogens and antigens. This sensor can rapidly, sensitively, and specifically detect infectious pathogens. Based on terahertz wave technology, combined with the specific capture of target pathogens by AuNP / BP and the synergistic enhancement effect of electromagnetic induction and bandgap changes, pathogens can be identified and detected through spectral information, providing the possibility for intelligent pathogen identification. This provides important evidence for the diagnosis and subsequent treatment of infectious diseases and is of great significance for treating patients, controlling the spread of epidemics, and reducing casualties.

[0039] Figure 2 This is a flowchart illustrating the steps of a pathogen detection method based on an AuNP / BP composite material biosensor provided in an embodiment of the present invention. Figure 2 As shown, the method includes: Step 101: Drop a solution of specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, and incubate at room temperature for 30-60 minutes to allow the gold nanoparticles of the biosensor to efficiently adsorb the antibody or aptamer solution; the AuNP / BP composite biosensor is... Figure 1 Biosensors in [the context of biosensors].

[0040] The target thickness is the AuNP / BP particle size selected by terahertz detection to produce the maximum absorbance response.

[0041] The purpose of incubating at room temperature for 30-60 minutes is to enable AuNP particles with a diameter of 5-50 nm to efficiently adsorb antibodies or aptamers.

[0042] Step 102: Add BSA solution to the surface of the biosensor and seal for 30 minutes to reduce non-specific adsorption. Rinse with PBS buffer to obtain AuNP / BP detection material.

[0043] BSA solution, or bovine serum albumin solution, was added dropwise for 30 min to block nonspecific adsorption. Unbound material was then removed by rinsing with PBS buffer to obtain the AuNP / BP detection material.

[0044] The purpose of steps 101-102 above is to fix and block antibodies or aptamers.

[0045] Step 103: Immerse the AuNP / BP detection material in a solution containing the pathogen to be detected and incubate for 30 minutes to obtain immune complexes.

[0046] The surface of the AuNP / BP detection material has been immobilized with a large number of specific antibodies or aptamers in the previous steps. Immersion allows each pathogen in the solution to diffuse to the surface of the composite material and come into contact with the antibodies or aptamers. A 30-minute incubation ensures that the vast majority of target pathogens are successfully captured.

[0047] Thus, we can obtain an “AuNP / BP-antibody or aptamer-pathogen” complex structure on the surface that has successfully bound to the target pathogen, which is called an “immune complex”.

[0048] Step 104: Rinse the immune complex with the PBS buffer to remove unbound pathogens and obtain the sample to be tested.

[0049] The solution still contains a large number of pathogens, other proteins, salt ions and impurities that have not bound to the antibody or aptamer. The flow of PBS will wash away the weakly adsorbed unbound pathogens, proteins and other molecules, but will not disrupt antibody or aptamer-antigen binding or damage biomolecules.

[0050] Generally, rinse three times with PBS buffer to remove unbound material.

[0051] Step 105: Scan the sample to be tested using a terahertz time-domain spectrometer to obtain target absorbance data, and identify whether the sample to be tested contains the target pathogen based on the target absorbance data.

[0052] When terahertz waves pass through a sample, they interact with substances in the sample, particularly immune complexes. Compared to "blank" or "control" sensors that do not bind pathogens, sensors that bind pathogens have the following effects: Absorption: Pathogens and their binding processes can alter the absorption characteristics of a sensor for terahertz energy.

[0053] Refractive index: The binding of biomolecules alters the dielectric environment of the sensor surface, leading to changes in the propagation speed of terahertz waves (i.e., changes in refractive index).

[0054] Time-domain waveform: The amplitude and phase of the directly measured terahertz electric field pulse will change (e.g., peak attenuation, time delay).

[0055] The instrument compares the signals of the "sample" and the "reference" (blank control), and after processing them through a series of algorithms, it ultimately outputs a core parameter—the absorbance spectrum. This spectrum shows the degree of energy absorption by the sample at different terahertz frequencies. This absorbance data is the "target absorbance data."

[0056] The absorbance spectrum of the test sample is compared with that of the control. If the absorbance of the test sample is significantly higher than that of the control at a specific frequency (e.g., the "fingerprint" characteristic peak of the pathogen), it can be determined that the sample contains the target pathogen.

[0057] As an optional embodiment, after step 105, the method further includes: Step 106: If it is determined that the sample to be tested does not contain the first pathogen, then select an AuNP / BP composite biosensor that uses a specific antibody or aptamer for the second pathogen as a biorecognition element, and test the sample to be tested according to the method described in steps 101-105; when the test result is positive, it is determined that the sample to be tested contains the second pathogen; when the test result is still negative, replace it with an AuNP / BP composite biosensor that targets specific antibodies or aptamers for other candidate pathogens and repeat the method described in steps 101-105 until the pathogen category in the sample to be tested is identified.

[0058] First, rinse the current sensor surface with mild PBS buffer to wash away sample matrix components that were previously bound to the antibody or aptamer, and clean the sensor surface in preparation for the next detection. PBS does not destroy the antibody or aptamer itself immobilized on the sensor, but it can remove non-specifically adsorbed impurities.

[0059] An antibody or aptamer of a second possible pathogen is dropped onto the sensor surface for detection. The gold nanoparticles on the AuNP / BP sensor surface can then fix these new antibodies or aptamers again through adsorption. If a positive signal is detected this time, it can be determined that the sample contains the second pathogen.

[0060] If the result is still negative, repeat the steps above and continue testing with antibodies or aptamers of a third pathogen, and so on, until a corresponding pathogen that can produce a positive signal is found.

[0061] As an optional embodiment, prior to step 101, the method further includes: Step 201: Drop a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor, and scan at multiple frequency points using a terahertz time-domain spectrometer to obtain absorbance data at each frequency point, thereby obtaining the first absorbance value at the multiple frequency points.

[0062] A solution of a specific antibody or aptamer for the target pathogen was dropped onto the surface of an AuNP / BP composite biosensor, and the sensor was scanned and detected at multiple different terahertz frequencies. At each frequency, a first absorbance value was measured. This value reflects the absorption of terahertz waves by the sensor containing only the target pathogen antibody or aptamer.

[0063] Step 202: Obtain the blank group curve based on the mapping relationship between the frequency point and the first absorption value.

[0064] Plot a curve with terahertz frequency on the x-axis and the first absorbance value measured at the corresponding frequency on the y-axis.

[0065] Step 203: The specific antibody or aptamer solution of the target pathogen and the solution containing the target pathogen are successively dropped onto the surface of the AuNP / BP composite biosensor, and the absorbance data are obtained by scanning multiple frequency points using a terahertz time-domain spectrometer to obtain the second absorbance value at the multiple frequency points.

[0066] A solution of a specific antibody or aptamer for the target pathogen and a solution containing the target pathogen were sequentially dropped onto the surface of the AuNP / BP composite biosensor. The AuNP / BP composite biosensor containing the antibody or aptamer and the target pathogen was scanned and detected using the same multiple frequency points of a terahertz time-domain spectrometer.

[0067] At each frequency point, a second absorbance value is measured. This value reflects the absorption of the target pathogen by the antibody or aptamer containing the target pathogen.

[0068] Step 204: Based on the mapping relationship between the frequency point and the second absorbance value, obtain the standard curve of the target pathogen corresponding to the target biometric element.

[0069] Similarly, plot a curve with terahertz frequency on the x-axis and the second absorbance value on the y-axis to obtain the standard curve.

[0070] As an optional embodiment, step 105 includes: Step 1051: Obtain the absorbance value corresponding to each frequency point from the target absorbance data; Step 1052: Obtain the detection curve based on the mapping relationship between the frequency points and the absorbance values; Step 1053: Compare the curve to be detected with the standard curve of the target pathogen, and determine whether the sample to be detected contains the target pathogen based on the similarity between the curve to be detected and the standard curve.

[0071] In steps 1051-1053, the absorbance values ​​corresponding to each specific frequency point are extracted from the "target absorbance data" obtained by the terahertz time-domain spectrometer.

[0072] Plotting terahertz frequency on the x-axis and the absorbance value extracted in step 1051 on the y-axis, all data points are connected to form a curve, resulting in the detection curve. This curve fully represents the optical "fingerprint" characteristics of the sample across the entire terahertz band (e.g., 0-2 THz).

[0073] Compare the overall shape and key features of the two curves. If the sample contains the target pathogen, it should show absorption peaks or valleys similar to the standard curve at the same frequency positions; if the sample does not contain the target pathogen, its curve shape is more similar to the blank control curve.

[0074] This determines whether the sample to be tested contains the target pathogen.

[0075] As an optional embodiment, prior to step 101, the method further includes: Step 301: Prepare multiple candidate composite materials consisting of black phosphorus substrates of different thicknesses and surface-modified gold nanoparticles; Step 302: Immobilize specific antibodies or aptamers of the target pathogen onto the multiple candidate composite materials respectively; Step 303: Select the thickness of the candidate composite material that produces the maximum absorption response as the target thickness by terahertz detection.

[0076] In steps 301-303, AuNP / BP of different thicknesses were prepared by electrochemical intercalation-exfoliation combined with in-situ electrochemical deposition. A target pathogen membrane protein or glycoprotein antibody or aptamer solution of a predetermined concentration was adsorbed onto gold nanoparticles on the surface of AuNP / BP of different thicknesses for 4 min. Then, a 0.1 wt% BSA solution in PBS was added, and incubation continued for 30 min to block non-specific binding sites and reduce non-specific adsorption. The surface was then gently rinsed three times with PBS to remove unbound BSA. A target pathogen solution of a predetermined concentration was added to the AuNP / BP surface and incubated at room temperature for 1 min. Using AuNP / BP without adsorbed antibody or aptamer and the target pathogen as a blank control, the absorbance of the target pathogen on AuNP / BP of different thicknesses was detected using terahertz detection technology. The thickness of AuNP / BP with the highest terahertz absorbance value was selected as the optimal terahertz response condition.

[0077] In this embodiment of the invention, the optimal AuNP / BP thickness is 5-30 nm and the AuNP particle size is 5-50 nm.

[0078] As an optional embodiment, step 105 involves scanning the sample to be detected using a terahertz time-domain spectrometer to obtain target absorbance data, including: Step 401: Place the sample to be tested in a terahertz time-domain spectrometer, remove the moisture from the sample, and continuously fill the spectrometer with nitrogen gas. Step 402: Using the transmission scanning mode, the spectrum at each frequency point is rapidly scanned 300 times, the absorbance data is collected each time, and the absorbance data is averaged to obtain the target absorbance data.

[0079] In steps 401-402, the sample to be tested is placed in the terahertz detection container, moisture is removed, nitrogen gas is continuously introduced, and analysis is performed using a stable transmission scanning mode. Each spectrum is scanned 300 times, and the experiment is repeated three times. Time-domain and frequency-domain data are collected and converted into absorbance values, extinction coefficients, refractive indices, and transmittance. The absorbance data from each test are collected and averaged to obtain the target absorbance data. The target absorbance data at frequencies of 0-2 THz is then analyzed.

[0080] In this embodiment of the invention, three experiments included: preparing the target pathogen at a concentration of 1×10⁻⁶. 3 ~ 1×10 7 PFU / mL HSV-1 solution, 1×10 3 ~ 1×10 7 CFU / mL E. coli solution, 1×10 3 ~ 1×10 7 CFU / mL C. albicans solution, 1×10 3 ~ 1×10 7 PFU / mL Leishmania solution was used to perform three terahertz measurements on each concentration of the sample solution.

[0081] As an optional embodiment, prior to step 101, the method further includes: Step 501: Use the AuNP / BP composite biosensor and terahertz time-domain spectrometer to detect target pathogen samples of different concentrations and obtain the absorbance-concentration logarithm plot of the AuNP / BP composite biosensor. Step 502: Calculate the detection limit of the AuNP / BP composite biosensor based on the absorbance-concentration logarithmic graph and the preset detection limit formula.

[0082] In steps 501-502, the detection limit is the lowest concentration of the target substance that the sensor can reliably detect.

[0083] Detect samples of target pathogens at different concentrations, plot standard curves (e.g., absorbance vs. logarithmic concentration graphs), and apply formula Y. LOD = Y Blank + 3 SD Calculation of detection limit (Y) LOD ), where Y BlankSD represents the average absorbance of the blank group and its standard deviation.

[0084] Calculate Y LOD After obtaining this absorbance value, substitute it into the formula of the previously plotted standard curve to calculate the corresponding concentration value. This concentration value is the detection limit of the sensor. The lower the detection limit, the higher the sensitivity of the sensor.

[0085] For example, the detection limit for HSV-1 is 5.75 × 10⁻⁶. 2 PFU / mL, E. coli The detection limit is 2.65 × 10⁻⁶. 2 CFU / mL C. albicans The detection limit is 3.52 × 10⁻⁶. 2 The detection limit for Leishmania is 4.75 × 10⁻⁶ CFU / mL. 2 CFU / mL.

[0086] The pathogen detection method based on AuNP / BP composite material biosensor provided in this invention can be applied in the following fields: Food safety: Rapid detection of bacterial pathogens (such as Escherichia coli and Staphylococcus aureus) in food.

[0087] Clinical diagnosis: Rapid detection of viruses (such as novel coronavirus, varicella-zoster virus), fungi (such as Candida albicans), and parasites (such as Leishmania) in patient samples for the early diagnosis of infectious diseases.

[0088] Public health surveillance: Detecting viruses and parasites during epidemiological investigations and monitoring the development of outbreaks.

[0089] Environmental monitoring: Screening for pathogen contamination in water or air.

[0090] This method outperforms existing technologies in terms of detection performance, specifically in the following ways: Speed: The entire detection process can be completed within 5 minutes, which is significantly faster than isolation and culture methods (48-96h), ELISA, or qPCR (≥2h).

[0091] High sensitivity: Detection limits (LOD) as low as 10² PFU / mL or CFU / mL (e.g., HSV-1 LOD 5.75 × 10² PFU / mL). E. coli The LOD (2.65×10² CFU / mL) overcomes the sensitivity limitation caused by the mismatch between the wavelength and virus size of free-space terahertz waves; the dense plasma "hot spots" and strong synergistic effect generated by the mosaic structure are the key to achieving high sensitivity.

[0092] High specificity: The interference degree (DI) is less than 10% (e.g., in the detection of HSV-1, the DI of HNTV, E. coli, L. monocytogenes and mixtures are all <9.2%), which can effectively distinguish between target pathogens and non-target pathogens and reduce the interference of impurities in complex biological samples.

[0093] Metamaterials not required: By utilizing the intrinsic electromagnetic enhancement properties of AuNP / BP composite materials (especially the synergistic effect of the mosaic structure), the complex and expensive manufacturing process of metamaterials in existing technologies and the performance degradation caused by gaps in the connection with biological interfaces are avoided.

[0094] In summary, the pathogen detection method based on an AuNP / BP composite biosensor provided by this invention involves dropping a specific antibody or aptamer solution of the target pathogen onto the surface of an AuNP / BP composite biosensor of a target thickness, incubating at room temperature for 30-60 minutes to allow the gold nanoparticles of the biosensor to efficiently adsorb the antibody or aptamer solution; the AuNP / BP composite biosensor is any one of the biosensors described in claims 1-3; adding BSA solution to the surface of the biosensor and blocking it for 30 minutes to reduce non-specific adsorption, rinsing with PBS buffer to obtain AuNP / BP detection material; immersing the AuNP / BP detection material in a solution containing the pathogen to be detected and incubating for 30 minutes to obtain an immune complex; rinsing with the PBS buffer to remove unbound pathogens from the immune complex to obtain the sample to be detected; scanning the sample to be detected using a terahertz time-domain spectrometer to obtain target absorbance data, and identifying whether the sample to be detected contains the target pathogen based on the target absorbance data.

[0095] This detection method is convenient and quick, and can identify Escherichia coli and various pathogens in a short time, so as to detect problems in time and take corresponding measures. It has the advantages of being fast, highly sensitive and specific.

[0096] Implementation Case 1: Detection of the target pathogen, herpes zoster virus HSV-1 HSV-1 gD mAb (glycoprotein antibody or aptamer) was selected as the specific adsorption antibody or aptamer.

[0097] AuNP / BP nanoparticles of varying thicknesses were prepared by electrochemical intercalation-exfoliation combined with in-situ electrochemical deposition. 20 μg / mL HSV-1 gD mAb was adsorbed onto gold nanoparticles of different thicknesses on the surface of AuNP / BP nanoparticles for 4 min. Then, a 0.1 wt% BSA solution in PBS was added, and incubation continued for 30 min to block non-specific binding sites and reduce non-specific adsorption. The nanoparticles were then gently rinsed three times with PBS to remove unbound BSA. 1×10⁻⁶ 5A PFU / mL HSV-1 solution was added dropwise to the surface of AuNP / BP and incubated at room temperature for 1 min. Using AuNP / BP without adsorbed antibodies or aptamers and the target pathogen as blanks, the HSV-1 absorbance of AuNP / BP at different thicknesses was detected using terahertz detection technology.

[0098] Figure 3 This is a schematic diagram illustrating the relationship between AuNP / BP thickness and absorbance provided in an embodiment of the present invention.

[0099] like Figure 3 The results showed that 5-30 nm AuNP / BP was the optimal terahertz response condition for detecting HSV-1.

[0100] Figure 4 This is a schematic diagram illustrating the relationship between sample concentration and absorbance provided in an embodiment of the present invention.

[0101] Prepare 1×10 3 ~ 1×10 7 PFU / mL HSV-1 solution was used to prepare 20 μg / mL HSV-1 gD mAb as a coating antibody or aptamer, and terahertz detection was performed three times on HSV-1 solutions of different concentrations.

[0102] like Figure 4 As shown, the terahertz absorption increases with increasing sample concentration between 0.1 and 1 THz, exhibiting a clear gradient relationship at 0.8 THz. The established calibration curve is Y = 0.105 LgC - 0.261, R 2 =0.99708. According to the guidelines of the International Union of Pure and Applied Chemistry (IUPAC). Y LOD = Y Blank + 3 SD, The LOD is 5.75 × 10 2 PFU / mL.

[0103] Figure 5 This is a comparison chart of absorbance values ​​for different samples provided in an embodiment of the present invention.

[0104] Mix1 (HNTV) E-coli , L. monocytogenes The mixture was added to the BP-AuNP-Anti-HSV-1-gD-Ab detection system, followed by the addition of Mix2 (HNTV, ...) to the HSV-1 reaction system. E-coli , L. monocytogenes(Mixture), and the procedure was performed according to the previously described standard experimental procedure. After the immunocomplex reaction, the product was enriched and then subjected to independent terahertz testing. The selectivity and sensitivity of the detection system can be evaluated by measuring and calculating the absorbance values ​​of the samples. Figure 5 HSV-1 exhibited significantly higher terahertz absorbance values ​​than the three interfering pathogens and mixture 1. Furthermore, the terahertz absorbance value of mixture 2 was very close to that of HSV-1, indicating that the biosensor could detect HSV-1 even under interference.

[0105] As shown in Table 1, the DI values ​​of the three interfering pathogens and mixture 1 were all below 10% (HNTV was 8%, Escherichia coli was 9.2%). L. monocytogenes The concentrations were 8.5% in mixture 1 and 7% in mixture 2. No cross-reactivity was observed between any of the interfering pathogens and Anti-HSV-1-gD mAb. Therefore, this AuNP / BP biosensor exhibits high specificity for HSV-1 recognition even in the presence of interference.

[0106] Table 1 Interference Analysis

[0107] For three groups of samples (2×10 3 2×10 4 Ten experiments were conducted using the method (PFU / mL) to further investigate its repeatability. As shown in Table 2, the estimated relative standard deviations (RSDs) for the three sample groups were 12.5%, 11.9%, and 10.2%, respectively, with accuracy exceeding 85%. Therefore, the obtained method for detecting HSV-1 based on the AuNP / BP terahertz biosensor exhibits good specificity and repeatability.

[0108] Table 2. Accuracy and precision of AuNP / BP terahertz biosensor in detecting HSV-1 QC samples.

[0109] Implementation Case 2: Target Pathogen Escherichia coli E. coli Detection Select E. coli FimH mAb (membrane protein antibody or aptamer) serves as a specific adsorption antibody or aptamer.

[0110] AuNP / BP of varying thicknesses was prepared by electrochemical intercalation-exfoliation combined with in-situ electrochemical deposition, using 20 μg / mL of... E. coliFimH mAb was adsorbed onto gold nanoparticles of varying thicknesses on AuNP / BP surfaces for 4 min, followed by the addition of 0.1 wt% BSA in PBS solution and incubation for another 30 min to block nonspecific binding sites and reduce nonspecific adsorption. The mixture was then gently rinsed three times with PBS to remove unbound BSA. 1×10 4 CFU / mL E. coli The solution was dropped onto the AuNP / BP surface and incubated at room temperature for 1 min. Using AuNP / BP without adsorbed antibodies or aptamers and the target pathogen as blanks, terahertz detection technology was used to detect AuNP / BP of different thicknesses. E. coli Absorption rate.

[0111] Figure 6 This is an embodiment of the present invention providing AuNP / BP of different thicknesses. E. coli Absorption rate comparison chart.

[0112] according to Figure 6 The results showed that AuNP / BP in the 5-30 nm range was the detection range. E. coli The optimal terahertz response conditions.

[0113] Prepare 1×10 3 ~ 1×10 7 CFU / mL E. coli Prepare a solution at 15 μg / mL. E. coli FimH mAb, as a coating antibody or aptamer, is effective against different concentrations. E. coli The solution underwent three terahertz measurements, as shown in Figure 6. Between 0.1 and 1 THz, the terahertz absorption increased with increasing sample concentration. At 0.8 THz, a clear gradient relationship was observed. The established calibration curve was Y = 0.38 LgC - 1.097, R0 2 =0.998. According to the guidelines of the International Union of Pure and Applied Chemistry (IUPAC). Y LOD = Y Blank + 3 SD, The LOD is 2.65 × 10 2 CFU / mL.

[0114] For the two groups of samples (2×10) 3 2×10 4 Ten experiments were conducted using the method (CFU / mL) to further investigate its repeatability. As shown in Table 3, the estimated relative standard deviations (RSDs) for the three sample groups were 11.5% and 8.9%, respectively, with accuracies all above 85%. Therefore, the method based on the AuNP / BP terahertz biosensor for detection... E. coliThe method has good specificity and reproducibility.

[0115] Table 3. Detection by AuNP / BP Terahertz Biosensor E. coli Accuracy and precision of QC samples

[0116] Implementation Case 3: Detection of the target pathogen Candida albicans Select C. albicans MM mAb (Candida albicans mannose antibody or aptamer) is used as a specific adsorption antibody or aptamer.

[0117] AuNP / BP of varying thicknesses was prepared by electrochemical intercalation-exfoliation combined with in-situ electrochemical deposition, using 20 μg / mL of... C. albicans MM mAb was adsorbed onto gold nanoparticles of varying thicknesses on AuNP / BP surfaces for 4 min, followed by the addition of 0.1 wt% BSA in PBS solution and incubation for another 30 min to block nonspecific binding sites and reduce nonspecific adsorption. The mixture was then gently rinsed three times with PBS to remove unbound BSA. 1×10 4 CFU / mL C. albicans The solution was dropped onto the AuNP / BP surface and incubated at room temperature for 1 min. Using AuNP / BP without adsorbed antibodies or aptamers and the target pathogen as blanks, terahertz detection technology was used to detect AuNP / BP of different thicknesses. C. albicans Absorption rate.

[0118] Figure 7 This is an embodiment of the invention providing different concentrations of... E. coli Absorbance comparison chart of solutions.

[0119] Prepare 1×10 3 ~ 1×10 7 CFU / mL C. albicans Prepare a solution at a concentration of 20 μg / mL. C. albicans MMmAb, as a coating antibody or aptamer, is effective against different concentrations of... E. coli The solution underwent three terahertz measurements, as shown in Figure 7. Between 0.1 and 1 THz, the terahertz absorption increased with increasing sample concentration. At 0.8 THz, a clear gradient relationship was observed. The established calibration curve was Y = 0.38X - 1.097, R0 2 =0.998. According to the guidelines of the International Union of Pure and Applied Chemistry (IUPAC). Y LOD = Y Blank + 3 SD, The LOD is 3.52 × 102 PFU / mL.

[0120] For the two groups of samples (2×10) 4 2×10 5 Ten experiments were conducted using the method (CFU / mL) to further investigate its repeatability. As shown in Table 4, the estimated relative standard deviations (RSDs) for the three sample groups were 8.6% and 9.5%, respectively, with accuracy rates all above 85%. Therefore, the method based on the AuNP / BP terahertz biosensor for detection... C. albicans The method has good specificity and reproducibility.

[0121] Table 4. Detection by AuNP / BP Terahertz Biosensor C. albicans Accuracy and precision of QC samples

[0122] Implementation Case 4: Detection of the target pathogen Leishmania Select Leishmania gp63 mAb (Leishmania surface glycoprotein antibody) is used as a specific adsorption antibody or aptamer.

[0123] AuNP / BP of varying thicknesses was prepared by electrochemical intercalation-exfoliation combined with in-situ electrochemical deposition, using 25 μg / mL of... Leishmania gp63 mAb was adsorbed onto gold nanoparticles of varying thicknesses on AuNP / BP surfaces for 4 min. Then, a 0.1 wt% BSA solution in PBS was added, and incubation continued for 30 min to block non-specific binding sites and reduce non-specific adsorption. The mixture was then gently rinsed three times with PBS to remove unbound BSA. 1×10 4 CFU / mL Leishmania The solution was dropped onto the AuNP / BP surface and incubated at room temperature for 1 min. Using AuNP / BP without adsorbed antibodies or aptamers and the target pathogen as blanks, terahertz detection technology was used to detect AuNP / BP of different thicknesses. Leishmania Absorption rate.

[0124] Figure 8 This is an embodiment of the present invention providing AuNP / BP of different thicknesses. Leishmania Absorption rate comparison chart.

[0125] according to Figure 8 The results showed that AuNP / BP in the 5-30 nm range was the detection range. C. albicans The optimal terahertz response conditions.

[0126] Prepare 1×10 3 ~ 1×10 7 particles / mL Leishmania Prepare a solution at a concentration of 20 μg / mL. Leishmania gp63 mAb, as a coating antibody or aptamer, is effective against different concentrations. E. coli The solution was subjected to three terahertz detections, such as Figure 8 As shown, the terahertz absorption increases with increasing sample concentration between 0.1 and 1 THz, exhibiting a clear gradient relationship at 0.8 THz. The established calibration curve is Y = 0.39 LgC - 1.12822, R 2 =0.992. According to the guidelines of the International Union of Pure and Applied Chemistry (IUPAC). Y LOD = Y Blank + 3 SD, The LOD is 4.75 × 10 2 Particles / mL.

[0127] For the two groups of samples (2×10) 5 2×10 6 Ten experiments were conducted using particles PFU / mL to further investigate the repeatability of the method. As shown in Table 5, the estimated relative standard deviations (RSDs) for the three sample groups were 7.5% and 8.5%, respectively, with accuracy exceeding 85%. Therefore, the method based on the AuNP / BP terahertz biosensor for detection... Leishmania The method has good specificity and reproducibility.

[0128] Table 5. Detection by AuNP / BP Terahertz Biosensor Leishmania Accuracy and precision of QC samples

[0129] The pathogen detection method based on a terahertz AuNP / BP biosensor provided by this invention can be applied to the following fields: Food safety: Rapid detection of Escherichia coli and other bacteria in food to ensure food quality and safety. Clinical diagnosis: Rapid detection of pathogens such as VZV and Candida albicans in patient samples, enabling early diagnosis and timely treatment.

[0130] Public health surveillance: Detecting viruses and parasites in epidemiological investigations helps monitor the development of epidemics. The terahertz AuNP / BP biosensor detection method provided by this invention can rapidly, sensitively, and specifically detect pathogens that threaten human safety. Based on terahertz wave technology, combined with specific antibody or aptamer capture, pathogens are identified and detected through spectral information, enabling intelligent pathogen identification. This provides important evidence for the diagnosis and subsequent treatment of infectious diseases, and is of great significance for treating patients, controlling the development of epidemics, and reducing casualties.

[0131] The method provided by this invention is convenient and quick, and can identify Escherichia coli and various pathogens in a short time, so as to promptly detect problems and take corresponding measures. It has the advantages of rapid detection, high sensitivity and good specificity.

[0132] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0133] The various embodiments in this specification are described in a related manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions of the method embodiments.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. An AuNP / BP composite biosensor, characterized in that, The biosensor includes an AuNP / BP composite material and a biometric element; The composite material has a multilayer structure, each layer consisting of a black phosphorus substrate of the target thickness and gold nanoparticles modified on the surface; the thickness of the black phosphorus is 5-30 nm, and the particle size of the gold nanoparticles is 5-50 nm. The biometric element is fixed on the surface of the gold nanoparticles for the specific capture of target pathogens and antigens.

2. The biosensor according to claim 1, characterized in that, The biometric element is an antibody or aptamer, which is fixed to the surface of the composite material through the adsorption of the gold nanoparticles.

3. The biosensor according to claim 1, characterized in that, The target pathogens include bacteria, viruses, fungi, and parasites.

4. A pathogen detection method based on an AuNP / BP composite material biosensor, characterized in that, The method includes: A solution of a specific antibody or aptamer for the target pathogen is dropped onto the surface of an AuNP / BP composite biosensor of the target thickness and incubated at room temperature for 30-60 minutes to allow the gold nanoparticles of the biosensor to efficiently adsorb the antibody or aptamer solution; the AuNP / BP composite biosensor is the biosensor according to any one of claims 1-3. BSA solution was added to the surface of the biosensor and sealed for 30 minutes to reduce non-specific adsorption. The material was then rinsed with PBS buffer to obtain the AuNP / BP detection material. The AuNP / BP detection material was immersed in a solution containing the pathogen to be detected and incubated for 30 minutes to obtain an immune complex; The unbound pathogens on the immune complex were washed away with the PBS buffer to obtain the sample to be tested. The sample to be tested is scanned using a terahertz time-domain spectrometer to obtain target absorbance data, and the target absorbance data is used to identify whether the sample to be tested contains the target pathogen.

5. The method according to claim 4, characterized in that, After identifying whether the target pathogen is present in the sample to be tested based on the target absorbance data, the method further includes: If it is determined that the sample to be tested does not contain the first pathogen, then an AuNP / BP composite biosensor using a specific antibody or aptamer for the second pathogen as a biorecognition element is selected to detect the sample to be tested according to the method of claim 4; when the test result is positive, it is determined that the sample to be tested contains the second pathogen; when the test result is still negative, the method of claim 4 is repeated by sequentially replacing the AuNP / BP composite biosensor with a specific antibody or aptamer for other candidate pathogens until the pathogen category in the sample to be tested is identified.

6. The method according to claim 4, characterized in that, Before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the procedure also includes: A solution of a specific antibody or aptamer for the target pathogen was dropped onto the surface of the AuNP / BP composite biosensor, and absorbance data was obtained by scanning multiple frequency points using a terahertz time-domain spectrometer to obtain the first absorbance value at the multiple frequency points. Based on the mapping relationship between the frequency points and the first absorbance value, a blank group curve is obtained; A solution of a specific antibody or aptamer for the target pathogen and a solution containing the target pathogen were sequentially dropped onto the surface of the AuNP / BP composite biosensor, and absorbance data were obtained by scanning multiple frequency points using a terahertz time-domain spectrometer to obtain a second absorbance value at the multiple frequency points. Based on the mapping relationship between the frequency points and the second absorbance value, a standard curve of the target pathogen is obtained.

7. The method according to claim 6, characterized in that, The step of identifying whether the target pathogen is present in the sample to be tested based on the target absorbance data includes: Obtain the absorbance value corresponding to each frequency point from the target absorbance data; Based on the mapping relationship between the frequency points and the absorbance values, the curve to be detected is obtained; The test curve is compared with the standard curve of the target pathogen, and the similarity between the test curve and the standard curve is used to determine whether the test sample contains the target pathogen.

8. The method according to claim 4, characterized in that, Before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the procedure also includes: Several candidate composite materials composed of black phosphorus substrates of different thicknesses and surface-modified gold nanoparticles were prepared. Specific antibodies or aptamers of the target pathogen are immobilized on the plurality of candidate composite materials, respectively; The thickness of the candidate composite material that produces the maximum absorbance response is selected as the target thickness using terahertz detection.

9. The method according to claim 4, characterized in that, The step of scanning the sample to be tested using a terahertz time-domain spectrometer to obtain target absorbance data includes: The sample to be tested is placed in a terahertz time-domain spectrometer to remove moisture from the sample, and nitrogen gas is continuously introduced into the spectrometer. Using a transmission scanning mode, the spectrum at each frequency point is rapidly and repeatedly scanned 300 times, and the absorbance data is collected each time. The absorbance data is then averaged to obtain the target absorbance data.

10. The method according to claim 4, characterized in that, Before dropping a solution of a specific antibody or aptamer for the target pathogen onto the surface of the AuNP / BP composite biosensor of the target thickness, the procedure also includes: The AuNP / BP composite biosensor and terahertz time-domain spectrometer were used to detect target pathogen samples of different concentrations, and the absorbance-concentration logarithm plot of the AuNP / BP composite biosensor was obtained. The detection limit of the AuNP / BP composite biosensor is calculated based on the absorbance-concentration logarithmic graph and the preset detection limit formula.

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