A surface sensor and a method of manufacturing and using the same

Abstract

The application belongs to the technical field of sensors, and particularly relates to a surface sensor and a preparation method and application thereof. The surface sensor comprises a sensing chip and a microfluidic channel; the sensing chip and the microfluidic channel are oppositely arranged with one side having a concave-convex surface to form a fluid channel; the microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a protruding structure; the bottom surface of the groove structure and the upper end surface of the protruding structure are both distributed with micropillars; and the protruding structure is perpendicular to the fluid direction and is arranged in an array. The protruding structure and the micropillars arranged in the microfluidic channel change the flow field in the microfluidic channel, increase the fluid flow rate, accelerate the material exchange rate in the microchannel, reduce the test error, and improve the detection sensitivity. When the surface sensor of the application is used to detect AFP antigen, the secondary antibody does not need to be introduced, the response of 1 ng / mL AFP antigen can be detected, and excellent sensitivity is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of sensors, and particularly relates to a surface sensor and a preparation method and application thereof. BACKGROUND

[0002] Plasmonic resonance biosensors are increasingly applied in medical diagnosis, food safety and environmental monitoring. Under the excitation of external light source, the surface plasmon resonance (SPR) between the noble metal layer and the medium produces a sensitive response to the refractive index (RI) change of the surrounding medium. By measuring the change of these resonance conditions (e.g. angle, wavelength, intensity or phase), the molecular interaction occurring on the surface of the sensor can be directly monitored in real time. This unique feature makes plasmonic resonance biosensors an indispensable tool for real-time and label-free analysis, and surface refractive index sensors have great application potential due to their high sensitivity and versatility.

[0003] Currently, in the SPR sensing technology, there are mainly propagating surface plasmon resonance (PSPR) and localized surface plasmon resonance (LSPR). The propagation electric field length of PSPR is large, and the electric field of LSPR is generally localized in a range of several nanometers, with high sensitivity. The fiber surface plasmon resonance biosensor is mainly based on the principle of fiber surface plasmon resonance sensing, and the characteristics of the measured biological molecules are analyzed by detecting the change of the refractive index in the evanescent field region of the fiber surface. In order to obtain high sensitivity, the above-mentioned method needs to be combined with other detection methods (fluorescence, magnetism, electricity, etc.), at this time the analyte needs to be functionalized (modified with labels), such as modification of fluorescent groups, magnetic groups, color groups, etc. However, although PSPR has good sensitivity, it needs to rely on complex equipment and high cost, and it is difficult to convert from a large system and equipment to a small integrated instrument. LSPR structure is relatively simple, but its resonance peak has a relatively large line width and a low quality factor, and it is difficult to realize industrialization of repeatability and reproducibility. Although the fiber sensor has certain advantages in volume, it has complex optical design, high-precision processing and assembly, and it is difficult to realize in terms of production cost, processing difficulty and repeatability, and the introduction of analyte modification labels increases the additional steps, and accordingly increases the detection time, test error and cost.

[0004] Therefore, it is of great significance to provide a surface sensor with high sensitivity, easy processing and low cost. SUMMARY

[0005] The present application aims to solve one or more technical problems in the prior art described above, and at least provide a beneficial choice or create conditions. Specifically, the present application provides a surface sensor with high sensitivity, easy processing and low cost, which can be converted into a small integrated instrument.

[0006] The application discloses a surface sensor.

[0007] Therefore, the first aspect of the application provides a surface sensor.

[0008] Specifically, the surface sensor comprises a microfluidic channel and a sensing chip; the sensing chip and the microfluidic channel have one side with concave-convex surfaces placed oppositely to form a fluid channel.

[0009] The microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a protruding structure.

[0010] The bottom surface of the groove structure and the upper end surface of the protruding structure are both distributed with microposts.

[0011] The protruding structure is perpendicular to the fluid direction and is arranged in an array.

[0012] Specifically, the structure area of the sensing chip and the structure area of the microfluidic channel with concave-convex surfaces are placed oppositely, the non-structure area of the sensing chip and the non-structure area of the microfluidic channel are attached together to form a fluid channel, thereby obtaining the surface sensor.

[0013] Preferably, the depth of the groove structure is 12-33 μm; the width of the groove structure is 200-1500 μm.

[0014] Further preferably, the depth of the groove structure is 10-30 μm; the width of the groove structure is 220-1000 μm.

[0015] More preferably, the depth of the groove structure is 10 μm; the width of the groove structure is 500 μm.

[0016] Preferably, the groove structure is a U-shaped groove.

[0017] Preferably, the height of the protruding structure is 4.5-10 μm; the width of the protruding structure is 18-42 μm; the length of the protruding structure is 100-1000 μm; the distance between adjacent protruding structures is 8-32 μm.

[0018] Further preferably, the height of the protruding structure is 5-10 μm; the width of the protruding structure is 20-40 μm; the length of the protruding structure is 100-1000 μm; the distance between adjacent protruding structures is 10-30 μm.

[0019] Still further preferably, the height of the protruding structure is 5 μm; the width of the protruding structure is 30 μm; the length of the protruding structure is 500 μm; the distance between adjacent protruding structures is 20 μm.

[0020] Preferably, the diameter of the micropillar is 0.9-3.3 μm; the height of the micropillar is 0.8-1.2 μm; further preferably, the diameter of the micropillar is 1-3 μm; the height of the micropillar is 0.9-1.1 μm.

[0021] Preferably, the micropillar is randomly distributed on the bottom surface of the groove structure and the upper end surface of the protruding structure.

[0022] Specifically, compared with a flat microfluidic channel without protruding structure, the present application changes the flow field in the microfluidic channel by setting protruding structures and micropillars of certain sizes in the microfluidic channel, increases the flow rate of the fluid, accelerates the exchange rate of substances in the channel, reduces the influence of FP interference effect on the test spectrum, reduces the test error, and improves the detection sensitivity.

[0023] Preferably, one hole is arranged at each of the two ends of the microfluidic channel along the length direction, respectively serving as the inlet and outlet of the detection solution.

[0024] Preferably, the diameter of the hole is 0.3-1 mm; further preferably, the diameter of the hole is 0.65-0.75 mm; still further preferably, the diameter of the hole is 0.7 mm.

[0025] Preferably, the sensing chip comprises, in sequence, a substrate, a dielectric layer, and a metal layer; the dielectric layer has a plurality of dielectric grooves, and the plurality of dielectric grooves are periodically and equidistantly distributed; the opening width of the dielectric groove gradually decreases in the direction from the groove opening to the groove bottom; and the metal layer is arranged on the dielectric layer and forms a metal groove corresponding to each dielectric groove.

[0026] Specifically, the difference between the sensing chip of the present application and the structure disclosed in the prior art with publication number CN113433094A is only that the types of the metal layer are different, the metal layer of the present application includes at least one of a gold layer, a silver layer, an aluminum layer, and a copper layer, while the metal layer in the prior art is selected from at least one of a gold layer, a silver layer, and a platinum layer, and the other is the same as the prior art with publication number CN113433094A.

[0027] Preferably, in the sensing chip, the metal layer can be a single-layer structure of the metal layer, or a laminated structure composed of different metal layers; for example, the metal layer can be a single-layer structure of a gold layer, an aluminum layer, a silver layer, or a copper layer, or a laminated structure composed of at least two of a gold layer, an aluminum layer, a silver layer, and a copper layer.

[0028] Specifically, gold has good chemical stability and biocompatibility, and is the preferred material for biosensing, but considering that gold is relatively expensive, and the nano-transfer printing technology and the working environment of the biosensing chip require a relatively thick metal layer (~ 300 nm), therefore, the present application preferably adopts a 50-100 nm Au and 200-250 nm other metal layer (Ag, Cu, Al or other low-priced, high-reflectivity metal) laminated manner, which reduces the processing cost of the biosensor under the premise of realizing the biosensing function, and facilitates the reduction of the cost of mass production.

[0029] The second aspect of the present application provides a preparation method of the surface sensor of the first aspect of the present application.

[0030] Specifically, the preparation method of the surface sensor comprises the following steps:

[0031] The microfluidic channel and the side of the sensing chip having the concave-convex surface are placed opposite to each other to obtain the surface sensor.

[0032] Specifically, because the sizes of the structural features of the microfluidic channel and the sensing chip belong to different size grades, it is equivalent to placing the sides with structural features of the two together, and the non-structural feature areas are attached together, and the structural feature areas form a space after cooperation, which serves as a fluid channel for the detection fluid to pass through.

[0033] Preferably, the preparation method of the microfluidic channel comprises the following steps:

[0034] (1) forming randomly distributed micropores on the template to obtain a template with micropores distributed thereon;

[0035] (2) covering a mask material on the surface of the template obtained in step (1), forming a mask material with a grating structure by photolithography, and using the mask material with the grating structure as a mask to etch the template to form an array of grating structures on the template;

[0036] (3) covering the surface of the template obtained in step (2) with a mask material, forming a mask material covering the grating structure by photolithography, taking the mask material covering the grating structure as a mask to etch the template, and forming an auxiliary protrusion on the template;

[0037] (4) covering the template obtained in step (3) with an organic silicon mixture, curing and separating to obtain a microfluidic channel.

[0038] Specifically, in step (1), the surface of the template is covered with a mask material, a circular hole is obtained on the mask material by photolithography, and then etching is performed to form a micropore at the position corresponding to the circular hole on the template.

[0039] Preferably, in step (1), the diameter of the micropore is 0.9-3.3 μm; the depth of the micropore is 0.8-1.2 μm; further preferably, in step (1), the diameter of the micropore is 1-3 μm; the depth of the micropore is 0.9-1.1 μm.

[0040] Specifically, according to the structural characteristics of the designed microfluidic channel, the micropores are randomly distributed in the template area corresponding to the grating structure and the vicinity thereof in step (2), and it is ensured that micropores are distributed between the grating structures and adjacent grating structures.

[0041] Specifically, in step (2), taking the mask material with the grating structure as a mask can protect the template position corresponding to the grating structure from being etched, while the material in other areas of the template is etched downward, and after removing the mask material, an array of grating structures is obtained on the template.

[0042] Specifically, in step (2), the grating structure is obtained on the template, the grating structure corresponds to the groove between the protruding structures of the finally obtained microfluidic channel, and the groove between the grating structures corresponds to the protruding structure of the finally obtained microfluidic channel.

[0043] Preferably, in step (2), the height of the grating structure is 4.5-10 μm; the width of the grating structure is 8-32 μm; the length of the grating structure is 100-1000 μm; and the distance between adjacent grating structures is 18-42 μm.

[0044] Preferably, in step (2), the height of the grating structure is 5-10 μm; the width of the grating structure is 10-30 μm; the length of the grating structure is 100-1000 μm; and the distance between adjacent grating structures is 20-40 μm.

[0045] Preferably, in step (2), the height of the grating structure is 5 μm; the width of the grating structure is 20 μm; the length of the grating structure is 500 μm; and the distance between adjacent grating structures is 30 μm.

[0046] Specifically, in step (3), the mask material covers the grating structure due to the effect of photolithography. The grating structure is not affected by subsequent etching due to the protection of the mask material, and the template position corresponding to the mask material covering the grating structure will not be etched, while the material in other areas of the template will be etched downward. After removing the mask material, an auxiliary protrusion is obtained on the template, and the grating structure is located above the auxiliary protrusion.

[0047] Specifically, the auxiliary protrusion formed in step (3) corresponds to the groove structure of the microfluidic channel finally prepared.

[0048] Preferably, the mask material covering the grating structure is in an I-shape, that is, the auxiliary protrusion obtained in step (3) is in an I-shape, and the grating structure is located on the auxiliary protrusion structure in the I-shape.

[0049] Preferably, in step (3), the height of the auxiliary protrusion is 12-33 μm; the width of the auxiliary protrusion is 200-1500 μm.

[0050] Further preferably, in step (3), the height of the auxiliary protrusion is 10-30 μm; the width of the auxiliary protrusion is 220-1000 μm.

[0051] Further preferably, in step (3), the height of the auxiliary protrusion is 10 μm; and the width of the auxiliary protrusion is 500 μm.

[0052] Preferably, before performing step (4), the template obtained in step (3) is cleaned with a cleaning agent to remove the mask material remaining on the surface of the template.

[0053] Preferably, the cleaning agent includes concentrated sulfuric acid and hydrogen peroxide.

[0054] Preferably, the mass fraction of the concentrated sulfuric acid is 95-98%, and the mass fraction of the hydrogen peroxide is 32-38%.

[0055] Further preferably, the mass fraction of the hydrogen peroxide is 35%.

[0056] Preferably, the volume ratio of the concentrated sulfuric acid and hydrogen peroxide is (6-8):3; further preferably, the volume ratio of the concentrated sulfuric acid and hydrogen peroxide is (6.5-7.5):3; further preferably, the volume ratio of the concentrated sulfuric acid and hydrogen peroxide is 7:3.

[0057] Preferably, the cleaning agent is further subjected to water rinsing, nitrogen blowing and drying after cleaning.

[0058] Preferably, in step (4), the silicone mixture comprises silicone and curing agent.

[0059] Preferably, the mass ratio of the silicone and the curing agent is (8-12):1; further preferably, the mass ratio of the silicone and the curing agent is (9-11):1; more preferably, the mass ratio of the silicone and the curing agent is 10:1.

[0060] Preferably, the silicone comprises polydimethylsiloxane (PDMS).

[0061] Specifically, the silicone mixture is Dow Corning Sylgard 184.

[0062] Preferably, the silicone is degassed in a vacuum dryer before curing.

[0063] Preferably, the curing temperature is 60-80℃, and the curing time is 1.5-2.5h; further preferably, the curing temperature is 65-75℃, and the curing time is 1.8-2.2h; more preferably, the curing temperature is 70℃, and the curing time is 2h.

[0064] Preferably, in step (4), in order to ensure the PDMS and the template obtained in step (3) to be separated smoothly, a release agent trichloro(1H, 1H, 2H, 2H-tridecafluorooctyl)silane (TFOCS) is evaporated on the template obtained in step (3), specifically, the release agent is coated on the template obtained in step (3), and then placed in a vacuum drying box for evaporation, and taken out.

[0065] Preferably, the evaporation time is 25-35min; further preferably, the evaporation time is 27-33min; more preferably, the evaporation time is 30min.

[0066] Specifically, step (4) corresponds to the process of transferring, and the template corresponding to the structural features of the microfluidic channel is prepared through steps (1)-(3), that is, the microfluidic channel template is obtained in step (3), and then the microfluidic channel is obtained through the transferring process in step (4) (for example, the micropore of the microfluidic channel template is transferred into micropillar of the microfluidic channel, the auxiliary protrusion of the microfluidic channel template is transferred into groove structure of the microfluidic channel, the grating structure of the microfluidic channel template is transferred into groove between the protrusions of the microfluidic channel, and the groove between the grating structures of the microfluidic channel template is transferred into protrusion structure of the microfluidic channel).

[0067] Preferably, after the step (4) of transferring is finished, the microfluidic channel is provided with a hole at each of the two ends along the length direction, which are respectively used as the entrance and exit of the detection solution.

[0068] Preferably, the diameter of the hole is 0.3-1mm; further preferably, the diameter of the hole is 0.65-0.75mm; more preferably, the diameter of the hole is 0.7mm.

[0069] Preferably, the microfluidic channels are arranged in an array.

[0070] The third aspect of the present application provides a surface sensor system.

[0071] Specifically, the surface sensor system comprises the surface sensor of the first aspect of the present application.

[0072] Preferably, the surface sensor system further comprises an injection pump, a light source generator, a half-transmission half-reflection mirror, a microscope, a detector, and a data output window; the injection pump is connected to the entrance of the microfluidic channel for detection solution.

[0073] Preferably, the detector comprises any one of a CCD photodetector, a CMOS photodetector, and a PIN photodetector.

[0074] The fourth aspect of the present application provides the application of the surface sensor of the first aspect of the present application or the surface sensor system of the third aspect of the present application in detection analysis for non-disease diagnosis.

[0075] Preferably, the detection analysis comprises antigen concentration detection.

[0076] Compared with the prior art, the technical solution provided by the present application has the following beneficial effects:

[0077] (1) The surface sensor of the present application comprises a microfluidic channel and a sensing chip; the microfluidic channel is a groove structure; the bottom surface of the groove structure is provided with micropillars and arrayed protruding structures; the upper end surface of the protruding structure is distributed with micropillars. Compared with the microfluidic channel without protruding structure, the arrangement of the protruding structure and the micropillars changes the flow field in the microfluidic channel; compared with the flow field without structure, the flow rate of the fluid is increased, the material exchange rate in the microchannel is accelerated, the influence of FP interference on the test spectrum is reduced, the influence of FP resonance is reduced or even eliminated, thereby reducing the test error and improving the detection sensitivity. When the surface sensor of the present application is used to detect AFP antigen, no secondary antibody is introduced, and 1ng / mL alpha-fetoprotein (AFP) response (clinical 7ng / mL is the critical value) can be detected under the condition of no label (without label signal amplification technology), and the S-type fitting is met in the range of 1-10000ng / mL.

[0078] (2) The metal layer of the sensing chip has a smooth surface, which can effectively improve the smoothness of the metal layer, further reduce the resonance peak line width, and improve the quality factor. At the same time, it can effectively avoid the problems of affecting the optical response of the biosensor caused by the change of the thickness of the metal layer and the asymmetry of the metal layer. The microfluidic channel and the sensing chip are combined, which can further reduce the test error and improve the detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 It is a structural schematic diagram of the microfluidic channel of the embodiment 1 of the present application;

[0080] Figure 2 It is a sectional view along the flow direction of the microfluidic channel of the embodiment 1 of the present application;

[0081] Figure 3 It is a sectional view along the flow direction of the surface sensor of the embodiment 1 of the present application;

[0082] Figure 4 It is a processing flow schematic diagram of the microfluidic channel of the embodiment 1 of the present application;

[0083] Figure 5 It is a structural schematic diagram of the microfluidic channel template of the embodiment 1 of the present application;

[0084] Figure 6 It is a top view schematic diagram of the microfluidic channel of the embodiment 1 of the present application;

[0085] Figure 7 It is a scanning electron microscope image of the microfluidic channel of the embodiment 1 of the present application;

[0086] Figure 8 It is a sectional view along the flow direction of the microfluidic channel of the comparative example 2;

[0087] Figure 9 It is a flow field simulation diagram of the microfluidic channel of the embodiment 1 of the present application and the microfluidic channel of the comparative example 2 when the flow field flow rate is 10 mm / s;

[0088] Figure 10 It is a structural schematic diagram of the surface sensing system;

[0089] Figure 11 It is a reflectivity spectrum diagram of the surface sensor of the embodiment 1 and the comparative example 1 of the present application;

[0090] Figure 12 It is an antibody binding response curve diagram of the surface sensor of the embodiment 1 and the comparative example 2 of the present application;

[0091] Figure 13 It is a real-time response curve diagram of the surface sensor of the embodiment 1 of the present application in the antigen concentration detection.

[0092] Figure 14 A fitting curve diagram of AFP antigen concentration and displacement response for AFP antigen concentration detection;

[0093] Figure 15 A trough displacement change and fixed wavelength reflectivity change diagram of surface plasmon resonance of the surface sensor of embodiment 1 of the application.

[0094] Wherein, 10 is a microfluidic channel, 101 is a groove structure, 102 is a convex structure, 103 is a micropost, 20 is a sensing chip, 30 is a grating structure, and 40 is an auxiliary convex. DETAILED DESCRIPTION

[0095] In order to make the skilled in the art more clearly understand the technical solutions described in the application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the application.

[0096] The raw materials, reagents or devices used in the following examples are commercially available or can be obtained by known methods unless otherwise specified.

[0097] Example 1

[0098] The structural schematic diagram of the microfluidic channel of embodiment 1 of the application is shown in Figure 1 .

[0099] The cross-sectional schematic diagram of the microfluidic channel of embodiment 1 of the application along the flow direction is shown in Figure 2 .

[0100] The cross-sectional schematic diagram of the surface sensor of embodiment 1 of the application along the flow direction is shown in Figure 3 .

[0101] Referring to Figure 2 and Figure 3 , a surface sensor comprises a microfluidic channel 10 and a sensing chip 20; the microfluidic channel 10 has a groove structure 101, and the groove structure is in a U-shaped form; the bottom surface of the groove structure 101 is provided with a strip-shaped convex structure 102 perpendicular to the fluid direction and arranged in an array; the upper end surface of the convex structure 102 is distributed with microposts 103, and the bottom surface of the corresponding groove structure 101 between the convex structures 102 is distributed with microposts 103.

[0102] The two ends of the groove structure 101 are provided with holes with a hole diameter of 0.7 mm as an inlet and an outlet of a detection solution; the depth of the groove structure 101 is 10 μm, and the size of the groove structure 101 in the width direction is 500 μm.

[0103] The height of the protruding structure 102 is 5 microns, the width of the protruding structure 102 is 30 microns, the length of the protruding structure 102 is 500 microns, and the distance between two adjacent protruding structures 102 is 20 microns.

[0104] The height of the micropillar 103 is 1 micron, the diameter of the micropillar 103 is not uniform, the diameter is 1-3 microns, and the micropillar is irregularly distributed.

[0105] The difference between the sensing chip in the embodiment 1 of the present application and the product in the embodiment 1 of the prior art with the publication number CN 113433094A is only that the metal layer of the embodiment 1 of the present application is formed by using a magnetron sputtering instrument to first deposit gold for 250 seconds and then deposit silver for 1000 seconds, to form a gold film with a thickness of 50 nanometers and a silver film with a thickness of 250 nanometers, and the other parts are the same as the product in the embodiment 1 of CN 113433094A.

[0106] The following is a preparation method of a surface sensor, mainly involving the preparation process of a microfluidic channel template, a transfer process, a microfluidic channel process, and a combination process of a sensing chip.

[0107] The processing flow diagram of the microfluidic channel in the embodiment 1 of the present application is shown in Figure 4 The photoresist 1 is a positive photoresist, and the photoresist 2 is a negative photoresist.

[0108] Referring to Figures 4-6 A preparation method of a surface sensor, comprising the following steps:

[0109] (1) Clean the silicon template (silicon wafer), cover the photoresist 1 AR P3740 (purchased from the German A1lresist GmbH company) on the surface of the silicon template, expose the laser to the randomly distributed circular hole structure with a diameter of 1-3 microns on the mask material through photolithography, then perform etching, form a circular hole with a depth of 1 micron on the silicon template at the position corresponding to the circular hole structure, and obtain a silicon wafer with a circular hole structure;

[0110] (2) Uniformly coat the photoresist 2 AZ2035 (purchased from the Merck company) on the surface of the silicon wafer with the circular hole structure obtained in step (1), and perform photolithography to expose the laser to the grating structure with an array distribution with a width size of 20 microns, to form a mask material with a grating structure, the distance between two adjacent mask materials with a grating structure is 30 microns, then use the mask material with a grating structure as a mask to etch the silicon wafer, the position of the silicon wafer corresponding to the mask material is not etched, and the other areas of the silicon wafer are etched downward, the etching depth is 10 microns, after removing the mask material, a silicon wafer with an array-arranged grating structure 30 is obtained, and the grating structure 30 has the circular hole structure obtained in step (1);

[0111] (3) Covering the surface of the silicon wafer with the grating structure 30 obtained in step (2) with photoresist 2AZ2035, and performing photolithography, laser exposure, and forming a mask material in the shape of a work structure covering the grating structure, the width of the work structure at the middle position being 500 μm, the length being 1.8 cm, the length dimension of the two ends of the work structure being 1500 μm, and the width dimension being 1500 μm, the mask material in the shape of a work structure forming a protective effect on the covered structure, ensuring that the structure will not be affected by the subsequent etching process; using the mask material in the shape of a work structure as a mask to etch the silicon wafer, the etching depth being 10 μm, to obtain a silicon wafer with an auxiliary protrusion 40, i.e. a microfluidic channel template, which is arranged in an array, and a structural diagram of the microfluidic channel template is shown in Figure 5

[0112] (4) Transfer (reproduction): using piranha solution (concentrated H2SO4:H2O2 volume ratio of 7:3) to clean the mask material remaining on the surface of the microfluidic channel template obtained in step (3), rinsing with deionized water, blowing dry with nitrogen, and baking on a hot plate for 10 min before use; in order to ensure that the PDMS and the microfluidic channel template are successfully separated, a release agent TFOCS is evaporated on the microfluidic channel template, specifically, 0.5 mL of release agent is applied to the surface of the microfluidic channel template, and placed in a vacuum drying oven, evaporated for 30 min, and then taken out; the silicone mixture (Dow Corning Sylgard 184) is poured onto the microfluidic channel template with the release agent evaporated, degassed in a vacuum dryer, and cured at a temperature of 70°C for 2 h, and then demoulded, and two holes (diameter 0.7 mm) are set at the two ends of the work structure as the inlet and outlet of the detection solution respectively, to obtain a microfluidic channel, and a top view of the microfluidic channel is shown in Figure 6

[0113] (5) In order to bond the microfluidic channel with the sensing chip to form a surface sensor, a film needs to be plated on the non-functional area (the area without structural features and bonded with the microfluidic channel) of the sensing chip, because the gold metal layer of the sensing chip cannot be bonded with the microfluidic channel, a layer of SiO2 needs to be plated as an adhesive layer, after the mask shields the structural area of the sensing chip, the sensing chip is placed in a film plating device, 5 nm of Cr / Al layer is sputtered (gold does not combine with SiO2, Cr / Al is needed as an intermediate adhesive layer, magnetron sputtering, direct current power (DC), argon gas (Ar) 45 sccm, 100 W, 30 s), and then 25 nm of SiO2 is sputtered (magnetron sputtering, radio frequency power (RF), argon gas (Ar) 60 sccm, oxygen gas (O2) 20 sccm, 150 W, 1200 s);

[0114] ​​(6) placing the microfluidic channel and the sensing chip processed in step (5) in a plasma cleaning machine, treating the microfluidic channel and the sensing chip with an oxygen plasma with a power of 80 W for 1 min, then placing the two sides of the microfluidic channel and the sensing chip with the concave-convex structure characteristics opposite to each other, and the non-structure characteristic areas are attached, forming a fluid channel, that is, forming a surface sensor, then heating at 70℃ for 3 min to enhance the bonding strength of the PDMS of the microfluidic channel and the interface of the sensing chip, and obtaining the surface sensor of the microfluidic channel and the sensing chip.

[0115] The scanning electron microscope image of the microfluidic channel prepared in Example 1 of the present application is shown in Figure 7 , wherein, Figure 7 Fig. (a) and Fig. (b) are scanning electron microscope images at different magnifications. The convex structure of the microfluidic channel corresponds to the long strip-shaped features in the scanning electron microscope image, and the micropillars correspond to the circle-shaped features in the scanning electron microscope image, and it can be seen from Figure 7 that the micropillars are randomly distributed on the convex structure and between the convex structures, and the diameters of the micropillars are not the same.

[0116] Example 2

[0117] The difference between Example 2 and Example 1 is only that the metal layer of the sensing chip in Example 2 is deposited with gold by a magnetron sputtering instrument for 1500 s to form a 300 nm gold film, and the others are the same as in Example 1.

[0118] Example 3

[0119] The difference between Example 3 and Example 1 is only that the metal layer of the sensing chip in Example 3 is first deposited with gold by a magnetron sputtering instrument for 500 s and then deposited with silver for 800 s to form a 100 nm gold film and a 200 nm silver film, and the others are the same as in Example 1.

[0120] Example 4

[0121] The difference between Example 2 and Example 1 is only that the metal layer of the sensing chip in Example 4 is first deposited with gold by a magnetron sputtering instrument for 500 s and then deposited with aluminum for 500 s to form a 100 nm gold film and a 200 nm aluminum film, and the others are the same as in Example 1.

[0122] Comparative Example 1

[0123] The difference between Comparative Example 1 and Example 1 is only that the microfluidic channel in Comparative Example 1 is only provided with a convex structure at the bottom of the groove structure, does not contain micropillars, and the upper end surface of the convex structure is also not provided with micropillars, and the others are the same as in Example 1.

[0124] Comparative Example 2

[0125] The difference between Comparative Example 2 and Example 1 is only that the microfluidic channel of Comparative Example 2 has no protruding structure and no micropillar, that is, the bottom surface of the groove is a flat plane, and the other is the same as Example 1.

[0126] The cross-sectional view of the microfluidic channel of Comparative Example 2 along the flow direction is shown in Figure 8 The bottom surface of the microfluidic channel is not distributed with protruding structures and micropillars, and the bottom surface of the groove is a flat plane.

[0127] Performance test

[0128] 1. Flow field simulation test

[0129] COMSOL software was used to simulate the static flow state of the fluid in the rectangular microfluidic channel without perturbation structure (Comparative Example 2) and the microfluidic channel with periodic groove perturbation structure (Example 1). The flow field simulation diagram of the microfluidic channel of Example 1 and Comparative Example 2 at a flow field flow rate of 10 mm / s is shown in Figure 9 , wherein, Figure 9 , wherein, Figure 9 , wherein,

[0130] It can be seen from Figure 9 that in the flat microfluidic channel of the prior art, the flow field is uniform and the flow field does not change, while compared with the microfluidic channel of the prior art, the flow field of the fluid changes due to the presence of the protruding structure and the micropillar in the microfluidic channel of the present application, the flow rate increases, the efficiency of material exchange can be increased, the influence of FP interference phenomenon on the test spectrum is reduced, and then the test error is reduced and the detection sensitivity is improved.

[0131] 2. Reflectance spectrum test

[0132] The reflectance spectrum of the surface sensor of Example 1 and Comparative Example 1 was tested by using a surface sensing system, and the structure diagram of the surface sensing system is shown in Figure 10 The surface sensor in the surface sensing system uses the surface sensor of Example 1 and Comparative Example 1, respectively. The surface sensor of Example 1 and Comparative Example 1 is placed on the sample stage, and phosphate buffered saline (PBS) is introduced at a flow rate of 1 mL / h. The reflectance spectrum is tested, specifically, the light source passes through the half-transmission half-reflection mirror to form a light spot with a diameter of 100 μm on the sample surface through the microscope objective lens, and the light spot is aligned with the sensing area Figure 3The yellow area in Figure 20) is used to transmit light reflected from the sample surface to the detector, which then provides a reflectance spectrum. A flexible tube is inserted into the inlet and outlet of the microfluidic channel. The inlet is connected to a syringe, which is mounted on a syringe pump. The sample delivery rate is set to 1 mL / h. The surface sensor is fixed to the microscope stage. The structural area of ​​the sensor chip is identified through a microscope. The system then switches to dark field mode and begins to continuously collect reflectance spectra. The reflectance spectra of the surface sensor of Example 1 and Comparative Example 1 are shown in Figure 2. Figure 11 As shown, the horizontal axis Wavelength represents the wavelength, and the vertical axis Reflection (%) represents the reflectivity. Figure 11 Curve a in FIG represents the reflectivity spectrum of the surface sensor of Comparative Example 1. Figure 11 Curve b in FIG. 1 represents the reflectance spectrum of the surface sensor of Example 1.

[0133] Depend on Figure 11 It can be seen that the reflectivity spectrum curve of the surface sensor in Comparative Example 1 exhibits significant fluctuations, particularly with increasing wavelengths. This fluctuation is caused by FP resonance between the microfluidic channel and the sensor chip. However, the introduction of micropillars in the surface sensor of Example 1 disrupts the resonance between the two reflective surfaces of the microfluidic channel and the sensor chip, resulting in a smooth reflectivity spectrum curve free of FP resonance, reducing test error and improving sensitivity.

[0134] 3. Antibody Binding Response Test

[0135] The antibody binding responsiveness of the surface sensors of Example 1 and Comparative Example 2 was tested using a surface sensing system. The specific method was as follows: the surface sensors of Example 1 and Comparative Example 2 were placed on a sample table, and PBS buffer was passed through at a flow rate of 1 mL / h for 15 min, and then 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mM N-hydroxysuccinimide (NHS) were passed through for activation for 15 min, and PBS buffer was washed for 15 min. Then, 100 μg / mL alpha-fetoprotein antibody (Anti-AFP) was passed through, and the structural area of ​​the sensor chip was identified by microscopy. The surface sensing system was then switched to dark field mode and began to continuously collect reflectance spectra. The reflectance spectrum (trough) shift light response of the surface sensors of Example 1 and Comparative Example 2 was monitored in real time to obtain the antibody binding response curve, as shown in FIG. Figure 12 As shown. Among them, Figure 12 Figure (a) is the antibody binding response curve of the surface sensor of Comparative Example 2. Figure 12 Figure (b) shows the antibody binding response curve of the surface sensor of Example 1, where the abscissa "Time (min)" represents time (minutes) and the ordinate "Δλ (nm)" represents response (nanometers).

[0136] Depend onFigure 12 It can be seen that the response curve of the surface sensor of Comparative Example 1 gradually slows down after about 30 min, while the response curve of the surface sensor of Example 1 gradually stabilizes after about 10 min, and the fluctuation of the red curve in (a) is greater than that of the black curve in (b), because the perturbation structure (the protruding structure and the micropillar in the microfluidic channel of Example 1) accelerates the exchange rate of substances in the microfluidic channel, and the introduction of the micropillar structure destroys the resonance of the two reflection planes of the microfluidic channel and the sensing chip, reducing the FP effect.

[0137] 4. Antigen concentration detection

[0138] The application of the surface sensor of Example 1 in antigen concentration detection specifically includes the following steps:

[0139] (1) Sensing chip placement: Place the sensing chip on the test bench, and use a syringe pump to inject phosphate buffered saline (PBS) into the microfluidic channel of Example 1 at a flow rate of 1 mL / h. Identify the structure area of the sensing chip through a microscope, then switch the surface sensing system to a dark field mode, and start continuous acquisition of the reflection spectrum to monitor the reflectance spectrum in real time;

[0140] (2) Microfluidic channel activation: Inject 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mM N-hydroxysuccinimide (NHS) 2-(N-morpholino)ethanesulfonic acid (MES) solution into the microfluidic channel from the inlet of the microfluidic channel for 15 min to activate the carboxyl terminal group;

[0141] (3) Antibody immobilization: Wash the activated microfluidic channel obtained in step (2) with PBS, and after washing for 15 min, inject 100 μg / mL of alpha-fetoprotein antibody (Anti-AFP) into the microfluidic channel to immobilize for 50 min, to obtain a treated surface sensor; -1

[0142] (4) Blocking: Wash the surface sensor treated in step (3) with PBS for 15 min, and then inject 10 g / L of bovine serum albumin (BSA) to block the vacant sites for 40 min; -1

[0143] (5) Testing: Wash the surface sensor treated in step (4) with PBS for 15 min, and then inject 1000 ng / mL of AFP antigen for 30 min;

[0144] ​​(6) Regeneration (elution) of the sensing chip: the surface sensor after step (5) is washed by PBS, after 15 min, dissociation solution (NaH2PO4 / H3PO4 buffer solution with PH=3) is injected, after 15 min, PBS is replaced, at this time, the analyte antigen is eluted;

[0145] (7) Test: the surface sensor is injected with 1000 ng / mL AFP antigen again for 30 min;

[0146] (8) Regeneration (elution) of the sensing chip: the surface sensor after step (7) is washed by PBS, after 15 min, dissociation solution (NaH2PO4 / H3PO4 buffer solution with PH=3) is injected, after 15 min, PBS is replaced, at this time, the analyte antigen is eluted.

[0147] The above modification and test process are monitored in real time, the AFP antigen concentration can be calculated by (5), (7) Δλ response, the real-time response curve of the surface sensor of Example 1 in the antigen concentration detection is shown in Figure 13 , wherein the abscissa Time (h) represents time (hour), and the ordinate Δλ (nm) represents response (nanometer).

[0148] It can be seen from Figure 13 that the analyte can be dissociated by the dissociation solution after the AFP antigen test, the previously fixed antibody Anti-AFP does not fall off and remains active, and the response can be generated again after the AFP antigen is injected again and the response curve is consistent with the previous one.

[0149] In the AFP antigen concentration detection, the AFP antigen concentration and displacement response are fitted, and the fitting curve is shown in Figure 14 , wherein the abscissa C [AFP Antigen] (ng / mL) represents the AFP antigen concentration (nanogram per milliliter), the ordinate Δλ (nm) represents the response (nanometer), and R 2 AFP =0.9943 represents the correlation coefficient after fitting is 0.9943, and AFP Antigen represents AFP antigen.

[0150] It can be seen from Figure 14 that the surface sensor of Example 1 can detect the response of 1 ng / mL AFP antigen without the introduction of the secondary antibody, and the fitting curve is consistent with the S-type fitting in the range of 1-10000 ng / mL -1 The current clinical detection limit of AFP antigen is 7 ng / mL, which indicates that the surface sensor prepared by the application has excellent sensitivity.

[0151] In addition, when performing the above data detection and analysis, the trough displacement of the surface plasmon resonance and the fixed wavelength reflectivity of the surface sensor at different time points are extracted. The changes in the trough displacement of the surface plasmon resonance and the fixed wavelength reflectivity of the surface sensor in Example 1 are as follows: Figure 15 As shown. Among them, Figure 15 Figure (a) shows the change of the trough displacement of the surface plasmon resonance. The horizontal axis Time (h) represents time (hours), and the vertical axis Δλ (nm) represents the response (nanometers); Figure 15 Figure (b) is a fixed wavelength reflectivity change diagram of surface plasmon resonance, where the horizontal axis Time (h) represents time (hours) and the vertical axis Reflection (%) represents reflectivity.

[0152] Depend on Figure 15 It can be seen that the change trend of the trough displacement of the surface plasmon resonance and the reflectivity at a fixed wavelength are consistent, so the analysis method can be to monitor the wavelength or the light intensity, which can provide more possibilities for sensing detection.

[0153] The surface sensors of Examples 2-4 of the present invention can also reduce the influence of FP interference on the test spectrum, reduce or even eliminate the influence of FP resonance, thereby reducing test errors and improving detection sensitivity; at the same time, they have excellent sensitivity in antigen concentration detection.

[0154] In summary, the present invention changes the flow field within the microfluidic channel by providing raised structures and micropillars in the microfluidic channel. Compared with an unstructured flow field, the flow velocity of the fluid is increased, the material exchange rate within the microchannel is accelerated, the impact of FP interference on the test spectrum is reduced, and the impact of FP resonance is reduced or even eliminated, thereby reducing test errors and improving detection sensitivity. When the surface sensor of the present invention is used to detect AFP antigen, no secondary antibody is required, and 1 ng mL -1 The response of AFP antigen was consistent with S-shaped fitting in the range of 1-10000 ng / mL.

[0155] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A surface sensor, characterized in that: It includes a sensor chip and a microfluidic channel; the sensor chip and the microfluidic channel are placed opposite to each other with the concave and convex sides thereof to form a fluid channel; The microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a strip-shaped protrusion structure perpendicular to the fluid direction and arranged in an array; The upper end surface of the protrusion structure is distributed with micron columns, and the bottom surfaces of the groove structures corresponding to the protrusion structures are distributed with micron columns, and the micron columns are randomly distributed on the bottom surfaces of the groove structures and the upper end surfaces of the protrusion structures; The structured area of ​​the sensor chip and the structured area of ​​the microfluidic channel having a concave and convex surface are placed opposite to each other, and the non-structured area of ​​the sensor chip and the non-structured area of ​​the microfluidic channel are attached together to form a fluid channel, thereby obtaining the surface sensor; The sensor chip comprises a substrate, a dielectric layer, and a metal layer in sequence, wherein the dielectric layer has a plurality of dielectric grooves, and the plurality of dielectric grooves are periodically distributed with equal intervals; The opening width of the dielectric groove gradually decreases in the direction from the groove opening to the groove bottom; the metal layer is arranged on the dielectric layer and forms a metal groove corresponding to each dielectric groove.

2. The surface sensor according to claim 1, wherein The depth of the groove structure is 12-33 μm; the width of the groove structure is 200-1500 μm.

3. The surface sensor according to claim 1, wherein The height of the protruding structure is 4.5-10 μm; the width of the protruding structure is 18-42 μm; the length of the protruding structure is 100-1000 μm; and the distance between adjacent protruding structures is 8-32 μm.

4. The surface sensor according to claim 1, wherein The diameter of the micron column is 0.9-3.3 μm; the height of the micron column is 0.8-1.2 μm.

5. The method for preparing a surface sensor according to any one of claims 1 to 4, characterized in that: The following steps are involved: The microfluidic channel and the side of the sensor chip having the concave and convex surfaces are placed opposite to each other to prepare the surface sensor.

6. The preparation method according to claim 5, characterized in that The method for preparing the microfluidic channel comprises the following steps: (1) forming randomly distributed micropores on the template to obtain a template with distributed micropores; (2) Covering the surface of the template obtained in step (1) with a mask material, forming a mask material having a grating structure by photolithography, etching the template using the mask material having a grating structure as a mask, and forming an array-arranged grating structure on the template; (3) covering the surface of the template obtained in step (2) with a mask material, forming a mask material covering the grating structure by photolithography, and etching the template using the mask material covering the grating structure as a mask to form auxiliary protrusions on the template; (4) Covering the organic silicon mixture on the template obtained in step (3), curing and separating, and obtaining a microfluidic channel.

7. The preparation method according to claim 6, characterized in that In step (1), the diameter of the micropore is 0.9-3.3 μm; the depth of the micropore is 0.8-1.2 μm; And / or, in step (2), the height of the grating structure is 4.5-10 μm; the width of the grating structure is 8-32 μm; the length of the grating structure is 100-1000 μm; and the distance between adjacent grating structures is 18-42 μm; And / or, in step (3), the height of the auxiliary protrusion is 12-33 μm; the width of the auxiliary protrusion is 200-1500 μm.

8. A surface sensor system, characterized in that: The surface sensor comprises the surface sensor according to any one of claims 1 to 4.

9. Use of the surface sensor according to any one of claims 1 to 4 or the surface sensor system according to claim 8 in detection and analysis for purposes other than disease diagnosis.

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