Optical biochip modified at specific sites, preparation method thereof, optical biodetection system and optical biodetection method

Abstract

The present disclosure provides an optical biochip modified at a specific site, a preparation method thereof, an optical biodetection system and an optical biodetection method. The optical biochip includes, from bottom to top, an optical microcavity; an intermediate connection layer formed at a specific site of the optical microcavity, the specific site being located in a resonant or resonant region on the surface of the optical microcavity; and a bioreceptor layer fixed on the intermediate connection layer, which can specifically bind to target molecules in a biological sample solution; the optical microcavity can convert the perturbation of the evanescent field on the surface of the optical microcavity caused by a specific binding event into a physical signal that can be read. The specific point is precisely positioned through a photolithography process, and an intermediate connection layer is arranged at the specific site, and a bioreceptor layer for detecting target molecules is fixed on the intermediate connection layer, thereby achieving precise modification of specific sites on the surface of the biological device, and effectively improving the detection limit and sensitivity of the optical biochip.

Description

Technical Field

[0001] The present application relates to the field of optical biosensing technology, and more specifically, to a site-specific modified optical biochip, a preparation method thereof, an optical biodetection system and an optical biodetection method. Background Art

[0002] A biosensor is a type of device that can detect and quantify the type or quantity of specific biological molecules or compounds in biological samples. Its structural composition can be divided into three parts: a bioreceptor layer (or bioreceptor), a transducer, and a signal processing unit. The bioreceptor layer, that is, the biologically sensitive material fixed on the surface of the sensor, is used to specifically identify target molecules in biological samples or interact specifically with target molecules. Its type determines the type of analyte detected by the biochip. The transducer in the biosensor converts molecular recognition or biomolecular binding events into readable and quantifiable physical signals, such as electricity, light, heat, sound, etc. The signal processing unit detects the physical signal generated by the transducer, thereby realizing the identification and detection of biological molecules or compounds.

[0003] Optical biochips are a type of biosensor that uses optical devices as transducers. They can detect the mutual recognition and binding between biomolecules under label-free and non-destructive conditions, and show extremely high sensitivity. The basic detection principle of this optical biochip relies on the evanescent wave mechanism, which is a wave whose intensity decays exponentially by penetrating the external medium on the surface of the optical sensor. The interaction between the bioreceptor layer on the surface of the optical device and specific molecules will cause the effective refractive index of the surface of the optical device to change, thereby affecting the characteristics of the evanescent wave. Since the intensity of the evanescent field decays exponentially, it only exists at the minimum distance from the sensor surface. Therefore, in order to enhance the detection sensitivity of the biochip, it is necessary to optimize the structure of the sensing surface of the optical device and concentrate the perturbation of the evanescent field caused by the specific recognition and interaction of biomolecules in the resonant cavity of the optical device. However, in the design of current optical biochips, the resonant cavity of the optical device and the waveguide part coupled to it are usually modified with the bioreceptor layer indiscriminately. This indiscriminate modification method may lead to a more dispersed distribution of binding events between target molecules and biological receptors, making it impossible to achieve a highly concentrated detection area on a high-sensitivity sensing surface, thereby limiting the detection limit and sensitivity improvement of the optical biochip. Summary of the invention

[0004] To overcome the above-mentioned problems, the present disclosure provides an optical biochip modified at a specific site, a preparation method thereof, an optical biodetection system and an optical biodetection method, which accurately locates specific points on the surface of an optical device through a photolithography process, arranges an intermediate connecting layer at the specific site, and fixes a biological receptor layer for detecting target molecules on the intermediate connecting layer, thereby achieving accurate modification of specific sites on the surface of the biological device, effectively improving the detection limit and sensitivity of the optical biochip.

[0005] In the first scheme of the present disclosure, a specific site modified optical biochip is provided. The optical biochip comprises, from bottom to top, an optical microcavity; an intermediate connection layer formed at a specific site of the optical microcavity, the specific site being located in a resonant or resonance region on the surface of the optical microcavity; and a bioreceptor layer fixed on the intermediate connection layer and configured to be able to specifically bind to a target molecule in a biological sample solution to be detected; wherein the optical microcavity is configured to be able to convert a perturbation of an evanescent field on the surface of the optical microcavity caused by a specific binding event into a physical signal that can be read.

[0006] In the second scheme of the present disclosure, a method for preparing an optical biochip according to the first scheme is provided. The preparation method comprises the following steps: step 1, forming a window in a photoresist layer coated on the surface of the optical microcavity by a photolithography process, wherein the window is aligned with the specific site to expose the specific site; step 2, forming the intermediate connection layer at the exposed specific site in the window, so that the intermediate connection layer is bound to the optical microcavity surface at the specific site; and step 3, forming the biological receptor layer capable of specifically binding to the target molecule on the intermediate connection layer.

[0007] In the third embodiment of the present disclosure, an optical bio-detection system is provided. The optical bio-chip comprises: at least one optical bio-chip according to the first embodiment; and a microfluidic chip, which is located above the optical bio-chip and bonded thereto, wherein at least one microfluidic channel of the microfluidic chip provides a flow channel for a biological sample solution containing the target molecule to be detected; wherein at least a portion of each of the microfluidic channels overlaps with the specific site of the corresponding optical bio-chip.

[0008] In a fourth scheme of the present disclosure, there is provided an optical bioassay method performed by the optical bioassay system of the third scheme, wherein the optical bioassay system comprises an optical biochip array composed of a plurality of the optical biochips, and is characterized in that the optical bioassay method comprises: measuring the initial resonant wavelength of the optical microcavity; introducing buffer solutions of standard target molecules of known concentrations from low to high into a plurality of the microfluidic channels, washing with a phosphate buffer solution after incubation, and recording the adjustment resonant wavelength at the corresponding concentration; calculating the adjustment resonant wavelength offset value at the corresponding concentration based on the adjustment resonant wavelength and the initial resonant wavelength, and obtaining a resonant wavelength offset-concentration curve; introducing the biological sample solution containing the target molecule to be detected into any one of the remaining microfluidic channels of the optical biochip array, and recording the test resonant wavelength of the optical microcavity; calculating the test resonant wavelength offset value corresponding to the biological sample solution based on the test resonant wavelength and the initial resonant wavelength; and obtaining the concentration of the target molecule in the biological sample solution based on the resonant wavelength offset-concentration curve and the test resonant wavelength offset value.

[0009] Compared with the prior art, the optical biochip modified at specific sites, the preparation method thereof, the optical biodetection system and the optical biodetection method provided by the present disclosure can achieve the fixation of bioreceptors at characteristic sites on the optical microcavity sensor surface through photolithography and vacuum deposition processes, and can accurately fix the bioreceptors at the most sensitive area of ​​the sensor surface. The sensitivity of the optical biochip is effectively improved, and the minimum concentration of biomolecule detection can also be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. When appropriate, the same reference numerals are used in all drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present device or method. In the drawings:

[0011] Figure 1 is a side view of an optical biochip according to an embodiment of the present disclosure;

[0012] Figure 2 is a schematic flow chart of a method for preparing an optical biochip according to an embodiment of the present disclosure;

[0013] Figure 3 A schematic diagram of preparing an optical biochip including a microring resonator according to an embodiment of the present disclosure;

[0014] Figure 4 A bioreceptor immobilization scheme on the surface of an optical biochip according to an embodiment of the present disclosure;

[0015] Figure 5 is a schematic structural diagram of a microring resonator array according to another embodiment of the present disclosure;

[0016] Figure 6 is a schematic structural diagram of an optical biological detection system (including a microfluidic chip and a microring resonator array bonded to each other) according to another embodiment of the present disclosure;

[0017] Figure 7 is a schematic flow chart of an optical biological detection method according to another embodiment of the present disclosure;

[0018] Figure 8 is a schematic diagram of the resonance wavelength shift of the microring resonator after the bioreceptor according to the embodiment of the present disclosure binds to the target molecule;

[0019] Fig. 9 is the resonant wavelength shift Δλ according to the embodiment of the present disclosure s -Concentration calibration working curve diagram. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiment of the present application clearer, the technical solution of the embodiment of the present application will be clearly and completely described in conjunction with the drawings of the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, not all of the embodiments. Based on the described embodiment of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0021] Unless otherwise defined, the technical terms or scientific terms used in this application should be understood by people with ordinary skills in the field to which this application belongs. "First", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0022] In order to keep the following description of the embodiments of the present application clear and concise, the present application omits detailed descriptions of known functions and known components.

[0023] Figure 1 FIG. 4 is a side view of an optical biochip according to an embodiment of the present disclosure. Figure 1 As shown, the optical biochip 100 includes a wafer 140, an optical microcavity 110, an intermediate connection layer 120 and a bioreceptor layer 130 from top to bottom. The optical microcavity 110 is formed on the wafer 140 as a substrate; the intermediate connection layer 120 is formed at a specific site 111 of the optical microcavity 110, and the specific site 111 is located in the resonance or resonant region of the surface of the optical microcavity 110; the bioreceptor layer 130 is fixed on the intermediate connection layer 120 and is configured to be able to specifically bind to the target molecule in the biological sample solution to be detected; the optical microcavity 110 is configured to be able to convert the perturbation of the evanescent field on the surface of the optical microcavity 110 caused by the specific binding event between the bioreceptor and the target molecule into a physical signal that can be read.

[0024] The present invention uses a photolithography process to carve out a window 112 (such as a photoresist layer coated on the surface of the optical microcavity 110) Figure 3 As shown in the figure, the window is located at a specific site 111 of the resonance / resonance region of the optical microcavity 110. Then, an ultra-thin intermediate connection layer 120 is deposited at the position of the window 112. Further, based on the bonding between the intermediate connection layer 120 and the bioreceptor layer 130, the bioreceptor molecules are fixed to the specific site 111 of the sensing surface of the optical microcavity 110. The optical microcavity 110 with specific site modification proposed in the present disclosure is conducive to further enhancing the interaction between the target molecules on the sensing surface and light, achieving the enhancement of the test signal on the sensing surface, thereby improving the test sensitivity of the optical biochip 100. In addition, the specific site modification scheme proposed in the present disclosure can effectively increase the fixed amount of bioreceptors on the sensing surface of the optical microcavity 110, which is conducive to improving the detection limit of the optical biochip 100.

[0025] Specifically, in the optical biochip 100 of the present disclosure, the optical microcavity 110 can be, for example, a whispering gallery mode optical microcavity, including a microsphere cavity, a microbottle cavity, a microbubble cavity, a microring cavity, a microdisk cavity, a microcore torus cavity, or a Fabry-Perot optical microcavity, a photonic crystal microcavity, or a surface plasmon optical microcavity.

[0026] In the optical biochip 100 of the present disclosure, the intermediate connection layer 120 plays a role in connecting the optical microcavity 110 and the bioreceptor layer 130 , so that the bioreceptor layer 130 is arranged at a specific site on the surface of the optical microcavity 110 .

[0027] On the one hand, the interaction force between the intermediate connection layer 120 and the surface of the optical microcavity 110 is a physical action or a chemical action. To this end, the intermediate connection layer 120 can be a thin film formed by magnetron sputtering, thermal evaporation deposition, electron beam evaporation deposition, chemical vapor deposition, pulsed laser deposition, spray thermal decomposition process or solution method. For example, the intermediate connection layer 120 is one of gold nanoparticles, silver nanoparticles and copper nanoparticles prepared by vacuum deposition process, or one of colloidal nanogold, gold nanorod solution, gold nanosphere solution, silver nanosphere solution, silver nanowire solution, silver nanorod, protein molecule, cell receptor, organic ligand molecule and bidentate ligand, multidentate ligand prepared by solution method. In addition, such metal nanospheres, nanorods, etc. with specific size and shape can further form a hybrid optical microcavity biosensor with the optical microcavity 110, which is an additional gain effect for the detection of the optical biosensor surface, including but not limited to surface plasmon resonance, surface enhanced Raman (SERS), fluorescence enhancement and other gain mechanisms.

[0028] Preferably, the intermediate connection layer 120 prepared by the solution method can be prepared by a spin coating method, a spray coating method, etc. During the preparation, the solution used should be careful not to damage the window formed by etching the photoresist.

[0029] Preferably, an adhesion layer (not shown) is further used between the intermediate connection layer 120 and the specific site 111 of the optical microcavity 110 to enhance the stability of the bioreceptor layer 130 on the specific site 111 of the optical microcavity 110. For example, a chromium layer, a metal titanium layer and other adhesion layers are deposited on the surface of the specific site before preparing the metal nanoparticles.

[0030] In addition, the intermediate connecting layer 120 used in the present disclosure can also be metal particles or metal clusters such as silver, copper, chromium, titanium, iron, cobalt, magnesium, zinc, molybdenum, etc. that can combine with biological protein molecules, antibodies, antigens, viruses, cells, etc.

[0031] On the other hand, the intermediate connection layer 120 and the biological receptor layer 130 are directly connected via a chemical covalent bond, or indirectly connected via at least one bridging molecule.

[0032] Specifically, the bioreceptor layer 130 is connected to the intermediate connection layer 120 through a chemical covalent bond (e.g., Au-S covalent bond), and can also be further reacted with the intermediate connection layer 120 on the specific site 111 on the surface of the optical microcavity 110 by, for example, performing a thiol reaction on the biomolecule. Preferably, the bioreceptor layer 120 is bonded to the intermediate connection layer 120 on the specific site 111 through a chemical covalent bond. In order to expand the scope of the bioreceptors, the bioreceptor layer 130 and the intermediate connection layer 120 can also be bonded using a bridging molecule.

[0033] The bioreceptor layer 130 provided by the present disclosure should be able to capture target molecules in biological samples, and the specific recognition of the detection of target molecules by the optical biochip 100 depends on the type of bioreceptors on the surface of the optical microcavity 110. Different types of bioreceptors specifically recognize and interact with specific target molecules, that is, the optical biochip 100 provided by the present disclosure, after being modified by the bioreceptor layer 130, can be used for the recognition and quantitative detection of specific target molecules in mixed biological samples, and has high selectivity. The bioreceptors may include, for example, one of biological protein molecules, enzymes, amino acids, peptides, antibodies, antigens, streptavidin, nucleic acid molecules, aptamers, cells, microorganisms, tissues and natural products, bacteria, bionics, biologically derived materials, thiol compounds containing thiol groups, and carbene compounds.

[0034] Figure 2 FIG. 2 shows a flow chart of a method 200 for preparing an optical biochip 100 according to an embodiment of the present disclosure. Figure 2 As shown, the preparation method 200 includes the following steps:

[0035] S210, forming a window 112 in the photoresist layer 150 coated on the surface of the optical microcavity 110 through a photolithography process, wherein the window 112 is aligned with the specific site 111 to expose the specific site 111;

[0036] S220, forming an intermediate connection layer 120 at the exposed specific site 111 in the window 112, so that the intermediate connection layer 120 is bonded to the surface of the optical microcavity at the specific site 111; and

[0037] S230 , forming a bioreceptor layer 130 capable of specifically binding to a target molecule on the intermediate connection layer 120 .

[0038] Before S210 , it is necessary to form an optical microcavity 110 (eg, a microring resonator) on a clean wafer 140 serving as a substrate.

[0039] In S210, a layer of photoresist (positive photoresist) is uniformly coated on the surface of the optical microcavity 110, and a window 112 of a specific size is etched at the specific site 111 where the biological receptor modification is required through deep ultraviolet photolithography, development and other processes, so that the specific site 111 is exposed through the window 112. Here, the photolithography process uses a positive photoresist process, but those skilled in the art should understand that the photolithography process can also use a negative photoresist process, as long as the specified window 112 can be formed, and the present disclosure is not limited here.

[0040] Next, in S220 , a thin intermediate connection layer 120 (eg, a gold nanoparticle layer) is deposited at the window 112 of the optical microcavity 110 , and then the photoresist is removed to obtain the optical microcavity 110 with the intermediate connection layer 120 deposited at the specific site 111 .

[0041] Finally, in S230 , a biological receptor layer solution (eg, a protein solution) is introduced into the surface of the optical microcavity 110 and incubated at a certain temperature for 1-2 hours, after which the microcavity surface is rinsed with phosphate buffered saline (PBS).

[0042] Figure 3 The figure shows a schematic diagram of the preparation process of an optical biochip using a microring resonator as an example of an optical microcavity. The microring resonator consists of at least one ring waveguide with a closed optical path and an edge-coupled waveguide. The optical microcavity mode formed inside the axisymmetric microring resonator is confined to the surface of the resonant cavity by near-total internal reflection. Although these microcavity mode fields are confined inside the resonator, they can still extend into the surrounding medium, and their intensity decays rapidly at a distance of about one wavelength of light, forming an evanescent field. The perturbation of the evanescent field on the surface of the optical microcavity will be converted into mode changes, which can be divided into the shift of the resonance frequency, the broadening of the full width at half maximum of the mode, and the splitting of the degenerate mode. The biological receptors fixed on the surface of the ring resonator can capture target molecules. Since the light waves coupled in the microring resonator circulate back and forth in the cavity, the interaction between the target molecules on its surface and the evanescent waves is enhanced, causing the optical microcavity mode perturbation, which is the basic mechanism of biosensing.

[0043] The mode change caused by the binding of target molecules on the surface of the ring waveguide of the microring resonator is one of the important factors affecting the sensitivity of the optical biochip to the determination of specific analytes. It can be concluded from the relevant research of the prior art that the most sensitive area for detecting target molecules is located on the surface of the ring waveguide, that is, the surface of the resonant cavity. In order to obtain a highly sensitive optical biochip, it is necessary to improve the fixation of target molecules on the surface of the ring waveguide as much as possible, and concentrate the binding events of target molecules and biological receptors in the biological sample on the ring waveguide, rather than indiscriminately fix the biological receptors on the surface of the microring resonator.

[0044] exist Figure 3 In the embodiment shown, the present disclosure first uses a photolithography process to carve a window at a specific location on the sensing surface of the microring resonator, and further deposits an ultra-thin intermediate connecting layer. When the intermediate layer is a metal layer, due to the low thickness of the metal film, a continuous film cannot be formed, and its morphology presents an island-like gold nanoparticle structure. For such island-like gold nanoparticles, surface plasmons can be further excited, thereby increasing the interaction strength between light and the sensing surface material. The gold nanoparticles deposited at specific locations can further bind to the bioreceptor layer through Au-S bonds, and are used to capture the biomolecules to be detected (such as antibodies, Figure 4As shown in Figure 2). When the bioreceptors fixed on the sensor surface bind to the analyte to be tested, the refractive index distribution on the surface of the microring resonator changes, affecting the propagation of the microring evanescent wave and causing the microcavity resonance wavelength to shift. When the content of biomolecules is higher, the shift of the microring resonance wavelength or angle will increase. Based on this principle, a standard curve is established using the relationship between different concentrations of standard analytes and the shift, which can be used to determine the analyte concentration in the biological sample to be tested.

[0045] Preferably, after step S230, a high concentration blocking protein solution may be used to incubate the optical biochip for 1-2 hours to block the residual active sites on the gold nanoparticles in the intermediate connection layer, ensuring that all active reaction sites on the intermediate connection layer are bonded to the blocking protein. Here, the blocking solution for blocking the active reaction sites on the intermediate connection layer is a protein or other blocking chemical small molecule solution that does not bind to biological receptors or target molecules, such as a bovine serum albumin solution (BSA, such as Figure 4 As shown), skimmed milk powder, goat serum protein solution, serum, fish skin gelatin, polyvinyl pyrrolidone PVP, etc.

[0046] The present disclosure also provides an optical bio-detection system 300 based on the optical bio-chip 100, which includes at least one optical bio-chip 100 and a microfluidic chip 310 matching the pattern thereof. For example, Figure 5 A highly integrated, high-throughput optical biochip array consisting of at least one optical biochip 100 is shown, where the optical microcavity 110 of the optical biochip 100 uses a microring resonator. The sensing surface of each microring resonator in the array can be fixed with different types of bioreceptor layers using the specific site modification method provided by the present disclosure. Based on the specific interaction between the bioreceptor layer and the target biomolecule, the optical biochip provided in the embodiment of the present disclosure can be designed for the determination of various analytes of interest.

[0047] Figure 6 FIG. 3 shows a schematic diagram of the structure of the optical biological detection system 300. Figure 6 As shown, the microfluidic chip 310 is located above and bonded to the at least one optical biochip 100 (including, for example, a microring resonator), and its at least one microfluidic channel 311 provides a flow channel for a biological sample solution containing target molecules to be detected, wherein at least a portion of each microfluidic channel 311 overlaps with a specific site 111 of the corresponding optical biochip 100.

[0048] Figure 7 FIG. 4 is a schematic flow chart of an optical bio-detection method 400 performed using the optical bio-detection system 300. Figure 7 As shown, the optical biological detection method 400 includes:

[0049] S410, measuring the initial resonance wavelength of the optical microcavity 110;

[0050] S420, passing buffer solutions of standard target molecules with known concentrations from low to high into the multiple microfluidic channels 311, washing with a phosphate buffer solution after incubation, and recording the adjustment resonance wavelength at the corresponding concentration;

[0051] S430, calculating a tuning resonant wavelength shift value at a corresponding concentration based on the tuning resonant wavelength and the initial resonant wavelength, and obtaining a resonant wavelength shift-concentration curve;

[0052] S440, introducing a biological sample solution containing target molecules to be detected into any one of the remaining microfluidic channels 311 of the optical biochip array, and recording the test resonance wavelength of the optical microcavity 110;

[0053] S450, calculating a test resonance wavelength shift value corresponding to the biological sample solution based on the test resonance wavelength and the initial resonance wavelength; and

[0054] S460, obtaining the concentration of the target molecule in the biological sample solution based on the resonance wavelength shift-concentration curve and the test resonance wavelength shift value.

[0055] The preparation method 200 and the optical biological detection method 400 of the present disclosure are described in detail below. In one embodiment, the optical microcavity 110 is a microring resonator, the intermediate connection layer 120 is an Au nanoparticle layer, and the biological receptor layer 130 is a protein solution.

[0056] (1) Fabricating a microring resonator on a clean wafer substrate;

[0057] (2) uniformly coating a layer of photoresist (positive photoresist) on the surface of the microring resonator to cover the silicon nitride optical microring; designing a pattern of the photoresist plate to expose the photoresist at specific sites of the silicon nitride microring resonator at specific sites where biological receptors need to be modified, and then carving out windows of specific sizes through deep ultraviolet lithography, development and other processes;

[0058] (3) using physical vapor deposition equipment to deposit a thin layer of gold nanoparticles as an intermediate connection layer at the window of the ring waveguide of the microring resonator, and then removing the photoresist to obtain a microring resonator with an intermediate connection layer deposited at a specific site;

[0059] (4) bonding the microring resonator to the microfluidic chip, with the microfluidic channel partially overlapping the annular waveguide of the microring resonator;

[0060] (5) After bonding the microfluidic chip, a biological receptor protein solution is introduced into the microfluidic channel and incubated at a certain temperature for 1-2 hours;

[0061] (6) After incubating the bioreceptor, rinse the sensor surface in step (5) using phosphate buffered saline (PBS);

[0062] (7) A high concentration of blocking protein solution (BSA, skimmed milk powder) is introduced into the microfluidic channel and incubated at a certain temperature for 1-2 hours to block the residual active sites on the gold nanoparticles in the intermediate connecting layer;

[0063] (8) Rinse the sensor surface with phosphate buffered saline (PBS);

[0064] (9) introducing buffer solutions of standard target molecules with different concentrations from low to high into the microfluidic channel, incubating for 30 minutes, and keeping the temperature of the system constant at a certain temperature;

[0065] (10) Pass phosphate buffered saline (PBS) into the microfluidic channel in step (9), flush the microfluidic channel, and record the resonant wavelength shift Δλ at the corresponding concentration. s ; The recorded resonance wavelength shift Δλ corresponding to the concentration s It is further processed and plotted as Δλ s -Concentration standard curve diagram, such as Fig. 9 As shown;

[0066] (11) a biological sample solution containing an analyte to be tested is introduced into another microfluidic channel, and after incubation for a certain period of time, a phosphate buffered saline (PBS) is introduced for cleaning, and a resonance wavelength shift value Δλ of the microring resonator is recorded;

[0067] (12) According to the resonant wavelength offset value Δλ of the microring resonator recorded in step (11), Figure 8 As shown, the concentration of the analyte in the biological sample solution to be tested is obtained by reading from the standard curve diagram in step (10).

[0068] Specifically, in the actual implementation process:

[0069] - In step (2), the process adopted to carve a window on the sensing surface of the optical microcavity can be a photolithography process for micro-nano processing devices. For the needs of larger or larger windows, a metal mask method can also be used. During preparation, the carved window position should be aligned with the specific site of the optical device as much as possible to achieve precise modification of the specific site;

[0070] -In step (3), the thickness of the gold nanolayer obtained by calibration and testing with a quartz microcrystal balance is 1 nm; this thickness can be adjusted by simulating the loss of the optical microcavity based on the simulation results. When a metal film grows on a substrate, it first needs to form nuclei before it can grow into a continuous film. The Au thin layer used in this case appears as a discontinuous film on the optical device, which is an island-shaped Au particle layer;

[0071] - In step (5), the bioreceptor protein solution introduced into the microfluidic channel contains at least one molecule with biological activity, and different types of protein solutions may be introduced into different channels to form an optical biochip with multiple analyte detection;

[0072] - The buffer solutions of the standard analytes of different concentrations from low to high described in step (9) are biological molecules that are close to and as homologous as possible to the analytes in the sample to be tested, and the concentration range covers the concentration of the sample to be tested.

[0073] The present disclosure uses a photolithography process to carve a window at a specific site of the optical microcavity, and then combines the vacuum deposition film preparation method to deposit an intermediate connection layer (ultra-thin gold nanoparticle layer) at a specific site of the sensing surface area of ​​the optical microcavity. Further, based on the bonding between the intermediate connection layer and the biological receptor layer, the biological receptor molecules are fixed at specific sites on the optical microcavity sensing surface. Specifically, in the embodiment provided by the present disclosure, gold nanoparticles are used as the intermediate connection layer, and physical vapor deposition equipment is used to deposit them at the carved window position. Next, the strong covalent binding effect (Au-S chemical bond) between metallic gold and thiol compounds is used to fix biological protein molecules, antibodies, antigens and other biologically active molecules or other chemical molecules in the sensing area of ​​the optical microcavity, thereby realizing the modification of the biological receptor layer on the optical biochip and constructing an optical biochip with biochemical sensing function. The method of using a photolithography process to carve a window and deposit an intermediate connection layer can accurately control the fixation of biological receptors at specific sites.

[0074] Taking the microring resonator as an example, the basic working principle of the optical biochip based on the microring resonator provided by the present disclosure is: the biological receptors fixed on the surface of the microring resonator are used to capture target molecules, or interact with other biological molecules and chemicals with specific binding capabilities. When the target molecule binds to the biological receptor layer at a specific site, the effective refractive index of the surface of the microring resonator changes, causing the resonant wavelength of the resonator to change, thereby achieving accurate detection of the target molecule. The optical biochip modified with the biological receptor layer at a specific site proposed in the present disclosure can concentrate the specific binding events between biological molecules in the high-sensitivity detection area of ​​the microring resonator, greatly improving the sensitivity and accuracy of target molecule detection.

[0075] In one embodiment, the optical biological detection system 300 provided by the present disclosure may also include an information processing unit (not shown), which is optically coupled to the optical microcavity 110 to detect a physical signal caused by the specific binding between the biological receptor and the target molecule, such as a test resonant wavelength of the optical microcavity, and calculate the concentration of the target molecule in the biological sample solution to be analyzed based on the physical signal.

[0076] It should be understood that in various embodiments of the present application, the information processing unit may be a processor. The processor may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application specific integrated circuits (ASIC), field programmable gate arrays (FPGA), or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components, etc. It should be noted that the processor may also be integrated with components for storage such as memory units and / or cache units.

[0077] In the implementation process, each step of the above method can be completed by an integrated logic circuit of hardware in a processor or an instruction in the form of software. The steps of the method disclosed in conjunction with the embodiment of the present application can be directly embodied as a hardware processor for execution, or a combination of hardware and software modules in a processor for execution. The software module can be located in a storage medium mature in the art such as a random access memory, a flash memory, a read-only memory, a programmable read-only memory or an electrically erasable programmable memory, a register, etc. The storage medium is located in a memory, and the processor reads the information in the memory and completes the steps of the above method in conjunction with its hardware. To avoid repetition, it is not described in detail here.

[0078] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of disclosure involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above disclosed concept. For example, the above features are replaced with the technical features with similar functions disclosed in this application (but not limited to) by each other to form a technical solution.

[0079] In addition, although each operation is described in a specific order, this should not be construed as requiring these operations to be performed in the specific order shown or in a sequential order. Under certain circumstances, multitasking and parallel processing may be advantageous. Similarly, although some specific implementation details are included in the above discussion, these should not be construed as limiting the scope of the present invention. Some features described in the context of a separate embodiment can also be implemented in a single embodiment in combination. On the contrary, the various features described in the context of a single embodiment can also be implemented in multiple embodiments individually or in any suitable sub-combination mode.

[0080] Although the subject matter has been described in language specific to structural features and / or methodological logical actions, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or actions described above. On the contrary, the specific features and actions described above are merely example forms of implementing the claims.

[0081] Several embodiments of the present application have been described in detail above, but the present application is not limited to these specific embodiments. Those skilled in the art can make various variations and modifications to the embodiments based on the concept of the present application, and these variations and modifications should all fall within the scope of protection claimed by the present application.

Claims

1. A site-specific modified optical biochip, characterized in that: From bottom to top they include: Optical microcavity; an intermediate connection layer formed at a specific site of the optical microcavity, the specific site being located in a resonant or resonance region of a surface of the optical microcavity; and a biological receptor layer, which is fixed on the intermediate connecting layer and is configured to be able to specifically bind to a target molecule in a biological sample solution to be detected; The optical microcavity is configured to convert the perturbation of the evanescent field on the surface of the optical microcavity caused by a specific binding event into a physical signal that can be read.

2. The optical biochip according to claim 1, characterized in that: The force between the intermediate connecting layer and the surface of the optical microcavity is a physical effect or a chemical effect; and The intermediate connection layer and the biological receptor layer are directly connected via a chemical covalent bond, or indirectly connected via at least one bridging molecule.

3. The optical biochip according to claim 2, characterized in that: The intermediate connecting layer is one of gold nanoparticles, silver nanoparticles and copper nanoparticles prepared by a vacuum deposition process, or one of colloidal nanogold, gold nanorod solution, gold nanosphere solution, silver nanosphere solution, silver nanowire solution, silver nanorod, protein molecules, cell receptors, organic ligand molecules, bidentate ligands and multidentate ligands prepared by a solution method.

4. The optical biochip according to claim 2, characterized in that: The biological receptor layer is one of biological protein molecules, enzymes, amino acids, peptides, antibodies, antigens, streptavidin, nucleic acid molecules, aptamers, cells, microorganisms, tissues and natural products, bacteria, bionic bodies, biologically derived materials, thiol compounds containing sulfhydryl groups and carbene compounds.

5. The optical biochip according to claim 1, characterized in that: The optical microcavity is one of a whispering gallery mode optical microcavity, a Fabry-Perot optical microcavity, a photonic crystal microcavity, and a surface plasmon optical microcavity.

6. A method for preparing an optical biochip according to any one of claims 1 to 5, characterized in that: The steps include: Step 1: forming a window in a photoresist layer coated on the surface of the optical microcavity by a photolithography process, wherein the window is aligned with the specific site to expose the specific site; Step 2, forming the intermediate connection layer at the exposed specific site in the window, so that the intermediate connection layer is bonded to the optical microcavity surface at the specific site; as well as Step 3: forming the bioreceptor layer capable of specifically binding to the target molecule on the intermediate connecting layer.

7. The preparation method according to claim 6, characterized in that: Step 1 specifically includes: Uniformly coating a layer of photoresist on the surface of the optical microcavity; The window is formed on the photoresist through deep ultraviolet photolithography, development and fixing.

8. The preparation method according to claim 6, characterized in that: Step 2 further includes: after forming the intermediate connection layer, removing the photoresist.

9. The preparation method according to any one of claims 6 to 8, characterized in that: In step 2, the intermediate connection layer is a thin film formed by magnetron sputtering, thermal evaporation deposition, electron beam evaporation deposition, chemical vapor deposition, pulsed laser deposition, spray thermal decomposition process or solution method.

10. The preparation method according to any one of claims 6 to 8, characterized in that: Further including: Step 4: incubating the optical biochip with a blocking solution to block the residual active sites on the intermediate connection layer.

11. The preparation method according to claim 10, characterized in that: The blocking solution is bovine serum albumin solution, skimmed milk powder, goat serum albumin solution, serum or fish skin gelatin.

12. An optical biological detection system, comprising: At least one optical biochip according to any one of claims 1 to 5; as well as A microfluidic chip, which is located above the optical biochip and bonded thereto, wherein at least one microfluidic channel of the microfluidic chip provides a flow channel for a biological sample solution containing the target molecule to be detected; Wherein, at least a portion of each of the microfluidic channels overlaps with the corresponding specific site of the optical biochip.

13. The optical biological detection system according to claim 12, characterized in that: The method further comprises a signal processing unit which is optically coupled to the optical microcavity to detect the physical signal caused by the specific binding.

14. An optical bio-detection method using the optical bio-detection system according to claim 12 or 13, wherein the optical bio-detection system comprises an optical bio-chip array composed of a plurality of the optical bio-chips, characterized in that: The optical bioassay method comprises: measuring an initial resonant wavelength of the optical microcavity; Passing buffer solutions of standard target molecules with known concentrations from low to high into the plurality of microfluidic channels, washing with a phosphate buffer solution after incubation, and recording the adjusted resonance wavelength at the corresponding concentration; Based on the adjusted resonant wavelength and the initial resonant wavelength, calculating the adjusted resonant wavelength shift value at the corresponding concentration, and obtaining a resonant wavelength shift-concentration curve; Passing the biological sample solution containing the target molecule to be detected into any one of the remaining microfluidic channels of the optical biochip array, and recording the test resonance wavelength of the optical microcavity; calculating a test resonance wavelength shift value corresponding to the biological sample solution based on the test resonance wavelength and the initial resonance wavelength; and Based on the resonance wavelength shift-concentration curve and the test resonance wavelength shift value, the concentration of the target molecule in the biological sample solution is obtained.

15. The optical biological detection method according to claim 14, characterized in that: Further including: The bioreceptor layer different from that of other optical biochips is fixed on at least one of the optical biochips in the optical biochip array to enable the optical biodetection system to detect more than two target molecules.

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