WO2022041584A1 - 病原体的自动化检测装置及自动化检测方法 - Google Patents

病原体的自动化检测装置及自动化检测方法 Download PDF

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WO2022041584A1
WO2022041584A1 PCT/CN2020/136912 CN2020136912W WO2022041584A1 WO 2022041584 A1 WO2022041584 A1 WO 2022041584A1 CN 2020136912 W CN2020136912 W CN 2020136912W WO 2022041584 A1 WO2022041584 A1 WO 2022041584A1
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biosensor
robotic arm
optical
porous silicon
layer
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French (fr)
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荣国光
萨万·默罕默德
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西湖大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators

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  • the present application belongs to the technical field of biological detection, and particularly relates to a pathogen detection device and a detection method.
  • New coronavirus pneumonia (Coronavirus Disease 2019, COVID-19), referred to as “new coronary pneumonia”, refers to the pneumonia caused by the 2019 new coronavirus infection, which is an acute respiratory infection.
  • the World Health Organization (WHO) named it "COVID-19" on February 11, 2020.
  • WHO World Health Organization
  • the coronavirus research group of the International Committee on Taxonomy of Viruses named the new coronavirus "SARS-CoV-2" in accordance with the taxonomic naming rules.
  • the detection of the new coronavirus SARS-CoV-2 is mainly limited to hospitals and CDCs, and the mainstream technology is the polymerase chain reaction based on nucleic acid amplification detection.
  • nucleic acid extraction and amplification is a complex process, and the technology of the testing personnel, the cleanliness of the environment, and the timeliness of sample delivery will all affect the accuracy of the results. Therefore, although the nucleic acid amplification detection method represented by PCR has high sensitivity, the detection rate is lower than 50%, the detection cycle is long, and it cannot be completed in many areas with limited conditions. Certain institutions have proposed rapid on-site testing solutions for the new coronavirus SARS-CoV-2.
  • nucleic acid amplification detection methods 30-minute automatic nucleic acid extraction NAAT and on-site NAAT in a "tent mobile laboratory” are proposed. These methods try to minimize human factors in nucleic acid extraction and amplification, improve reliability, or create a relatively clean environment at the detection site to improve reliability, but these new methods still face the long detection cycle and the long detection period brought by the transfer of samples. The problem of sample contamination.
  • IgG and IgM antibodies In addition to the nucleic acid amplification detection method, some researchers have proposed a method to detect IgG and IgM antibodies by colloidal gold immunoassay without nucleic acid extraction. Although immunoassay methods based on IgG and IgM antibodies do not require nucleic acid extraction and amplification, they are not easily contaminated by impurities in the environment, and are more suitable for on-site detection than nucleic acid detection methods; however, the problem faced by antibody methods is that many Infected people do not produce antibodies, and this part of the population cannot be detected by antibody methods. Moreover, people who are able to produce antibodies generally take at least 7 days after infection to develop detectable antibodies in serum, during which time these infected people have begun to spread the virus. In addition, IgG and IgM antibodies are not specific. Influenza and other viral infections can also produce IgG and IgM antibodies in patients. These factors limit the application of antibody detection methods in the prevention and control of the new crown epidemic.
  • nucleic acid amplification method requires nucleic acid extraction, purification and amplification of clinical samples, which requires well-trained personnel to operate the instrument;
  • the antibody method requires the extraction of serum from blood samples, and then the reaction with lateral chromatography test strips The result judgment also requires human operation. These operating procedures are not only prone to errors, but also often face the risk of infection when handling clinical samples, which limits the number of samples that can be detected per unit time, that is, the detection throughput.
  • the purpose of the present application is to provide an automated pathogen detection device and detection method in view of the above-mentioned deficiencies of the prior art.
  • a first aspect of the present application provides an automatic detection device for pathogens, including a control system, a first robotic arm, a second robotic arm, a biosensor and an optical fiber spectrometer, wherein: the biosensor is provided in the on the second robotic arm; the biosensor includes a single crystal silicon substrate and a nanoporous silicon thin film optical device disposed on the single crystal silicon substrate, and the nanoporous silicon thin film optical device includes a porous silicon monolayer interference film, Porous silicon Bragg reflector or porous silicon micro-resonator; a composite metal thin film is deposited on the surface of the nanoporous silicon thin film optical device; the control system controls the first robotic arm, the second robotic arm and the optical fiber
  • the spectrometer completes the following detection operations: a first robotic arm: adding a liquid sample to the biosensor surface, and cleaning the sensor surface after the liquid sample reacts on the biosensor surface; a second robotic arm: adding the The surface of the biosensor is aligned with
  • the biosensor includes at least 4 sensor units, the sensor units adopt a micro-well structure, and a polyimide isolation layer is provided between the micro-well structures.
  • the porous silicon Bragg mirror when the structure of the nanoporous silicon thin film optical device is a porous silicon Bragg mirror, the porous silicon Bragg mirror includes alternately stacked high refractive index layers and low refractive index layers.
  • the numerical contrast between the high refractive index and the low refractive index should be as large as possible.
  • the number of the high-refractive index layers and the low-refractive index layers in the porous silicon Bragg mirror is equal to and greater than 10 respectively.
  • the porous silicon micro-resonator when the structure of the nanoporous silicon thin film optical device is a porous silicon micro-resonator, the porous silicon micro-resonator includes: a first porous silicon Bragg mirror, a second porous silicon Bragg mirror, and a The porous silicon thin film defect layer between the first porous silicon Bragg mirror and the second porous silicon Bragg mirror; the first porous silicon Bragg mirror and the second porous silicon Bragg mirror independently include Alternately stacked high-refractive index layers and low-refractive index layers.
  • the numerical contrast between the high refractive index and the low refractive index should be as large as possible.
  • the number of high-refractive-index layers and low-refractive-index layers in the first porous silicon Bragg mirror are equal and are respectively 6; the high-refractive index layers in the second porous silicon Bragg mirror
  • the number of low-refractive index layers is equal to 6 or more, so that the reflectivity of the optical biosensor in the visible light wavelength range changes greatly, the corresponding characteristic peaks and characteristic valleys are sharper, and the quality factor is high. .
  • the higher the quality factor the stronger the detection ability for small red shifts, which means that the sensor has an enhanced ability to detect the reflectance spectrum changes caused by the combination of trace biological substances, that is, the higher the sensitivity of the sensor.
  • the single crystal silicon substrate is selected from N-type phosphorus-doped single-crystal silicon, N-type arsenic-doped single-crystal silicon, N-type antimony-doped single-crystal silicon, or P-type boron-doped single crystal silicon; preferably P boron-doped monocrystalline silicon.
  • the P-type single-crystal silicon substrate has a smaller pore size and less scattering effect on light. Using P-type single-crystal silicon as the substrate is conducive to obtaining sharp characteristic peaks in the detection process, improving the Detection Sensitivity of Optical Biosensors.
  • the pore size of the porous silicon nanopores in the porous silicon single-layer interference film, porous silicon Bragg mirror or porous silicon micro-resonator is 1-100 nanometers; preferably 20-30 nanometers.
  • the pore size of porous silicon is within the above preferred range, the light scattering effect is small, which is beneficial to obtain sharp characteristic peaks in the detection process and improve the detection sensitivity of the optical biosensor; and the porous silicon film with the pore size within the above preferred range It is easy to process and has good reliability.
  • the surface of the nanoporous silicon thin film optical device is thermally oxidized or carbonized before depositing the composite metal thin film. Since the prepared porous silicon film contains silicon-hydrogen bonds, it is easy to be slowly oxidized to silicon-oxygen bonds in the air. It is thermally oxidized or carbonized to achieve the effect of stabilizing the porous silicon film.
  • the composite metal film comprises a transition layer provided on the surface of the nanoporous silicon thin film optical device and a gold film layer provided on the surface of the transition layer; the transition layer is selected from a nickel layer, a chromium layer and a titanium layer one or more of them.
  • the function of the composite metal film layer is that since the composite metal film layer is deposited on the porous silicon thin film, the nanoporous structure of the porous silicon thin film will not be affected.
  • the nanoporous structure of the gold film causes Localized Surface Plasmon Resonance (LSPR) to occur on the surface of the gold film, and LSPR will have a strong electric field energy on the surface of the gold film or
  • the concentration of the electric field strength makes the binding energy of the biomolecules on the surface of the gold film layer to interact strongly with the electric field, so that the reflection spectrum changes greatly, so the sensitivity of the optical biosensor of the present application can be improved.
  • the present application adds a transition layer between the nanoporous silicon thin film optical device and the gold film layer, and the transition layer is selected from nickel layer, chromium layer or titanium layer, the transition layer can enhance the bonding force between the nanoporous silicon thin film optical device and the gold layer, improve the stability of the gold layer on the surface of the nanoporous silicon thin film optical device, and improve the stability of the optical biosensor sex.
  • the addition of the composite metal film also solves the technical difficulties in the detection of large particle viruses (such as the SARS-CoV-2 virus).
  • large particle viruses such as the SARS-CoV-2 virus.
  • the detection of optical biosensors based on nanoporous materials requires pathogens to enter their nanopores to ensure better detection sensitivity.
  • large particles of viruses such as SARS-CoV-2 virus particles with a diameter of 100-150 nanometers
  • the increase in the pore size of nanopores can also enhance light scattering and reduce optical Due to the sensitivity of biosensors, it is not wise to blindly enlarge the pore size of porous silicon nanopores in order to allow large particles of viruses to enter the nanopores.
  • LSPR localized plasmon resonance
  • the thickness of the transition layer in the composite metal thin film is 3-8 nanometers, preferably 5 nanometers; the thickness of the gold film layer is 10-20 nanometers, preferably 15 nanometers.
  • the thickness of the transition layer and the gold film layer has the following effects: first, the transition layer is too thin to stably and effectively attach the gold film to the porous silicon film, and the transition layer is too thick to weaken the underlying Bragg reflector. The reflected light (because the metal absorbs the energy of electromagnetic waves, light is also an electromagnetic wave), the reflected light energy can further enhance the LSPR excited by the surface of the gold film, so too thick will affect the sensitivity of the sensor.
  • the gold film is too thin to effectively excite LSPR, and it is also difficult to effectively support the bioprobe to be covalently cured on the sensor surface; too thick gold film will weaken the light reflected by the Bragg reflector below it (because the metal will absorb electromagnetic waves. energy, light is also an electromagnetic wave), the reflected light energy could have further enhanced the LSPR excited by the surface of the gold film, so too thick will affect the sensitivity of the sensor.
  • the thicknesses of the transition layer and the gold film layer are within the range provided in this application, the gold film on the sensor surface and the bioprobe cured on the gold film are relatively stable, and will not be affected by movement or surface washing during the detection operation. As a result, the gold film or the bioprobe falls off together with the gold film. At the same time, the electric field intensity of the excited LSPR mode on the surface of the gold film is the strongest, and the sensitivity of the sensor is the highest.
  • the application also provides an automated detection method for pathogens, comprising the steps of:
  • Biosensor coated bioprobe the first robotic arm adds the bioprobe liquid sample to the biosensor surface, and the second robotic arm keeps the bioprobe liquid sample reacting on the biosensor surface for 0.5 to 2 hours ; a first robotic arm sucks the bioprobe liquid sample from the biosensor surface, and uses a buffer to clean the biosensor surface;
  • the biosensor performs the first optical detection: the first robotic arm adds buffer to the surface of the biosensor, and covers the sensor surface with a cover glass; the second robotic arm aligns the biosensor with the optical fiber spectrometer The optical fiber probe is used for the first optical detection; the optical fiber spectrometer collects the first optical reflection spectrum data;
  • Clinical sample loading the first robotic arm adds the clinical liquid sample to the surface of the biosensor, and the second robotic arm keeps the clinical liquid sample reacting on the surface of the biosensor for 5-20 minutes; aspirating the clinical fluid sample from the biosensor surface and washing the biosensor surface with a buffer;
  • the second optical detection of the biosensor the first robotic arm adds buffer to the surface of the biosensor, and covers the sensor surface with a cover glass; the second robotic arm aligns the biosensor with the optical fiber.
  • the optical fiber probe of the spectrometer performs the second optical detection; the optical fiber spectrometer collects the second optical reflection spectrum data;
  • the biological probes include antibodies, binding proteins or nucleic acid aptamers; more preferably, the biological probes are SARS-CoV-2 virus antibodies.
  • the present application provides an automatic detection device and automatic detection method for pathogens, including automatic control of sample addition to biosensors based on robotic arms, automatic detection of optical reflectance spectra of biosensors, and analysis and output of results.
  • the entire detection process is automatically completed after one-click click, without manual intervention, and can be used for rapid on-site automatic detection of pathogens such as new coronaviruses. It can give results quickly and avoid cross-infection caused by a large number of people waiting for the results for a long time.
  • an optical biosensor based on nanomaterial-porous silicon is also used, and the optical reflection spectrum of the sensor element has characteristic peaks and characteristic valleys.
  • the pathogen binds to the sensor surface, it changes the optical refractive index near the sensor surface (refractive index increase), so that the characteristic peaks and valleys of the reflectance spectrum of the sensor are shifted to the long wavelength direction (red shift).
  • red shift The amount of red shift can be used to Quantify pathogens.
  • coating the surface of the sensor with antibodies, aptamers or ACE2-binding proteins can specifically capture specific pathogens (such as SARS-CoV-2 virus) without binding other biological substances;
  • pathogens such as SARS-CoV-2 virus
  • the optical signal change of the biosensor reflected by the capture event can be specifically directed to a specific pathogen.
  • the present application also designs the specific structure of the nanoporous silicon thin film optical device and deposits a composite metal thin film on the surface of the nanoporous silicon thin film optical device, so that the optical biosensor of the present application has extremely high detection sensitivity.
  • the optical biosensor of the present application to detect SARS-CoV-2 virus, although the new coronavirus particles are large (100-150 nanometers in diameter), it is difficult to enter the inside of the nanopores of porous silicon. Bonding near the metal layer achieves higher detection sensitivity.
  • Fig. 1 is the detection spectrum of S-ECD protein of SARS-CoV-2 virus detected by the pathogen automatic detection device based on device 1;
  • Fig. 2 is the detection spectrum of S-ECD protein of SARS-CoV-2 virus detected by the pathogen automatic detection device based on device 2;
  • FIG. 3 is a detection spectrum of the S-ECD protein of SARS-CoV-2 virus detected by an automated pathogen detection device based on device 7.
  • Nanoporous silicon thin film optical devices were fabricated on the surface of single crystal silicon wafers using anodization method (according to routine procedures in the art).
  • the nanoporous silicon thin film optical device has three structures: porous silicon monolayer interference film, porous silicon Bragg mirror or porous silicon micro-resonator.
  • the preparation of the nanoporous silicon thin film in the nanoporous silicon thin film optical device can be carried out by conventional methods in the art. Taking the anodic oxidation method as an example in this embodiment, the steps are briefly described as follows:
  • a constant current can be applied between the anode and the cathode, and the silicon wafer can be etched to form porous silicon.
  • the porosity of porous silicon that is, the refractive index, can be controlled by the applied current density per unit silicon area; the thickness of porous silicon can be controlled by etching time.
  • the multi-layer structure of porous silicon includes the corresponding thickness and refractive index of each layer, and each layer in the multi-layer porous silicon can be continuously etched by switching the current density and etching time required for each layer.
  • the thermal oxidation method is as follows: oxidizing at 700-800° C. for 10-30 minutes to form a silicon dioxide covering layer with a thickness of 1-2 nanometers on the inner surface of the pores of the porous silicon.
  • the carbonization method is as follows: the surface of the nanoporous silicon thin film optical device is carbonized by a low pressure chemical vapor deposition method. First, the prepared porous silicon samples were soaked in 2.5% HF solution for 90 s, the soaked porous silicon samples were rinsed several times with ethanol solution, and the cleaned porous silicon samples were blown dry with N2 . Then, the blow-dried porous silicon sample was immediately placed into the quartz tube of the tube furnace, and the porous silicon sample was located at the heating center of the tube furnace. Next, the air pressure in the quartz tube was evacuated to a vacuum, and the furnace temperature was raised to 650 °C.
  • a physical vapor deposition (PVD) method or a magnetron sputtering method are used to deposit a transition layer and a gold film layer in sequence.
  • resistive vacuum thermal evaporation equipment for example: ZD-400 single-chamber high-vacuum resistive evaporation equipment purchased from Shenyang Kecheng Vacuum Technology Co., Ltd.
  • the deposition steps follow the device instruction manual.
  • Bioprobes including antibodies, binding proteins, aptamers, etc.
  • the biosensor has specificity for coronaviruses.
  • the steps of immobilizing biological probes on the surface of nanoporous silicon thin film optical devices are as follows:
  • the steps of immobilizing biological probes on the surface of nanoporous silicon thin film optical devices are as follows:
  • the modified sulfhydryl group (SH-) at the 5' end of the aptamer can directly bind to the gold surface.
  • the reducing agent TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • S-S disulfide bond
  • Thiol and gold form Au-S bond to immobilize the aptamer on the sensor surface, and then add 6-mercapto-1-hexanol to cover the surface of the unbound probe to prevent non-specific adsorption.
  • the aptamer-immobilized porous silicon biosensor is obtained. After optimization, it is expected that it can be stored in a low temperature environment of 4 degrees Celsius for one month for rapid detection of the new coronavirus in the field.
  • the device 1 includes a P-type boron-doped monocrystalline silicon and a nanoporous silicon thin film optical device disposed on the P-type boron-doped monocrystalline silicon, and the nanoporous silicon thin film optical device is a porous silicon single-layer interference film.
  • the thickness of the porous silicon single-layer interference film is 1 micrometer, and the diameter of the nanopore is 20-30 nanometers.
  • the surface of the nanoporous silicon thin film optical device is thermally oxidized, and a composite metal thin film is sequentially deposited and coated with biological probes.
  • the composite metal film includes a 5-nanometer nickel film layer and a 15-nanometer gold film layer, and the biological probe is a SARS-COV-2 antibody (purchased from Yiqiao Shenzhou).
  • the device 2 includes a P-type boron-doped monocrystalline silicon and a nanoporous silicon thin film optical device disposed on the P-type boron-doped monocrystalline silicon, and the nanoporous silicon thin film optical device is a porous silicon Bragg mirror.
  • the surface of the nanoporous silicon thin film optical device is thermally oxidized, and a composite metal thin film is sequentially deposited and coated with biological probes.
  • the composite metal film includes a 5-nanometer nickel film layer and a 15-nanometer gold film layer, and the biological probe is a SARS-COV-2 antibody (purchased from Yiqiao Shenzhou).
  • the porous silicon Bragg mirror includes alternately stacked high-refractive index layers and low-refractive index layers.
  • Devices 3 to 10 include P-type boron-doped monocrystalline silicon and a nanoporous silicon thin film optical device disposed on the P-type boron-doped monocrystalline silicon, and the nanoporous silicon thin film optical device is a porous silicon micro-resonator.
  • the surface of the nanoporous silicon film optical device is thermally oxidized, and a composite metal film is sequentially deposited and coated with a biological probe, and the biological probe is a SARS-COV-2 antibody (purchased from Yiqiao Shenzhou).
  • the porous silicon micro-resonator includes: a first porous silicon Bragg mirror, a second porous silicon Bragg mirror, and a second porous silicon Bragg mirror provided on the first porous silicon Bragg mirror and the second porous silicon Bragg mirror
  • the porous silicon thin film defect layer between them; the first porous silicon Bragg mirror and the second porous silicon Bragg mirror independently include alternately stacked high refractive index layers and low refractive index layers.
  • the number of high-refractive-index layers and low-refractive-index layers in the first porous silicon Bragg mirror represented by “number of first layers” in Table 1
  • the number of the second porous silicon Bragg mirror The number of high-refractive index layers and low-refractive index layers in the mirror (represented by “the second number of layers” in Table 1)
  • the thickness of the transition layer in the composite metal film layer represented by “the second number of layers” in Table 1
  • first layer second layer transition layer thickness Gold film thickness Device 3 5 5 5nm 15nm Device 4 5 10 5nm 15nm Device 5 7 10 5nm 15nm Device 6 7 5 5nm 15nm Device 7 (preferred embodiment) 6 10 5nm 15nm Device 8 6 10 5nm 5nm Device 9 6 10 5nm 25nm Device 10 6 12 10nm 15nm
  • the structure of the comparative device 1 is basically the same as that of the device 1, the difference is only that the composite metal film is not deposited on the surface of the comparative device 1.
  • the structure of the comparative device 2 is basically the same as that of the device 2, the difference is only that the composite metal film is not deposited on the surface of the comparative device 2.
  • the structure of the comparative device 3 is basically the same as that of the device 7, except that no composite metal film is deposited on the surface of the comparative device 3.
  • An automated detection device for pathogens includes a control system, a first robotic arm, a second robotic arm, a biosensor and an optical fiber spectrometer, wherein: the biosensor is arranged on the second robotic arm; the biosensor is a second implementation Various optical biosensors in the modalities.
  • first robotic arm adding a liquid sample to the surface of the biosensor , and clean the surface of the sensor after the liquid sample reacts on the surface of the biosensor
  • second robotic arm align the surface of the biosensor with the fiber probe of the fiber optic spectrometer for optical detection
  • fiber optic spectrometer detect And collect the optical reflectance spectrum data of the biosensor.
  • the first robotic arm adds the biological probe liquid sample (the biological probe can be an antibody, binding protein or nucleic acid aptamer; for example: Aptamer solution, ACE2 solution, etc.) into the microwell on the surface of the biosensor, and the second robotic arm keeps all The biological probe liquid sample is reacted on the biosensor surface for 0.5 to 2 hours; the first robotic arm sucks the biological probe liquid sample from the biosensor surface, and uses a buffer to clean the biosensor surface;
  • the biological probe liquid sample can be an antibody, binding protein or nucleic acid aptamer; for example: Aptamer solution, ACE2 solution, etc.
  • the first robotic arm adds buffer to the surface of the biosensor, and covers the sensor surface with a cover glass; the second robotic arm aligns the biosensor with the optical fiber probe of the optical fiber spectrometer to perform the first optical detection; the optical fiber The spectrometer collects the first optical reflection spectrum data;
  • the first robotic arm adds clinical fluid samples (such as sputum, saliva, throat swab sampling fluid, bronchoalveolar lavage fluid, etc.) to the biosensor surface, and the second robotic arm keeps the clinical fluid samples in the The biosensor surface reacts for 5-20 minutes; the first robotic arm sucks the clinical liquid sample from the biosensor surface, and uses a buffer to wash the biosensor surface;
  • clinical fluid samples such as sputum, saliva, throat swab sampling fluid, bronchoalveolar lavage fluid, etc.
  • the first robotic arm adds buffer to the surface of the biosensor, and covers the sensor surface with a cover glass; the second robotic arm aligns the biosensor with the optical fiber probe of the optical fiber spectrometer to perform the second optical detection;
  • the optical fiber spectrometer collects the second optical reflection spectrum data;
  • the first optical reflectance spectrum data and the second optical reflectance spectrum data are compared, and the pathogen is quantified according to the red shifts of the characteristic peaks and valleys of the reflectance spectrum.
  • the fourth embodiment of the present application relates to an example of automated detection of the SARS-CoV-2 virus.
  • the first robotic arm added 20 microliters of bioprobe Aptamer solution into the microwells of the biosensor, the concentration of Aptamer solution in each microwell may be different, and kept for 1 hour; the second robotic arm slowly vibrated the sample within 1 hour to promote the surface Homogeneous reaction; the first robotic arm aspirates the Aptamer solution from the microwells.
  • the first robotic arm adds PBS buffer to the microwell, sucks it into the pipette, and then pushes it into the microwell. This operation is repeated three times to absorb the Aptamer that has not reacted on the sensor surface to the maximum extent; the first robotic arm adds new PBS buffer To the microwell, repeat the above cleaning steps twice, for a total of three cleaning procedures.
  • the first robotic arm adds new PBS buffer to the microwells, and the automatic suction cup covers the sensor surface with a coverslip, covering all the microwells; the second robotic arm numbers the sensor microwells from 1 to N, and aligns the optical fibers in sequence
  • the optical fiber probe of the spectrometer automatically collects the reflectance spectrum data of each microwell after alignment, which is the first optical reflectance spectrum data; the automatic suction cup removes the cover glass and sucks off the PBS buffer in the microwell .
  • the first robotic arm adds 20 microliters of throat swab sampling solution into the microwells on the surface of the biosensor, and the other two microwells respectively add negative samples and positive samples (specific steps for negative samples and positive samples are omitted).
  • the second robotic arm holds or vibrates the biosensor for 10 minutes to promote a uniform response on the biosensor surface; the first robotic arm sucks the throat swab sample solution from the biosensor surface;
  • the first robotic arm adds PBS buffer to the microwell on the surface of the biosensor, sucks it into the pipette, and then pushes it into the microwell. This operation is repeated three times to absorb the biological substances that have not reacted on the surface of the sensor to the maximum extent; New PBS buffer was added to the microwells, and the above washing steps were repeated two more times, for a total of three washing procedures.
  • the automatic suction cup covers the sensor surface with a coverslip, covering all the microwells;
  • the second robotic arm numbers the sensor microwells from 1 to N, and aligns them with the optical fiber probe of the optical fiber spectrometer. , and automatically collect the reflectance spectrum data of each microwell after alignment, which is the second optical reflectance spectrum data; the automatic suction cup removes the coverslip, and sucks off the PBS buffer in the microwell.
  • the first optical reflectance spectrum data and the second optical reflectance spectrum data are compared, and the pathogen is quantified according to the red shifts of the characteristic peaks and valleys of the reflectance spectrum.
  • the S-ECD protein is the S protein on the surface of the SARS-CoV-2 virus, including S1 and S2 proteins. It is the specific functional region of the ACE2-binding protein that the virus attacks and binds to human cells.
  • the following shows the detection of S-ECD protein on the surface of SARS-CoV-2 virus using the automated detection device and automated detection method of the present application (devices 1 to 10 and comparison device 1 to comparison device 3 are used in the device respectively) The effect is to show the detection sensitivity of the automatic detection device and detection method provided by the present application to the SARS-CoV-2 virus.
  • the characteristic valley position before S-ECD protein binding is 615 nm, not 830 nm at the center wavelength (wavelength in vacuum) of the designed total reflection band of DBR, which is due to the thermal After oxidation, many silicon materials are transformed into silicon dioxide, and the refractive index drops significantly (from about 3.5 for silicon to about 1.5 for silicon dioxide), which makes the total reflection band of the porous silicon Bragg reflector appear blue as a whole. And after the composite metal film is coated on the surface of the Bragg mirror device, the total reflection band of the Bragg mirror (from 565 nm to 615 nm) is transformed into a characteristic peak (565 nm) and a characteristic valley (615 nm).
  • Comparing device 2 cannot effectively detect S-ECD protein at a concentration of 0.5 nM, and the red shift of characteristic peaks or characteristic valleys is not obvious.
  • Devices 3 to 10 can effectively detect low concentrations of S-ECD proteins in the range of 1 fM to 1 nM.
  • the comparison device 3 could not effectively detect the low concentration S-ECD protein in the concentration range of 1fM to 1nM, and the red shift of the characteristic peak or characteristic valley was not obvious.
  • device 7 shows extremely high detection sensitivity, and the binding of S-ECD protein at a concentration of 0.5 nM to the antibody on the surface of device 7 will bring about a red shift of characteristic peaks or characteristic valleys of about 10 nanometers (the detection spectrum is shown in Figure 3). shown).
  • the detection spectrum is shown in Figure 3). shown.
  • the device 7 generates local plasmon resonance on the surface of the gold film layer on the basis of the porous silicon micro-resonant cavity and through the setting of the composite metal film layer, which improves the stability of the optical biosensor and makes the SARS with larger particles.
  • -CoV-2 virus does not need to enter the nanopores of porous silicon, and it also has high detection sensitivity when it binds to the antibody near the composite metal film layer.
  • the quality factor (Quality Factor, defined as the resonance center wavelength divided by the half-peak width) of device 7 at the resonance valley of 635.14 nm is about 100, while that of device 2 is located at about 100.
  • the figure of merit for the feature valley at 615 nm is around 10, which is an order of magnitude lower than Device 7. Therefore, the device 7 can identify a small red shift when detecting a low content of the target biological substance, thereby having a lower detection limit (Limit of Detection). From the comparison of detection limits, the detection limit of device 7 can reach 1 fM, while the detection limit of device 2 is 10 fM.
  • the device 7 is further optimized in structure.
  • the number of high-refractive-index layers and low-refractive-index layers in the first porous silicon Bragg mirror are equal and are respectively 6; the high-refractive index layers in the second porous silicon Bragg mirror
  • the number of the low-refractive-index layers is equal to six or more (10 in the device 7).
  • the detection limits of devices 3 to 6 are not as good as that of device 7.
  • the detection limit of device 3 is 100fM
  • the detection limit of devices 4-6 are all 10fM.
  • the thickness of the transition layer in the composite metal film is between 3 and 8 nanometers; the thickness of the gold film layer is between 10 and 20 nanometers, which further reduces the light generated in the detection process by the metal.
  • the effect of absorption is beneficial to increase the LSPR field strength on the gold surface, and to obtain sharp characteristic peaks during the detection process, thereby improving the detection sensitivity of the optical biosensor.
  • the detection sensitivity of devices 8-10 is lower than that of device 7.
  • the detection limits of devices 8-10 are all 10fM.
  • the automatic detection device and detection method provided in this application not only improve the detection throughput, but also avoid the risks of mistakes and cross-infection caused by personnel detection operations, and because of the use of nanomaterial-based porous silicon
  • the optical biosensor has high detection sensitivity.

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Abstract

本申请属于医学生物检测技术领域，公开了一种病原体的自动化检测装置及自动化检测方法。本申请所提供的自动化检测装置包括控制系统、第一机械臂、第二机械臂、生物传感器和光纤光谱仪，其中：所述生物传感器设于所述第二机械臂上；在控制系统的控制下，第一机械臂将液体样品加入所述生物传感器表面和清洗所述传感器表面；第二机械臂将生物传感器表面对准光纤光谱仪的光纤探头进行光学检测；光纤光谱仪检测并采集生物传感器的光学反射谱数据。

Description

病原体的自动化检测装置及自动化检测方法 技术领域
本申请属于生物检测技术领域，特别涉及一种病原体检测装置及检测方法。
背景技术
新型冠状病毒肺炎(Coronavirus Disease 2019，COVID-19)，简称“新冠肺炎”，是指2019新型冠状病毒感染导致的肺炎，是一种急性的呼吸系统感染。世界卫生组织(WHO)于2020年2月11日将其命名为“COVID-19”。同日，国际病毒分类委员会冠状病毒研究小组按照分类学的命名规则，将此次新冠病毒命名为“SARS-CoV-2”。
目前，新型冠状病毒SARS-CoV-2的检测主要局限于医院和疾病预防控制中心，主流技术是基于核酸扩增检测的聚合酶链式反应。然而，核酸提取和扩增是一个复杂的过程，检测人员的技术、环境的洁净度和送样的及时性都会影响结果的准确性。因此，以PCR为代表的核酸扩增检测方法虽然灵敏度高，但是检出率低于50％、检测周期长，且很多条件受限地区无法完成。某些机构提出了新冠病毒SARS-CoV-2快速现场检测解决方案。在核酸扩增检测法方面，提出了30分钟自动核酸提取NAAT、以及“帐篷式移动实验室”内的现场NAAT。这些方法试图在核酸提取和扩增方面尽量减少人为因素，提高可靠性，或者在检测现场营造一个相对洁净的环境以提高可靠性，但这些新的方法仍面临传递样品带来的检测周期长和样品污染的问题。
除核酸扩增检测方法之外，有研究者提出采用无需核酸提取的胶体金免疫法对IgG、IgM抗体进行检测的方法。虽然，基于IgG、IgM抗体的免 疫分析方法不需要核酸的提取和扩增，不容易被环境中的杂质污染，比核酸检测法更适于进行现场检测；但是，抗体法面临的问题是，很多被感染的人群体内不产生抗体，这部分人群无法用抗体法检出。而且，能够产生抗体的人群一般在感染后需要至少7天时间才会在血清中产生可检出的抗体，而在此期间这些感染者已开始传播病毒。此外，IgG、IgM抗体并不具有特异性，流感和其他病毒感染也会使病人体内产生IgG、IgM抗体，这些因素限制了抗体检测法在防控新冠疫情方面的应用。
另一方面，不论是核酸扩增法，还是抗体检测法，样品(包括痰液、唾液、咽拭子采样液、支气管肺泡灌洗液)的准备和检测操作都需要人工来完成。例如，核酸扩增法需要临床样本的核酸提取、纯化和扩增，都需要训练有素的人员操作仪器来完成；抗体法需要从血液样本中提取血清，之后用侧向层析试纸条反应结果判断也都需要人员操作。这些操作过程不仅容易出错，而且人员操作临床样品也常面临被感染的风险，限制了单位时间内能够检测的样品数，即检测通量。
发明内容
本申请的目的在于针对上述现有技术的不足，提供一种病原体自动化检测装置及检测方法。
为解决上述技术问题，本申请的第一方面提供一种病原体的自动化检测装置，包括控制系统、第一机械臂、第二机械臂、生物传感器和光纤光谱仪，其中：所述生物传感器设于所述第二机械臂上；所述生物传感器包括单晶硅基底及设于所述单晶硅基底上的纳米多孔硅薄膜光学器件，所述纳米多孔硅薄膜光学器件包括多孔硅单层干涉膜、多孔硅布拉格反射镜或多孔硅微谐振腔；所述纳米多孔硅薄膜光学器件的表面沉积有复合金属薄膜；所述控制系统控制所述第一机械臂、所述第二机械臂和所述光纤光谱仪完成下述检 测操作：第一机械臂：将液体样品加入所述生物传感器表面，并于所述液体样品在所述生物传感器表面反应之后清洗所述传感器表面；第二机械臂：将所述生物传感器表面对准所述光纤光谱仪的光纤探头，进行光学检测；光纤光谱仪：检测并采集生物传感器的光学反射谱数据。
优选地，所述生物传感器包括至少4个传感器单元，所述传感器单元采用微井结构，在各微井结构间设有聚酰亚胺隔离层。
本申请中，当纳米多孔硅薄膜光学器件的结构为多孔硅布拉格反射镜时，所述多孔硅布拉格反射镜包括交替堆叠的高折射率层和低折射率层。
所述高折射率层和所述低折射率层满足：n 1d 1＝n 2d 2＝λ/4；其中，n 1和d 1分别为高折射率层的折射率和厚度；n 2和d 2分别为低折射率层的折射率和厚度；λ为所设计多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。为了提高布拉格反射镜的反射率，所述高折射率和低折射率的数值对比应尽可能大。
优选地，所述多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为10个以上。
优选地，当纳米多孔硅薄膜光学器件的结构为多孔硅微谐振腔时，所述多孔硅微谐振腔包括：第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜及设于所述第一多孔硅布拉格反射镜和第二多孔硅布拉格反射镜之间的多孔硅薄膜缺陷层；所述第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜各自独立地包括交替堆叠的高折射率层和低折射率层。
所述高折射率层和低折射率层满足：n 1d 1＝n 2d 2＝λ/4；其中，n 1和d 1分别为高折射率层的折射率和厚度；n 2和d 2分别为低折射率层的折射率和厚度；λ为第一或第二多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。为了提高布拉格反射镜的反射率，所述高折射率和低折射率的数值对比应尽可能大。
所述多孔硅薄膜缺陷层满足：n 3d 3＝Nλ/4，其中，n 3和d 3分别为多孔硅薄膜缺陷层的折射率和厚度，其中N>0且N≠1，n 3可以是硅折射率(3.5)和空气折射率(1.0)之间的任意值，λ为第一或第二多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。
优选地，所述第一多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个；所述第二多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个或6个以上，从而使得光学生物传感器在可见光波长范围内的反射率变化较大，对应的特征峰和特征谷较为尖锐，品质因数较高。品质因数越高，对于微小红移的检测能力越强，意味着传感器对于微量生物物质结合带来的反射谱变化检测能力增强，也就是传感器灵敏度越高。
优选地，所述单晶硅基底选自N型磷掺杂单晶硅、N型砷掺杂单晶硅、N型锑掺杂单晶硅或P型硼掺杂单晶硅；优选为P型硼掺杂单晶硅。相对于N型单晶硅基底，P型单晶硅基底的孔径比较小，对光的散射作用较小，采用P型单晶硅作为基底，有利于在检测过程中获得尖锐的特征峰，提高光学生物传感器的检测灵敏度。
优选地，所述多孔硅单层干涉膜、多孔硅布拉格反射镜或多孔硅微谐振腔中的多孔硅纳米孔的孔径为1～100纳米；优选20～30纳米。当多孔硅的孔径在上述优选范围内，对光的散射作用较小，有利于在检测过程中获得尖锐的特征峰，提高光学生物传感器的检测灵敏度；并且孔径在上述优选范围内的多孔硅薄膜易于加工、具有较好的可靠性。
优选地，所述纳米多孔硅薄膜光学器件的表面在沉积复合金属薄膜之前被热氧化或碳化。由于制备好的多孔硅薄膜中包含硅氢键，在空气中易于被缓慢氧化为硅氧键，本申请中在对纳米多孔硅薄膜光学器件的表面沉积复合金属薄膜、包被生物探针之前，对其进行热氧化或碳化，从而达到稳定多 孔硅薄膜的效果。
优选地，所述复合金属薄膜包括设于所述纳米多孔硅薄膜光学器件表面的过渡层和设于所述过渡层表面的金膜层；所述过渡层选自镍层、铬层和钛层中的一种或几种。
所述复合金属膜层的作用为：由于该复合金属膜层沉积在多孔硅薄膜之上，因而不会影响多孔硅薄膜的纳米孔结构。当入射光照在金膜层时，金膜的纳米孔结构使得金膜层表面会有局部等离子体共振(Localized Surface Plasmon Resonance,LSPR)发生，LSPR在金膜层表面会有很强的电场能量或电场强度的聚集，使生物分子在金膜层表面的结合能与电场发生强相互作用，从而使反射谱发生较大变化，因此可提高本申请的光学生物传感器的灵敏度。然而，金膜层本身具有不稳定性，易于从纳米多孔硅薄膜光学器件表面脱落，因此，本申请在纳米多孔硅薄膜光学器件与金膜层之间增设了过渡层，该过渡层选自镍层、铬层或钛层，过渡层可增强纳米多孔硅薄膜光学器件与金膜层之间的结合力，提高金膜层在纳米多孔硅薄膜光学器件表面的稳定性，提高光学生物传感器的稳定性。
更重要的是，复合金属薄膜的增设还解决了大颗粒病毒(例如SARS-CoV-2病毒)检测中的技术困难。一般来说，基于纳米多孔材料的光学生物传感器的检测需要病原体进入其纳米孔中，才可保证较好的检测灵敏度。然而，大颗粒病毒(例如SARS-CoV-2毒颗粒较大，直径为100～150纳米)无法进入小孔径的纳米孔内；同时，纳米孔孔径的增大亦可增强光散射作用、降低光学生物传感器的灵敏度，因而，为了使大颗粒病毒能进入纳米孔内而盲目扩大多孔硅纳米孔的孔径也是不明智的做法。本申请通过复合金属薄膜的增设，使得在金膜层表面产生局部等离子体共振(LSPR)，显著提高检测灵敏度。因而，大颗粒病毒不需要进入多孔硅的纳米孔内，即使在多孔硅表面的金属层附近结合也会有较高的敏感度，可显著提高SARS-CoV-2 病毒的检测灵敏度。
进一步地，所述复合金属薄膜中的过渡层的厚度为3～8纳米，优选5纳米；金膜层的厚度为10～20纳米，优选15纳米。复合金属薄膜中，过渡层和金膜层的厚度影响作用如下：首先，过渡层太薄不能稳定有效地将金膜附着在多孔硅薄膜之上，过渡层太厚则会削弱其下布拉格反射镜反射回来的光(因金属会吸收电磁波的能量，光也是一种电磁波)，该反射回来的光能量本来可以进一步增强金膜表面激发的LSPR，因此太厚会影响传感器的灵敏度。其次，金膜太薄难以有效激发LSPR，也难以有效支撑生物探针以共价键方式固化在传感器表面；金膜太厚会削弱其下布拉格反射镜反射回来的光(因金属会吸收电磁波的能量，光也是一种电磁波)，该反射回来的光能量本来可以进一步增强金膜表面激发的LSPR，因此太厚会影响传感器的灵敏度。当过渡层和金膜层的厚度在本申请所提供的范围内时，传感器表面的金膜和金膜之上固化的生物探针都比较稳定，不会因检测操作中的移动或表面冲洗等造成金膜或者生物探针连同金膜一起脱落，同时金膜表面的激发的LSPR模式的电场强度最强，传感器的灵敏度最高。
本申请还提供一种病原体的自动化检测方法，包括如下步骤：
(1)生物传感器包被生物探针：第一机械臂将生物探针液体样品加在生物传感器表面，第二机械臂保持所述生物探针液体样品在所述生物传感器表面反应0.5～2小时；第一机械臂从所述生物传感器表面吸走所述生物探针液体样品，并使用缓冲液清洗所述生物传感器表面；
(2)生物传感器进行第一次光学检测：第一机械臂向所述生物传感器表面加入缓冲液，将盖玻片盖在所述传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第一次光学检测；光纤光谱仪采集第一光学反射谱数据；
(3)临床样本加样：第一机械臂将临床液体样本加入所述生物传感器 表面，第二机械臂保持所述临床液体样本在所述生物传感器表面反应5～20分钟；第一机械臂从所述生物传感器表面吸走所述临床液体样本，并使用缓冲液清洗所述生物传感器表面；
(4)生物传感器进行第二次光学检测：第一机械臂向所述生物传感器表面加入缓冲液，并将盖玻片盖在所述传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第二次光学检测；光纤光谱仪采集第二光学反射谱数据；
(5)光学反射谱数据分析：将所述第一光学反射谱数据和所述第二光学反射谱数据进行比对，根据反射谱特征峰和谷的红移量对病原体进行定量。
优选地，所述生物探针包括抗体、结合蛋白或核酸适配体；更优选地，所述生物探针为SARS-CoV-2病毒抗体。
相对于现有技术而言，本申请提供了一种病原体的自动化检测装置和自动检测方法，包括基于机械臂自动控制样品加入生物传感器，生物传感器光学反射谱自动检测，以及结果的分析和输出。整个检测流程一键点击后全自动完成，无需人工干预，可用于新冠病毒等病原体的快速现场自动检测，不但可避免人员检测操作带来的失误、交叉感染等风险，且提高了检测通量，能够快速给出结果，避免大量人员长时间等待结果而产生交叉感染。
同时，本申请所提供的病原体的自动化检测装置中，还使用了基于纳米材料-多孔硅的光学生物传感器，该传感器元件的光学反射谱具有特征峰和特征谷。当病原体在传感器表面结合后，改变传感器表面附近的光学折射率(折射率增大)，使传感器的反射谱特征峰和谷向长波长方向移动(红移)，该红移的量可以用来对病原体进行定量。为保证生物传感器的特异性，在传感器表面包被抗体、适配体或者ACE2结合蛋白，即可特异性捕获特定的病原体(如SARS-CoV-2病毒)，而不会结合其他生物物质；该捕获事件反应 出来的生物传感器的光学信号变化即可特异性的指向特定的病原体。
更重要的是，本申请还通过对纳米多孔硅薄膜光学器件具体结构的设计、以及在纳米多孔硅薄膜光学器件表面沉积复合金属薄膜，从而使得本申请的光学生物传感器具有极高的检测灵敏度。当使用本申请的光学生物传感器对SARS-CoV-2病毒进行检测时，虽然新冠病毒颗粒较大(100～150纳米直径)，难以进入多孔硅的纳米孔内部，仍然可以通过在多孔硅表面的金属层附近结合获得较高的检测敏感度。
附图说明
图1为基于器件1的病原体自动化检测装置检测SARS-CoV-2病毒的S-ECD蛋白的检测光谱图；
图2为基于器件2的病原体自动化检测装置检测SARS-CoV-2病毒的S-ECD蛋白的检测光谱图；
图3为基于器件7的病原体自动化检测装置检测SARS-CoV-2病毒的S-ECD蛋白的检测光谱图。
具体实施方式
为了能够更清楚理解本申请的目的、特点和优势，下面结合附图对本申请的实施方式进行详细描述。所用材料未注明生产厂商者，均为可以通过市售购买获得的常规产品。对示例性实施方式的描述仅仅是出于示范目的，而非对本申请及其应用的限制。
一、光学生物传感器的制备
本申请的第一实施方式涉及光学生物传感器的制备：
(1)制备纳米多孔硅薄膜光学器件:
使用阳极氧化法(按照本领域的常规步骤)在单晶硅片表面制备纳米多孔硅薄膜光学器件。所述纳米多孔硅薄膜光学器件有三种结构：多孔硅单层干涉膜、多孔硅布拉格反射镜或多孔硅微谐振腔。
纳米多孔硅薄膜光学器件中的纳米多孔硅薄膜的制备，可以采用本领域内的常规方法进行。本实施例中以阳极氧化法为例，简述步骤如下：
使用P型硼掺杂(或者N型磷/砷/锑掺杂)、0.01欧姆厘米电阻率、<100>晶向的硅片，在15％氢氟酸(N型用5.5％)中进行阳极氧化法电化学腐蚀，整个腐蚀槽由聚四氟乙烯(特氟龙)制作，以容纳氢氟酸溶液。硅片浸泡在氢氟酸中，硅片背面为阳极，可与铂或铝片接触；另用铂金电极作为阴极也浸泡在氢氟酸中，但不与硅片正面接触。通过恒定电流源，可在阳极和阴极之间施加恒定的电流，即可在硅片上腐蚀生成多孔硅。多孔硅的多孔率，也就是折射率，可由单位硅面积上施加的电流密度来控制；多孔硅的厚度可由腐蚀时间来控制。多孔硅的多层结构，包含每一层对应的厚度和折射率，可通过切换每一层所需要的电流密度和腐蚀时间连续腐蚀形成多层多孔硅中的每一层。
(2)对纳米多孔硅薄膜光学器件进行热氧化或者热碳化：
热氧化方法如下：在700～800℃下氧化10～30分钟，即可在多孔硅的孔内表面形成一层1～2纳米厚的二氧化硅覆盖层。
碳化方法如下：采用低压化学气相沉积法对纳米多孔硅薄膜光学器件表面进行碳化。首先，将制备好的多孔硅样品放入2.5％的HF溶液中浸泡90s，用乙醇溶液对浸泡好的多孔硅样品进行多次冲洗，用N 2吹干清洗干净的多孔硅样品。然后，将吹干的多孔硅样品立即放入管式炉的石英管中，多孔硅样品位于管式炉加热中心处。接着，将石英管内的气压抽至真空，并将炉温升至650℃，期间通入20sccm的H 2和1sccm的Ar对多孔硅片进行退火处理，温度到达650℃时通入1sccm的C 2H 2并将温度缓慢升至750℃，保温 10分钟，再将温度缓慢升至850℃，保温10分钟后关闭C 2H 2，保持H 2和Ar流量不变，开始降温，待炉温冷却至室温后将多孔硅样品从炉中取出，得到表面被碳化的多孔硅。
(3)在纳米多孔硅薄膜光学器件上沉积复合金属薄膜：
在纳米多孔硅薄膜光学器件上采用物理气相沉积(PVD)法或磁控溅射法依次沉积过渡层和金膜层。
以物理气相沉积法为例，可采用电阻式真空热蒸镀设备(例如：购自沈阳科诚真空技术有限公司的ZD-400单室高真空电阻式蒸镀设备)进行膜层沉积，沉积步骤按照设备使用说明书进行。
(4)在纳米多孔硅薄膜光学器件表面固化生物探针：
在纳米多孔硅薄膜光学器件表面固化生物探针，这些生物探针包括抗体、结合蛋白、适配体等。例如，在传感器表面通过共价键、静电吸附、物理吸附等方法固化冠状病毒抗体、适配体或者ACE2结合蛋白，则使生物传感器具备针对冠状病毒的特异性。
以在金的表面固化抗体为例，在纳米多孔硅薄膜光学器件表面固化生物探针的步骤如下：
首先羧基(-COOH)化金表面，后使用EDC/NHS激活羧基表面形成氨基(-NH 2)，形成的氨基与单克隆抗体Fc端的羧基(-COOH)高特异性结合，从而固定抗体于传感器表面。添加牛血清白蛋白(BSA)等覆盖未结合探针的表面，防止非特异性吸附的发生。同理，获得抗体固定的多孔硅传感器，优化后，期待可于4摄氏度低温环境保存一个月，用于现场新冠病毒的快速检测。
以在金表面固化核酸适配体为例，在纳米多孔硅薄膜光学器件表面固化生物探针的步骤如下：
核酸适配体的5’端修饰的巯基(SH-)可以与金表面直接结合。固定适配体前，添加还原剂TCEP(三(2-羧乙基)膦盐酸盐)使巯基处于活性状态，而不相互交联形成双硫键(S-S)。巯基与金形成Au-S键而固定适配体至传感器表面，后添加6-巯基-1-己醇等覆盖未结合探针的表面，防止非特异性吸附的发生。获得适配体固定的多孔硅生物传感器，优化后，期待可于4摄氏度低温环境保存一个月，用于现场新冠病毒的快速检测。
二、光学生物传感器器件
本申请的第二实施方式涉及光学生物传感器的结构：
器件1(P型单晶硅基底+多孔硅单层干涉膜+复合金属膜+生物探针)
器件1包括P型硼掺杂单晶硅及设于所述P型硼掺杂单晶硅上的纳米多孔硅薄膜光学器件，所述纳米多孔硅薄膜光学器件为多孔硅单层干涉膜。所述多孔硅单层干涉膜的厚度为1微米，纳米孔的孔径为20-30纳米。所述纳米多孔硅薄膜光学器件的表面经热氧化，并依次沉积有复合金属薄膜、包被有生物探针。所述复合金属薄膜包括5纳米镍膜层和15纳米金膜层，所述生物探针为SARS-COV-2抗体(购自义翘神州)。
器件2(P型单晶硅基底+多孔硅布拉格反射镜+复合金属膜+生物探针)
器件2包括P型硼掺杂单晶硅及设于所述P型硼掺杂单晶硅上的纳米多孔硅薄膜光学器件，所述纳米多孔硅薄膜光学器件为多孔硅布拉格反射镜。所述纳米多孔硅薄膜光学器的表面经热氧化，并依次沉积有复合金属薄膜、包被有生物探针。所述复合金属薄膜包括5纳米镍膜层和15纳米金膜层，所述生物探针为SARS-COV-2抗体(购自义翘神州)。
其中，所述多孔硅布拉格反射镜包括交替堆叠的高折射率层和低折射率层。其中，所述高折射率层和低折射率层满足：n 1d 1＝n 2d 2＝λ/4；其中，n 1和d 1分别为高折射率层的折射率和厚度；n 2和d 2分别为低折射率层的折射 率和厚度；λ为多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。
在本器件示例中，多孔硅布拉格反射镜中的高折射率层和低折射率层的个数均为10个。n 1＝2.08；d 1＝100纳米；n 2＝1.38；d 2＝150纳米；λ＝830纳米。
器件3～10(P型单晶硅基底+多孔硅微谐振腔+复合金属膜+生物探针)
器件3～10包括P型硼掺杂单晶硅及设于所述P型硼掺杂单晶硅上的纳米多孔硅薄膜光学器件，所述纳米多孔硅薄膜光学器件为多孔硅微谐振腔。所述纳米多孔硅薄膜光学器的表面经热氧化，并依次沉积有复合金属薄膜、包被有生物探针，所述生物探针为SARS-COV-2抗体(购自义翘神州)。
其中，所述多孔硅微谐振腔包括：第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜及设于所述第一多孔硅布拉格反射镜和第二多孔硅布拉格反射镜之间的多孔硅薄膜缺陷层；所述第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜各自独立地包括交替堆叠的高折射率层和低折射率层。
所述高折射率层和低折射率层满足：n 1d 1＝n 2d 2＝λ/4；其中，n 1和d 1分别为高折射率层的折射率和厚度；n 2和d 2分别为低折射率层的折射率和厚度；λ为第一或第二多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。
所述多孔硅薄膜缺陷层满足：n 3d 3＝Nλ/4，其中，n 3和d 3分别为多孔硅薄膜缺陷层的折射率和厚度，其中N>0且N≠1，n 3可以是硅折射率(3.5)和空气折射率(1.0)之间的任意值，λ为第一或第二多孔硅布拉格反射镜的全反射带的中心波长(真空中波长)。
器件3～10中，多孔硅薄膜层的参数如下：n 1＝2.08；d 1＝100纳米；n 2＝1.38；d 2＝150纳米；n 3＝n 2,d 3＝300纳米；λ＝830纳米。此外，器件3～10中，第一多孔硅布拉格反射镜中的高折射率层和低折射率层的个数(表1中以“第一层数”表示)、第二多孔硅布拉格反射镜中的高折射率层和低折射率 层的个数(表1中以“第二层数”表示)、复合金属膜层中的过渡层的厚度、复合金属膜层中的金膜层的厚度，如表1所示。
表1
  第一层数 第二层数 过渡层厚度 金膜层厚度
器件3 5 5 5纳米 15纳米
器件4 5 10 5纳米 15纳米
器件5 7 10 5纳米 15纳米
器件6 7 5 5纳米 15纳米
器件7(最优实施例) 6 10 5纳米 15纳米
器件8 6 10 5纳米 5纳米
器件9 6 10 5纳米 25纳米
器件10 6 12 10纳米 15纳米
对比器件1
对比器件1的结构与器件1基本相同，不同之处仅在于，对比器件1的表面未沉积复合金属薄膜。
对比器件2
对比器件2的结构与器件2基本相同，不同之处仅在于，对比器件2的表面未沉积复合金属薄膜。
对比器件3
对比器件3的结构与器件7基本相同，不同之处在于，对比器件3的表面未沉积复合金属薄膜。
三、病原体的自动化检测装置
本申请的第三实施方式涉及病原体的自动化检测装置：
病原体的自动化检测装置其包括控制系统、第一机械臂、第二机械臂、生物传感器和光纤光谱仪，其中：所述生物传感器设于所述第二机械臂上；所述生物传感器为第二实施方式中的各种光学生物传感器。
在所述控制系统的控制下，所述第一机械臂、所述第二机械臂、生物 传感器和所述光纤光谱仪完成下述检测操作：第一机械臂：将液体样品加入所述生物传感器表面，并于所述液体样品在所述生物传感器表面反应之后清洗所述传感器表面；第二机械臂：将所述生物传感器表面对准所述光纤光谱仪的光纤探头，进行光学检测；光纤光谱仪：检测并采集生物传感器的光学反射谱数据。
四、病原体的自动化检测方法
本申请的第四实施方式涉及病原体的自动化检测方法：
(1)生物传感器包被生物探针：
第一机械臂将生物探针液体样品(生物探针可以为抗体、结合蛋白或核酸适配体；例如：Aptamer溶液、ACE2溶液等)加在生物传感器表面的微井中，第二机械臂保持所述生物探针液体样品在所述生物传感器表面反应0.5～2小时；第一机械臂从所述生物传感器表面吸走所述生物探针液体样品，并使用缓冲液清洗所述生物传感器表面；
(2)生物传感器进行第一次光学检测：
第一机械臂向所述生物传感器表面加入缓冲液，将盖玻片盖在所述传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第一次光学检测；光纤光谱仪采集第一光学反射谱数据；
(3)临床样本加样：
第一机械臂将临床液体样本(例如可以为痰液、唾液、咽拭子采样液、支气管肺泡灌洗液等)加入所述生物传感器表面，第二机械臂保持所述临床液体样本在所述生物传感器表面反应5～20分钟；第一机械臂从所述生物传感器表面吸走所述临床液体样本，并使用缓冲液清洗所述生物传感器表面；
(4)生物传感器进行第二次光学检测：
第一机械臂向所述生物传感器表面加入缓冲液，并将盖玻片盖在所述 传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第二次光学检测；光纤光谱仪采集第二光学反射谱数据；
(5)光学反射谱数据分析：
将所述第一光学反射谱数据和所述第二光学反射谱数据进行比对，根据反射谱特征峰和谷的红移量对病原体进行定量。
五、对SARS-CoV-2病毒的自动化检测示例
本申请的第四实施方式涉及对SARS-CoV-2病毒的自动化检测示例。
(1)生物传感器包被生物探针：
第一机械臂将20微升生物探针Aptamer溶液加入生物传感器的微井中，每个微井中的Aptamer溶液浓度可能不同，保持1小时；第二机械臂在1小时内缓慢振动样品，以促进表面均匀反应；第一机械臂吸掉微井中的Aptamer溶液。
第一机械臂加PBS缓冲液至微井中，吸入移液管内，再推入微井中，如此反复操作三次，以最大限度吸走未在传感器表面反应的Aptamer；第一机械臂加新的PBS缓冲液至微井中，将上面的清洗步骤再操作两次，总计清洗流程为三次。
(2)生物传感器进行第一次光学检测：
第一机械臂加新的PBS缓冲液至微井中，自动吸盘将盖玻片盖在传感器表面，覆盖所有的微井；第二机械臂将传感器的微井编号为1至N，依次对准光纤光谱仪的光纤探头，并在每个微井对准后自动采集该微井的反射光谱数据，为第一光学反射谱数据；自动吸盘将盖玻片取走，将微井中的PBS缓冲液吸掉。
(3)临床样本加样：
第一机械臂将20微升咽拭子采样液加入生物传感器表面的微井中， 另有两个微井分别加阴性样本和阳性样本(阴性样本和阳性样本的具体步骤省略)。第二机械臂保持或振动生物传感器10分钟，以促进生物传感器表面均匀反应；第一机械臂从所述生物传感器表面吸走咽拭子采样液；
第一机械臂加PBS缓冲液至生物传感器表面的微井中，吸入移液管内，再推入微井中，如此反复操作三次，以最大限度吸走未在传感器表面反应的生物物质；第一机械臂加新的PBS缓冲液至微井中，将上面的清洗步骤再操作两次，总计清洗流程为三次。
(4)生物传感器进行第二次光学检测：
加新的PBS缓冲液至微井中，自动吸盘将盖玻片盖在传感器表面，覆盖所有的微井；第二机械臂将传感器的微井编号为1至N，依次对准光纤光谱仪的光纤探头，并在每个微井对准后自动采集该微井的反射光谱数据，为第二光学反射谱数据；自动吸盘将盖玻片取走，将微井中的PBS缓冲液吸掉。
(5)光学反射谱数据分析：
将所述第一光学反射谱数据和所述第二光学反射谱数据进行比对，根据反射谱特征峰和谷的红移量对病原体进行定量。
六、SARS-CoV-2病毒表面的S-ECD蛋白检测结果：
S-ECD蛋白是SARS-CoV-2病毒表面的S蛋白，包含S1、S2蛋白，是病毒攻击与结合人体细胞的ACE2结合蛋白的特异性功能区。以下所示为采用本申请的自动化检测装置和自动化检测方法(装置中分别使用器件1～器件10、对比器件1～对比器件3)，检测SARS-CoV-2病毒表面的S-ECD蛋白的检测效果，以示出本申请所提供的自动化检测装置及检测方法对SARS-CoV-2病毒的检测灵敏度。
(1)使用器件1、对比器件1的装置的检测效果：
器件1可对浓度为1nM(1nM＝10 -9mol/L)的S-ECD蛋白有效检测：1nM浓度的S-ECD蛋白在传感器表面与抗体的结合会带来6纳米左右的特征峰或特征谷的红移(检测光谱图如附图1所示)。但是，当S-ECD蛋白浓度低于1pM时(1pM＝10 -12mol/L)，特征峰或特征谷的红移不明显，可出现假阴性结果。
对比器件1无法对浓度为1nM的S-ECD蛋白进行有效检测，特征峰或特征谷的红移不明显。
(2)使用器件2、对比器件2的装置的检测效果：
器件2可对浓度为0.5nM的S-ECD蛋白有效检测：0.5nM浓度的S-ECD蛋白在传感器表面与抗体的结合会带来5纳米左右的特征峰或特征谷的红移(检测光谱图如附图2所示)。但是，当S-ECD蛋白浓度低于10fM(1fM＝10 -15mol/L)时，特征峰或特征谷的红移不明显，可出现假阴性结果。
(注：图2中，在S-ECD蛋白结合前的特征谷位置是615纳米，而不是DBR设计全反射带中心波长(真空中波长)的830纳米，这是由于多孔硅布拉格反射镜经过热氧化后，很多硅材料转变为二氧化硅，折射率大幅下降(从硅折射率3.5左右变为二氧化硅折射率1.5左右)，使得多孔硅布拉格反射镜的全反射带整体发生很大的蓝移。并且在布拉格反射镜器件表面镀复合金属膜后，布拉格反射镜的全反射带(从565纳米至615纳米)转变为一个特征峰(565纳米)和一个特征谷(615纳米)。
对比器件2无法对浓度为0.5nM的S-ECD蛋白进行有效检测，特征峰或特征谷的红移不明显。
(3)使用器件3～10、对比器件3的装置的检测效果：
器件3～10可对浓度为1fM～1nM范围内的低浓度S-ECD蛋白有效检测。对比器件3无法对浓度1fM～1nM范围内的低浓度S-ECD蛋白进行有效 检测，特征峰或特征谷的红移不明显。
其中，器件7表现出极高检测灵敏度，0.5nM浓度的S-ECD蛋白在器件7表面与抗体的结合会带来10纳米左右的特征峰或特征谷的红移(检测光谱图如附图3所示)。这是因为，器件7通过在多孔硅微谐振腔的基础上，通过复合金属膜层的设置，在金膜层表面产生局部等离子体共振、提高光学生物传感器的稳定性，使得颗粒较大的SARS-CoV-2病毒不需要进入多孔硅的纳米孔内，在复合金属膜层附近与抗体结合也有较高的检测灵敏度。
将器件7和器件2的反射谱进行对比后可以发现，器件7位于635.14纳米的谐振谷的品质因数(Quality Factor，定义为谐振中心波长除以半峰宽)在100左右，而器件2的位于615纳米的特征谷的品质因数在10左右，比器件7低了一个数量级。因此，器件7在检测低含量的目标生物物质时能够辨识很小的红移，从而具有较低的检测限(Limit of Detection)。从检测限对比来看，器件7检测限可达1fM，而器件2检测限为10fM。
除此之外，器件7还进一步在结构上进行了优化。
一方面，器件7中，第一多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个；第二多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个或6个以上(器件7中为10个)。申请人发现，上述结构设计使得传感器在检测低浓度病毒时，在可见光波长范围内的反射谱特征峰和特征谷红移最大，对应的特征峰和特征谷最为尖锐、品质因数最高，对于微小红移的检测能力最强，检测限较低，相应的传感器灵敏度也最高。
相比之下，器件3～器件6的检测限不如器件7。其中：器件3的检测限为100fM，而器件4-6的检测限均为10fM。
另一方面，器件7中，复合金属薄膜中的过渡层的厚度在3～8纳米之间；使得金膜层的厚度在10～20纳米之间，进一步减小检测过程中发生的光 被金属吸收的作用，有利于提高金表面LSPR场强，并在检测过程中获得尖锐的特征峰，提高光学生物传感器的检测灵敏度。
相比之下，器件8～10的检测灵敏度低于器件7。其中：器件8-10的检测限均为10fM。
综上所述，本申请所提供的自动化检测装置和检测方法，不但提高了检测通量，还可避免人员检测操作带来的失误和交叉感染等风险，并且由于使用了基于纳米材料-多孔硅的光学生物传感器，具有较高的检测灵敏度。
上述实例只为说明本申请的技术构思及特点，其目的在于让熟悉此项技术的人员能够了解本申请的内容并据以实施，并不能以此限制本申请的保护范围。凡根据本申请精神实质所做的等效变换或修饰，都应涵盖在本申请的保护范围之内。

Claims (10)

  1. 一种病原体的自动化检测装置，其特征在于，包括控制系统、第一机械臂、第二机械臂、生物传感器和光纤光谱仪，其中：
    所述生物传感器设于所述第二机械臂上；所述生物传感器包括单晶硅基底及设于所述单晶硅基底上的纳米多孔硅薄膜光学器件，所述纳米多孔硅薄膜光学器件包括多孔硅单层干涉膜、多孔硅布拉格反射镜或多孔硅微谐振腔；所述纳米多孔硅薄膜光学器件的表面沉积有复合金属薄膜；
    所述控制系统控制所述第一机械臂、所述第二机械臂、所述生物传感器和所述光纤光谱仪完成下述检测操作：
    第一机械臂：将液体样品加入所述生物传感器表面，并于所述液体样品在所述生物传感器表面反应之后清洗所述传感器表面；
    第二机械臂：将所述生物传感器表面对准所述光纤光谱仪的光纤探头，进行光学检测；
    光纤光谱仪：检测并采集生物传感器的光学反射谱数据。
  2. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，所述生物传感器包括至少4个传感器单元，所述传感器单元采用微井结构，在各微井结构间设有聚酰亚胺隔离层。
  3. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，所述多孔硅布拉格反射镜包括交替堆叠的高折射率层和低折射率层；优选地，所述高折射率层和所述低折射率层的个数相等且分别为10个以上。
  4. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，所述多孔硅微谐振腔包括：第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜及设于所述第一多孔硅布拉格反射镜和第二多孔硅布拉格反射镜之间的多 孔硅薄膜缺陷层；所述第一多孔硅布拉格反射镜、第二多孔硅布拉格反射镜各自独立地包括交替堆叠的高折射率层和低折射率层；
    优选地，所述第一多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个；所述第二多孔硅布拉格反射镜中的高折射率层和低折射率层的个数相等且分别为6个或6个以上。
  5. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，
    所述单晶硅基底选自N型磷掺杂单晶硅、N型砷掺杂单晶硅、N型锑掺杂单晶硅或P型硼掺杂单晶硅；优选为P型硼掺杂单晶硅。
  6. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，所述多孔硅单层干涉膜、多孔硅布拉格反射镜或多孔硅微谐振腔中的多孔硅纳米孔的孔径为1～100纳米；优选20～30纳米。
  7. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，所述纳米多孔硅薄膜光学器件的表面在沉积复合金属薄膜之前被热氧化或碳化。
  8. 根据权利要求1所述的病原体的自动化检测装置，其特征在于，
    所述复合金属薄膜包括设于所述纳米多孔硅薄膜光学器件表面的过渡层和设于所述过渡层表面的金膜层；
    所述过渡层选自镍层、铬层或钛层中的一种或几种；
    所述过渡层的厚度为3～8纳米，优选5纳米；所述金膜层的厚度为10～20纳米，优选15纳米。
  9. 一种病原体的自动化检测方法，其特征在于，包括如下步骤：
    (1)生物传感器包被生物探针：
    第一机械臂将生物探针液体样品加到生物传感器表面，第二机械臂保 持所述生物探针液体样品在所述生物传感器表面反应0.5～2小时；第一机械臂从所述生物传感器表面吸走所述生物探针液体样品，并使用缓冲液清洗所述生物传感器表面；
    (2)生物传感器进行第一次光学检测：
    第一机械臂向所述生物传感器表面加入缓冲液，将盖玻片盖在所述传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第一次光学检测；光纤光谱仪采集第一光学反射谱数据；
    (3)临床样本加样：
    第一机械臂将临床液体样本加入所述生物传感器表面，第二机械臂保持所述临床液体样本在所述生物传感器表面反应5～20分钟；第一机械臂从所述生物传感器表面吸走所述临床液体样本，并使用缓冲液清洗所述生物传感器表面；
    (4)生物传感器进行第二次光学检测：
    第一机械臂向所述生物传感器表面加入缓冲液，并将盖玻片盖在所述传感器表面；第二机械臂将所述生物传感器对准光纤光谱仪的光纤探头，进行第二次光学检测；光纤光谱仪采集第二光学反射谱数据；
    (5)光学反射谱数据分析：
    将所述第一光学反射谱数据和所述第二光学反射谱数据进行比对，根据反射谱特征峰和谷的红移量对病原体进行定量。
  10. 根据权利要求9所述的病原体的自动化检测方法，其特征在于，所述生物探针包括抗体、结合蛋白或核酸适配体；优选地，所述生物探针为SARS-CoV-2病毒抗体。
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