Fluorescence imaging sensing detection system and method

Abstract

The present invention relates to the technical field of optical sensing detection, and provides a fluorescence imaging sensing detection system and method. The fluorescence imaging sensing detection system of the present invention comprises a dark box, an excitation light source module, a sensing module, an optical path adjustment module and an image acquisition module. The excitation light source module is arranged in the dark box and is used to provide an excitation light source; the sensing module is arranged on the top of the dark box, and a cover glass for bulk solution detection or a biosensor chip for solid surface detection is arranged in the sensing module, and a plurality of sample delivery pipelines are connected to the sensing module and are used to provide a sample solution to be detected to the cover glass or the biosensor chip; the optical path adjustment module is used to filter and steer the output light of the excitation light source, guide the output light of the excitation light source to the sample solution to be detected in the sensing module to make it emit fluorescence, and receive the fluorescence; the image acquisition module is used to collect the fluorescence image transmitted through the optical path adjustment module and convert it into a corresponding image data signal.

Description

Technical Field

[0001] The present invention relates to the field of optical sensing detection technology, and in particular to a fluorescence imaging sensing detection system and method. Background Art

[0002] Fluorescence sensing technology is widely used in many fields such as clinical diagnosis, pharmaceutical development, food safety and environmental monitoring due to its advantages of high sensitivity, good specificity and diverse methods. Fluorescence sensing technology can be roughly divided into two categories according to the spatial distribution of fluorescence: bulk solution and solid surface. Current research mainly focuses on the development of fluorescence sensing technology itself, but it requires the support of supporting optoelectronic systems and signal processing methods to realize sensing applications.

[0003] Traditional fluorescence sensing detection usually requires laser excitation and the use of an independent microscope. Although the technology is mature and advanced and can detect some analytes, such instruments are usually expensive and bulky, and the detection process requires professionals to operate under laboratory conditions, which seriously affects the speed and convenience of the test and cannot meet the growing detection needs of all sectors of society, especially in poor areas with limited resources. In addition, traditional fluorescence analyzers find it difficult to detect multiple samples simultaneously, and the actual detection efficiency is low. Summary of the invention

[0004] The present invention provides a fluorescence imaging sensor detection system and method, which are used to solve the defects of poor portability and low detection efficiency of fluorescence sensor detection equipment in the prior art, and improve the convenience and efficiency of detection.

[0005] In a first aspect, the present invention provides a fluorescence imaging sensing detection system, comprising:

[0006] A dark box is provided with a containing cavity;

[0007] An excitation light source module, disposed in the accommodating cavity, and used to provide an excitation light source;

[0008] A sensor module is arranged on the top of the dark box, wherein a cover glass for bulk solution detection or a biosensor chip for solid surface detection is arranged in the sensor module, and the sensor module is connected with a sample delivery pipeline for providing a sample solution to be detected to the cover glass or the biosensor chip;

[0009] An optical path adjustment module, the optical path adjustment module is connected to the bottom of the sensor module, and one side of the optical path adjustment module is connected to the excitation light source module, and is used to filter and steer the output light of the excitation light source, guide the output light of the excitation light source to the sample solution to be detected in the sensor module to make it emit fluorescence, and receive the fluorescence;

[0010] An image acquisition module is arranged in the accommodating cavity. The image acquisition module is arranged at the bottom of the optical path adjustment module and is used to acquire a fluorescent image passing through the optical path adjustment module and convert it into a corresponding image data signal.

[0011] According to the fluidic multi-channel fluorescence imaging sensor detection system provided by the present invention, the sensor module includes:

[0012] Fixture cover;

[0013] The fixture bottom plate is provided with a receiving groove, a through hole for light transmission is opened at the bottom of the receiving groove, the through hole is covered with the cover glass or the biosensor chip, the receiving groove is connected with the sample delivery pipeline all around, and the fixture bottom plate is detachably connected to the fixture cover plate;

[0014] The fluidic chip has a bottom surface in contact with the top surface of the cover glass or the biosensor chip, and the fluidic chip is provided with a plurality of micro flow channels for storing sample solutions to be detected.

[0015] According to the fluidic multi-channel fluorescence imaging sensor detection system provided by the present invention, the fluidic chip comprises, from bottom to top:

[0016] A flow channel layer, wherein the flow channel layer is provided with four micro flow grooves at intervals, the areas where the four micro flow grooves are located correspond to the through holes, and micro flow inlets and micro flow outlets are provided around the flow channel layer;

[0017] The connecting layer is provided with four pairs of liquid-conducting grooves, one pair of the liquid-conducting grooves is correspondingly distributed at both ends of the micro-circulation groove, and each of the liquid-conducting grooves is respectively connected with the micro-circulation groove, the micro-flow inlet or the micro-flow outlet;

[0018] The bonding layer covers the top of the connecting layer, and the flow channel layer, the connecting layer and the bonding layer are bonded and connected.

[0019] According to the fluidic multi-channel fluorescence imaging sensor detection system provided by the present invention, the optical path adjustment module includes:

[0020] A mirror base is provided with a cavity, and a first light hole corresponding to the through hole is opened at the top of the mirror base, a second light hole corresponding to the image acquisition module is opened at the bottom of the mirror base, and a third light hole is opened on the side of the mirror base connected to the excitation light source module, wherein the first light hole, the second light hole and the through hole are coaxially arranged;

[0021] A dichroic beam splitter prism is disposed in the cavity and is used to filter and steer the outgoing light of the excitation light source, and guide the outgoing light of the excitation light source in a direction perpendicular to the cover glass or the biosensor chip to the sample solution to be detected in the sensor module to make it emit fluorescence;

[0022] A mirror base bracket, wherein the top end of the mirror base bracket is connected to the mirror base, and the bottom end of the mirror base bracket is connected to the bottom of the accommodating cavity.

[0023] According to the fluidic multi-channel fluorescence imaging sensor detection system provided by the present invention, the mirror base bracket comprises:

[0024] Base;

[0025] A first connecting rod, wherein the first connecting rod is movably connected to the first connecting rod in a vertical direction, and a top end of the first connecting rod is connected to a transfer block;

[0026] A second connecting rod, wherein the second connecting rod is arranged perpendicular to the first connecting rod, a first end of the second connecting rod is movably connected to the adapter block along a horizontal direction, and a second end of the second connecting rod is connected to the mirror seat.

[0027] According to the flow control multi-channel fluorescence imaging sensor detection system provided by the present invention, the excitation light source module includes the excitation light source, converging lens, pinhole diaphragm and neutral density filter arranged in sequence; the centers of the light source, converging lens, pinhole diaphragm and neutral density filter are arranged coaxially.

[0028] According to the fluidic multi-channel fluorescence imaging sensing detection system provided by the present invention, the excitation light source is used to provide narrow-band output light with a wavelength range of 470nm-510nm, so as to excite fluorescence with a wavelength range of 500nm-560nm for the solution on the side to be detected.

[0029] According to the fluidic multi-channel fluorescence imaging sensor detection system provided by the present invention, the image acquisition module includes:

[0030] A camera, wherein the camera is connected to the bottom of the accommodating cavity through a camera fixing member;

[0031] A lens barrel is arranged on the lens of the camera, the top of the lens barrel is connected to the bottom of the lens holder, a relay lens is arranged in the lens barrel, and the relay lens, the lens of the camera and the second light hole are coaxially arranged;

[0032] The fixing bracket is arranged between the lens base and the camera, the top end of the fixing bracket is connected to the lens base, and the bottom end of the fixing bracket is connected to the camera.

[0033] In a second aspect, the present invention provides a flow control multi-channel fluorescence imaging sensor detection method, which adopts the fluorescence imaging sensor detection system described in the first aspect, and includes the following steps:

[0034] Determine the excitation light source parameters of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0035] Place the cover glass in the receiving slot of the sensor module, make sure the excitation light source is turned off, and obtain a dark background image through the image acquisition module;

[0036] preparing a negative sample solution containing a fluorescent probe, providing the negative sample solution with an incubation environment required for the fluorescent probe to recognize the analyte, and introducing the negative sample solution into the micro flow channel of the sensor module;

[0037] Turn on the light source and obtain the image of the negative sample through the image acquisition module;

[0038] Turn off the excitation light source, remove the negative sample solution in the micro flow-through slot, prepare a sample solution to be detected containing a fluorescent probe, provide the solution with an incubation environment required for the fluorescent probe to identify the analyte, and introduce the sample solution to be detected containing the fluorescent probe into the micro flow-through slot;

[0039] Turn on the light source and obtain the image of the sample to be detected through the image acquisition module.

[0040] In a third aspect, the present invention further provides a flow control multi-channel fluorescence imaging sensor detection method, which uses the fluorescence imaging sensor detection system described in the first aspect, and includes the following steps:

[0041] Determine the parameters of the pinhole aperture and neutral density filter of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0042] Modifying a suitable first antibody on the biosensor chip for identifying the analyte, placing the biosensor chip functionalized with the first antibody in a receiving slot of the sensor module, confirming that the excitation light source is in an off state, and obtaining a dark background image through an image acquisition module;

[0043] preparing a negative sample solution, introducing the negative sample solution into the micro flow channel of the sensor module, providing the sensor module with an incubation environment required for antigen-antibody recognition, and then removing the negative sample solution;

[0044] Prepare a second antibody solution containing a fluorescent group label, introduce the second antibody solution into the micro flow channel of the sensor module, provide the sensor module with an incubation environment required for antigen-antibody recognition, and then remove the second antibody solution;

[0045] Turn on the light source and obtain the image of the negative sample through the image acquisition module;

[0046] Turn off the excitation light source, clean the micro flow channel, prepare the sample solution to be detected, introduce the sample solution to be detected into the micro flow channel, provide the sensor module with the incubation environment required for antigen-antibody recognition, and then remove the sample solution to be detected;

[0047] Prepare a second antibody solution containing a fluorescent group label, introduce the second antibody solution into the micro flow channel of the sensor module, provide the sensor module with an incubation environment required for antigen-antibody recognition, and then remove the second antibody solution;

[0048] Turn on the light source and obtain the image of the sample to be detected through the image acquisition module.

[0049] The fluorescence imaging sensing detection system provided by the present invention comprises a dark box, an excitation light source module, a sensing module, an optical path adjustment module and an image acquisition module. By arranging each module in the dark box and reasonably setting the position of each module according to the optical path transmission route, the volume of the optical structure is reduced, thereby reducing the volume of the fluorescence imaging sensing detection system and improving portability. Furthermore, according to actual conditions, by arranging a cover glass for bulk solution detection or a biosensor chip for solid surface detection in the sensing module, a fluorescence sensing detection mode suitable for bulk solution and solid surface is formed, and multiple samples are detected simultaneously through multiple micro flow slots on the fluidic chip, thereby realizing multi-sample detection, saving detection time and improving detection efficiency.

[0050] The present invention also provides a fluorescence imaging sensing detection method, which uses the fluorescence imaging sensing detection system described in the first aspect to realize fluorescence sensing detection of bulk solution and solid surface. The derivation process of this beneficial effect is generally similar to the derivation process of the beneficial effect brought by the above-mentioned fluorescence imaging sensing detection system, and will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1 A schematic diagram of the overall assembly of a fluorescence imaging sensor detection system provided by an embodiment of the present invention;

[0053] Figure 2 A schematic diagram of the decomposition structure of a fluorescence imaging sensor detection system provided by an embodiment of the present invention;

[0054] Figure 3A schematic diagram of an excitation light source module of a fluorescence imaging sensing detection system provided in an embodiment of the present invention;

[0055] Figure 4 A schematic diagram of an optical path adjustment module of a fluorescence imaging sensor detection system provided in an embodiment of the present invention;

[0056] Figure 5 A schematic diagram of a sensor module of a fluorescence imaging sensor detection system provided in an embodiment of the present invention;

[0057] Figure 6 A schematic diagram of an image acquisition module of a fluorescence imaging sensor detection system provided in an embodiment of the present invention;

[0058] Figure 7 A partial schematic diagram of an image acquisition module of a fluorescence imaging sensor detection system provided in an embodiment of the present invention;

[0059] Figure 8 A schematic diagram of the optical path of the fluorescence imaging sensor detection system provided by an embodiment of the present invention when it is working;

[0060] Fig. 9 A flow chart of a fluorescence imaging sensing detection method provided by an embodiment of the present invention;

[0061] Fig.10 This is a flow chart of a fluorescence imaging sensing detection method provided by another embodiment of the present invention.

[0062] Reference numerals:

[0063] 1. Dark box; 11. Top plate; 111. Square mounting hole; 12. Wall plate; 121. Light source power supply socket; 122. Camera socket; 123. Window plate; 13. Bottom plate; 14. Optical breadboard;

[0064] 2. Excitation light source module; 211. Excitation light source; 212. Converging lens; 213. Lamp holder; 214. Fixed lens barrel; 215. Fixed cage plate; 216. Fixed cage rod; 217. Lamp bead power supply line; 218. Pinhole aperture; 219. Fixed clamp ring; 221. Neutral density filter; 222. Filter fixed cage plate; 223. Filter fixed cage rod; 224. Filter fixed lens barrel; 225. Filter fixed clamp ring;

[0065] 3. Optical path adjustment module; 31. Dichroic beam splitter prism; 32. Mirror base; 33. Light shielding cover; 34. Second connecting rod; 35. Adapter block; 36. First connecting rod; 37. Base;

[0066] 4. Sensor module; 411. First sample inlet pipeline; 412. First sample outlet pipeline; 421. Second sample inlet pipeline; 422. Second sample outlet pipeline; 431. Third sample inlet pipeline; 432. Third sample outlet pipeline; 441. Fourth sample inlet pipeline; 442. Fourth sample outlet pipeline; 45. Pipeline connector; 46. Biosensor chip; 47. Fluidic chip; 471. Flow channel layer; 472. Connecting layer; 473. Bonding layer; 481. Fixture bottom plate; 482. Fixture cover plate;

[0067] 5. Image acquisition module; 511. Camera; 512. Camera fixing part; 513. Lens; 514. Lens flange; 521. Relay lens; 522. Lens barrel; 531. Fixing bracket; 54. Camera power supply line. DETAILED DESCRIPTION

[0068] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] Combine the following Figures 1 to 10 A fluorescence imaging sensing detection system and method provided in an embodiment of the present invention are described.

[0070] The fluidic multi-channel fluorescence imaging sensor detection system provided in this embodiment includes: a dark box 1, an excitation light source module 2, an optical path adjustment module 3, a sensor module 4 and an image acquisition module 5.

[0071] Among them, the dark box 1 is provided with a accommodating cavity, and the excitation light source module 2 is arranged in the accommodating cavity for providing an excitation light source; the sensor module 4 is arranged on the top of the dark box 1, and a cover glass for bulk solution detection or a biosensor chip 46 for solid surface detection is arranged in the sensor module 4, and the sensor module 4 is connected with multiple sample delivery pipelines for providing the cover glass or the biosensor chip 46 with the sample solution to be detected; the optical path adjustment module 3 is connected to the bottom of the sensor module 4, and one side of the optical path adjustment module 3 is connected to the excitation light source module 2, which is used to filter and steer the output light of the excitation light source, guide the output light of the excitation light source to the sample solution to be detected in the sensor module 4 to make it emit fluorescence, and receive the fluorescence; the image acquisition module 5 is arranged in the accommodating cavity, and the image acquisition module 5 is arranged at the bottom of the optical path adjustment module 3, which is used to collect the fluorescence image passing through the optical path adjustment module 3 and convert it into a corresponding image data signal.

[0072] In this embodiment, the dark box 1 is a closed structure with overall dimensions of 170 mm in length, 127 mm in width, and 132 mm in height, which meets the requirements of portability and is used to provide the dark environment required for the experiment. Each module is arranged in the dark box 1, and the dark box 1 can also provide shockproof protection.

[0073] Reference Figure 2 In some embodiments, the dark box 1 includes a top plate 11, a wall plate 12 and a bottom plate 13, which are connected to each other by screws to form a cubic structure. A window plate 123 is detachably connected to the front surface of the wall plate 12, which is used to adjust the internal modules after opening. The window plate 123 can be adsorbed in the window on the front surface of the wall plate 12 by magnetic buckles, or can be fastened by screws. A light source power supply socket 121 and a camera socket 122 can also be provided on one side of the wall plate 12. The top plate 11 is connected to the wall plate 12 by screws. The top plate 11 has a square mounting hole 111, and the sensor module 4 is embedded in the square mounting hole 111. The bottom of the sensor module 4 is connected to the optical path adjustment module 3 by screws, so that the sensor module 4 is only partially located outside the dark box 1, and multiple sample delivery pipelines are also located outside the dark box 1, which is convenient for delivering samples to be tested. The exposed design of the sensor module 4 relative to the dark box 1 provides convenience for the placement or removal of the cover glass or the biosensor chip 46 and the fluidic chip 47, as well as the sample entry and exit operations.

[0074] In a further embodiment, a signal processing module is also included. The signal processing module can be a computer and can be connected to the camera socket 122 to receive and store real-time image signals from the image acquisition module 5, and obtain the fluorescence sensing characteristics of the sample solution to be tested located on the biosensor chip 46 or the coverslip through a data analysis method.

[0075] Reference Figure 5 In this embodiment, the sensor module 4 includes: a fixture cover plate 482, a fixture bottom plate 481 and a fluidic chip 47. The material of the fluidic chip 47 is PDMS (Polydimethylsiloxane). The fixture bottom plate 481 is provided with a receiving groove. A through hole for light transmission is opened at the bottom of the receiving groove. The through hole is covered with a cover glass or a biosensor chip 46.

[0076] Exemplarily, the diameter of the through hole is 19 mm, and the cover glass or biosensor chip 46 is arranged coaxially with the through hole, so that the fluorescence can be acquired by the image acquisition module 5 directly below through the through hole. The receiving groove is connected with sample delivery pipelines on all sides, and the clamp base plate 481 and the clamp cover plate 482 are detachably connected. For example, the clamp cover plate 482 is placed on the clamp base plate 481, and the two can be positioned by four pairs of magnetic buckles and fastened by screws.

[0077] The bottom surface of the fluidic chip 47 contacts the top surface of the cover glass or the biosensor chip 46 . The fluidic chip 47 is provided with a plurality of micro flow grooves for storing the sample solution to be detected. Since the micro flow grooves run through the fluidic chip 47 , the solution in the micro flow grooves will contact the cover glass or the biosensor chip 46 .

[0078] It should be noted that the coverslip or biosensor chip 46 is close to the surface of the fluidic chip 47, but does not form a bonding connection. They are placed together in the receiving groove of the fixture bottom plate 481. The depth of the receiving groove is slightly smaller than the total thickness of the fluidic chip 47 and the coverslip or biosensor chip 46, and can be set to a difference of 1 mm. Based on this, the elasticity of the PDMS material is utilized to make the fluidic chip 47 tightly connected to the fixture bottom plate 481 and the coverslip or biosensor chip 46, respectively, and the through hole is sealed to prevent solution leakage here.

[0079] Reference Figure 5 In a further embodiment, the fluidic chip 47 includes, from bottom to top, a flow channel layer 471 , a connecting layer 472 and a bonding layer 473 .

[0080] The flow channel layer 471 is provided with four micro-circulation grooves at intervals, which may be in the shape of long strips with a width of 2 mm and a length of 16 mm. The distance between adjacent micro-circulation grooves is 2 mm, and the depth of the micro-circulation grooves is 1.5 mm. The areas where the four micro-circulation grooves are located correspond to the through holes, and micro-flow inlets and micro-flow outlets are provided around the flow channel layer 471. The connecting layer 472 is provided with four pairs of liquid-conducting grooves, one pair of liquid-conducting grooves is correspondingly distributed at both ends of the micro-circulation grooves, and each liquid-conducting groove is respectively connected to the micro-circulation groove, the micro-flow inlet or the micro-flow outlet. The bonding layer 473 covers the top of the connecting layer 472, and the flow channel layer 471, the connecting layer 472 and the bonding layer 473 are bonded and connected.

[0081] Reference Figure 5In the present embodiment, pipe connectors 45 connected to the containing grooves are arranged around the fixture bottom plate 481 for connecting the sample delivery pipeline. Eight sample delivery pipelines are arranged, which are distributed in groups of two around the fixture bottom plate 481, including a first sample inlet pipeline 411, a first sample outlet pipeline 412, a second sample inlet pipeline 421, a second sample outlet pipeline 422, a third sample inlet pipeline 431, a third sample outlet pipeline 432, a fourth sample inlet pipeline 441 and a fourth sample outlet pipeline 442. Each pipe connector 45 is located below the flow channel layer 471, corresponding to the microflow inlet and the microflow outlet around the flow channel layer 471. With such an arrangement, a four-channel inlet and outlet sample sensing module 4 can be realized, and eight pipe connectors 45 are arranged in sequence around the chip fixture. After the solution enters the receiving tank through four sample inlet pipes, it flows into the liquid guide groove of the connecting layer 472 through the microflow inlet of the flow channel layer 471, and then enters the corresponding microcirculation groove from one end. Since the bottom and top of the microcirculation groove are blocked, when the solution in the microcirculation groove is filled, it flows out through the corresponding liquid guide groove at the other end, and then flows out of the receiving tank from the corresponding sample outlet pipe through the microflow outlet.

[0082] With such a configuration, a multi-channel flow control effect can be achieved through the above scheme, and experimental detection of a single target analyte or multiple target analytes can be achieved. This embodiment uses four micro-circulation slots. Under the standard fluorescence intensity-concentration function relationship of the sample solution to be detected, multiple samples can be detected at the same time, and the concentration of the sample solution to be detected is obtained by the fluorescence intensity. Compared with only one micro-circulation slot, the detection efficiency is significantly improved. If the concentration function relationship is not perfect, one of the micro-circulation slots can be an unknown sample, and the other three micro-circulation slots can be standard solutions of gradient concentrations. Then, based on the comparison of the fluorescence intensities of the four micro-circulation slots, the concentration of the unknown sample can be estimated.

[0083] In this embodiment, four target analytes can be detected simultaneously. On the premise that standard functional relationships are established for the four target analytes, the four micro-circulation channels can be used to detect the four target analytes respectively. Usually, in this case, the same sample solution to be detected (such as a person's serum) is used and divided into four parts for separate detection. Compared with a single micro-circulation channel that can only detect one target analyte in a sample at a time, this detection has a higher throughput.

[0084] In some embodiments, the cover glass or the biosensor chip 46 is square with a side length of 20 mm, wherein a nanostructure sensor array is arranged on the biosensor chip 46, and the nanostructure sensor array is a metal nanostructure sensor array, and the metal includes one or more of gold, silver or copper, and gold is used in the embodiment of the present invention. The array of the nanostructure sensor array is a dot matrix, a hole array or a grating array. In this embodiment, the nanostructure pattern of the biosensor chip 46 is arranged with four columns, each column corresponds to a micro flow slot, and each column has 6 circular precious metal nanostructure film attachment areas with a diameter of 1.5 mm, which can realize multi-point detection, and its nanostructure can realize fluorescence enhancement after proper design, which is conducive to improving the accuracy of the system.

[0085] Reference Figure 4 In this embodiment, the optical path adjustment module 3 includes a lens holder 32, a dichroic beam splitter prism 31 and a lens holder bracket.

[0086] Among them, the mirror base 32 is provided with a cavity, and a first light hole corresponding to the through hole is opened on the top of the mirror base 32, a second light hole corresponding to the image acquisition module 5 is opened at the bottom of the mirror base 32, and a third light hole is opened on the side where the mirror base 32 is connected to the excitation light source module 2, and a light shielding cover 33 is arranged on the side of the mirror base 32 opposite to the third light hole. The first light hole, the second light hole and the through hole are coaxially arranged to realize optical coaxiality of the sensor module 4 and the optical path adjustment module 3 in the vertical direction.

[0087] The dichroic beam splitter prism 31 is disposed in the cavity, and is used to filter and steer the outgoing light of the excitation light source, and guide the outgoing light of the excitation light source to the sample solution to be detected in the sensor module 4 in a direction perpendicular to the cover glass or the biosensor chip 46 .

[0088] The top of the mirror base bracket is connected to the mirror base 32, and the bottom of the mirror base bracket is connected to the bottom of the accommodating cavity. In this arrangement, since the optical path adjustment module 3 is at the optical path turning position and has a large mass, a mirror base bracket is designed for it to achieve center of gravity support, which can effectively increase the structural stability of the system; the dichroic beam splitter prism 31 can realize 90° deflection of the incident light beam of the excitation light source with a wavelength of 470nm-510nm band to the sensor module 4 directly above, and realize non-deflection transmission of the fluorescent light beam with a wavelength of 500nm-560nm band incident from above, thereby having the functions of a 90° reflector and a filter at the same time, effectively saving space and improving portability; compared with the general dichroic beam splitter parallel plate, the dichroic beam splitter prism 31 can avoid the offset and virtual shadow phenomenon of the optical axis, which is conducive to ensuring the accuracy of fluorescent image recognition and extraction.

[0089] Reference Figure 4In a further embodiment, the mirror base bracket includes a base 37, a first connecting rod 36 and a second connecting rod 34. The first connecting rod 36 is movably connected to the base 37 in a vertical direction, and a transfer block 35 is connected to the top of the first connecting rod 36; the second connecting rod 34 is arranged perpendicular to the first connecting rod 36, and a first end of the second connecting rod 34 is movably connected to the transfer block 35 in a horizontal direction, and a second end of the second connecting rod 34 is connected to the mirror base 32; in some embodiments, the base 37 may be vertically connected with a sleeve, and the first connecting rod 36 is inserted into the sleeve. The first connecting rod 36 can be connected to the sleeve to realize vertical movement, and a locking bolt can be provided on the outside of the sleeve to position the first connecting rod 36; the adapter block 35 can be provided with a socket in the horizontal direction, the second connecting rod 34 is inserted in the socket, and a locking bolt is also provided at the corresponding socket on the adapter block 35, so that the second connecting rod 34 can be positioned, and the length of the second connecting rod 34 passing through the socket is adjusted, and the distance of the mirror seat 32 relative to the first connecting rod 36 is changed in turn, so as to realize the vertical adjustment of the first connecting rod 36 and the horizontal adjustment of the second connecting rod 34. With such a configuration, the vertical movement adjustment of the mirror seat 32 is realized by the movable connection between the first connecting rod 36 and the base 37 in the vertical direction, and the horizontal movement adjustment of the mirror seat 32 is realized by the movable connection between the second connecting rod 34 and the adapter block 35.

[0090] Reference Figure 3 In this embodiment, the excitation light source module 2 includes an excitation light source 211, a converging lens 212, a pinhole diaphragm 218, and a neutral density filter 221, which are sequentially arranged; the excitation light source 211, the converging lens 212, the pinhole diaphragm 218, and the neutral density filter 221 are arranged coaxially. Such an arrangement is to solve the problem of excessive power of the excitation light source 211. The pinhole diaphragm 218 and the neutral density filter 221 can be used to achieve power attenuation, thereby improving the signal-to-noise ratio of the collected image and avoiding the phenomenon of photobleaching of the fluorescent probe too quickly; at the same time, the opening size of the pinhole diaphragm 218 and the transmittance of the neutral density filter 221 can be adjusted according to the needs of the fluorescent probe to meet the experimental requirements.

[0091] The neutral density filter 221 (attenuation filter) is made into a sheet by utilizing the light absorption property of a substance and is arranged in the light path to attenuate the light intensity.

[0092] Specifically, the excitation light source 211, the converging lens 212, the pinhole diaphragm 218, the neutral density filter 221 and the third light hole are arranged in parallel and coaxially in sequence; in order to ensure the compactness of the structure and the optical coaxial requirements, the excitation light source 211 and the converging lens 212 are coaxially connected through a lamp holder 213, and the lamp holder 213 is set as a cylindrical structure. The lamp holder 213 is sleeved in a fixed lens barrel 214, and the fixed lens barrel 214 is connected to the fixed cage plate 215 by threads. The pinhole diaphragm 218 is fixed in the lamp holder 213 by a fixed clamping ring 219, and the neutral density filter 221 is fixed by a filter fixing clamping ring 2 25 is fixed in the filter fixing lens barrel 224, and the filter fixing lens barrel 224 is fixed on the filter fixing cage plate 222 by threads. Four filter fixing cage rods 223 are arranged on both sides of the filter fixing cage plate 222. A fixed cage rod 216 is also arranged on the side of the fixed cage plate 215 corresponding to the filter fixing cage plate 222, and the fixed cage rod 216 corresponds to and is connected to the filter fixing cage rod 223 one by one. The filter fixing cage plate 222 and the lens holder 32 are connected by the filter fixing cage rod 223, thereby realizing the optical coaxiality of the excitation light source module 2 and the optical path adjustment module 3 in the horizontal direction.

[0093] In some embodiments, the diameter of the lamp holder 213 can be selected to be 24 mm, the total divergence angle of the emitted light of the converging lens 212 is 15°, and the internal and external thread specifications of the fixed lens barrel and the fixed clamp ring used are

[0094] In this embodiment, the excitation light source module 2 is used to provide narrow-band output light with a spectral wavelength range of 470nm-510nm, appropriate power and beam diameter, so as to excite fluorescence with a spectral wavelength range of 500nm-560nm for the solution on the detection side.

[0095] In some embodiments, the excitation light source module 2 selects an ice blue LED lamp bead with an output power of 1W as the excitation light source. Compared with the excitation method using a 488nm central wavelength laser diode, LED (Light Emitting Diode) can not only provide multiple wavelengths of emitted light, but also has low energy consumption, long life, high level of integration, and simple driving, which can effectively reduce costs and reduce volume. The LED lamp bead is connected to the light source power supply socket 121 fixed to the wall panel 12 through the lamp bead power supply line 217. The light source power supply socket 121 can be designed as a USB jack, which can be connected to an external 5V USB power supply, including a computer USB interface and a smart phone charger.

[0096] It should be noted that appropriate power means that the power finally irradiated on the cover glass or biosensor chip 46 will not be too large or too small. On the one hand, excessive power will cause photobleaching of fluorescent substances, and on the other hand, the excitation light will penetrate the dichroic beam splitter prism 31 to reach the image acquisition module 5 and reduce the signal-to-noise ratio; too small power will cause weak fluorescence, which will also reduce the signal-to-noise ratio. The appropriate beam diameter allows the beam finally irradiated on the cover glass or biosensor chip 46 to be only slightly larger than the area where the sample to be detected is located, which can prevent too much stray light from being received by the image acquisition module 5 and improve the signal-to-noise ratio.

[0097] Reference Figure 6 , Figure 7 , Figure 8 In this embodiment, the image acquisition module 5 includes: a camera 511 , a lens barrel 522 and a fixing bracket 531 .

[0098] Specifically, the camera 511 can adopt a CMOS (Complementary Metal Oxide Semiconductor) camera, and the camera 511 is connected to the optical breadboard 14 at the bottom of the accommodating cavity through the camera fixing part 512; since there are connection holes arrayed on the optical breadboard 14, waist-shaped countersunk holes can be used on both sides of the camera fixing part 512, and the waist-shaped countersunk holes are connected to the connection holes by bolts, and the position of the image acquisition module 5 on the optical breadboard 14 can be fine-tuned by tightening the bolts; the camera 511 is connected to the camera socket 122 fixed on the wall panel 12 through the camera power supply line 54, and the computer can be connected to the camera socket 122 through a USB data cable for power supply and data transmission.

[0099] The camera 511 is connected to the lens 513 via the lens flange 514. The lens barrel 522 is arranged above the lens 513 of the camera 511. The top of the lens barrel 522 is connected to the bottom of the lens holder 32. A relay lens 521 is arranged in the lens barrel 522. The relay lens 521, the lens 513 of the CMOS camera and the second light hole are coaxially arranged to realize optical coaxiality of the optical path adjustment module 3 and the image acquisition module 5 in the vertical direction.

[0100] The fixed bracket 531 is arranged between the lens base 32 and the camera 511, and can be arranged as four mutually parallel supporting rods, which are respectively connected to the four sides of the camera 511, the top end of the fixed bracket 531 is connected to the lens base 32, and the bottom end of the fixed bracket 531 is connected to the camera 511. In this arrangement, a larger field of view is provided for the camera 511 through the relay lens 521, so that the images of the four micro flow slots can be completely collected.

[0101] In some embodiments, the diameter of the relay lens 521 is 25.4 mm, and the focal length is 60 mm. The focal length of the lens 513 of the CMOS camera is 12 mm, the F value can be adjusted within the range of 2.8 to 32, and the interface is C-type; the size of the CMOS chip in the CMOS camera 511 is 1 / 2.9 inches, and the configuration of the two can give full play to the field of view and resolution performance of the CMOS camera, while providing the system with flexibility in optical adjustment.

[0102] The fluorescence imaging sensor detection system provided in the embodiment of the present invention integrates the advantages of LED light source, multi-channel fluidic technology, CMOS camera and personal computer, and then utilizes the beam splitting and filtering principles of dichroic prism to form a portable, multi-channel, large field of view, imaging sensor detection system for bulk and solid phase dual-mode fluorescence sensing detection applications. It can effectively reduce the size of the instrument, realize simultaneous detection of multiple channels or multiple samples, save detection time, and significantly improve detection efficiency.

[0103] like Fig. 9 As shown, an embodiment of the present invention further provides a flow control multi-channel fluorescence imaging sensing detection method, which adopts the above-mentioned fluorescence imaging sensing detection system and is applied to the fluorescence detection of the bulk solution, comprising the following steps:

[0104] Step 1: Determine the excitation light source parameters of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0105] Step 2: Determine the parameters of the pinhole aperture and neutral density filter of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0106] Step 3: Place the cover glass and the PDMS fluidic chip at the set position of the receiving groove in the sensor module 4, close and fasten the fixture cover plate 482 and the fixture bottom plate 481, and obtain a dark background image through the CMOS camera when the excitation light source is turned off;

[0107] Step 4: prepare a negative sample solution, mix it with the fluorescent probe solution, and provide the mixed negative sample solution with an incubation environment required for the fluorescent probe to identify the analyte, such as long-term heat preservation; introduce the negative sample solution through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441, so that the negative sample solution fills the micro flow channel of the PDMS fluidic chip;

[0108] Step 5: Turn on the excitation light source and obtain the negative sample image through the CMOS camera;

[0109] Step 6: Turn off the excitation light source, remove the negative sample solution in the micro flow channel, prepare the sample solution to be detected, fully mix it with the fluorescent probe solution, provide the solution with the incubation environment required for the fluorescent probe to identify the analyte, and then introduce the fully reacted sample solution to be detected into the micro flow channel through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441;

[0110] Step 7: Turn on the excitation light source and obtain the image of the sample to be detected through the CMOS camera;

[0111] Step 8: Analyze and calculate the acquired dark background image, negative sample image and sample image to be detected through the signal processing module to obtain the fluorescence sensing characteristics of the sample to be detected on the cover glass.

[0112] like Fig.10 As shown, the embodiment of the present invention also discloses a flow control multi-channel fluorescence imaging sensing detection method, which adopts the above-mentioned fluorescence imaging sensing detection system and is applied to solid surface fluorescence detection, comprising the following steps:

[0113] Step 1: Determine the excitation light source parameters of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0114] Step 2: Determine the parameters of the pinhole aperture and neutral density filter of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements;

[0115] Step 3: Modify the biosensor chip 46 with a suitable first antibody for identifying the analyte, place the biosensor chip 46 functionalized with the first antibody and the PDMS fluidic chip in the set position of the receiving groove in the sensor module 4, close and fasten the fixture cover 482 and the fixture bottom plate 481, and obtain a dark background image through the CMOS camera when the excitation light source is turned off;

[0116] Step 4: prepare a negative sample solution, introduce the negative sample solution through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441, so that the negative sample solution fills the micro flow groove of the PDMS fluidic chip, provides the incubation environment required for antigen-antibody recognition for the sensor module, such as long-term heat preservation, and then removes the negative sample solution;

[0117] Step 5: prepare a second antibody solution labeled with a fluorescent group, introduce the second antibody solution through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441, so that the second antibody solution fills the micro flow groove of the PDMS fluidic chip, provides the incubation environment required for antigen-antibody recognition for the sensor module, and then removes the second antibody solution;

[0118] Step 6: Turn on the excitation light source and obtain the negative sample image through the CMOS camera;

[0119] Step 7: Turn off the excitation light source, clean the micro flow slot, prepare the sample solution to be detected, fill the micro flow slot with the sample solution to be detected through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441, provide the incubation environment required for antigen-antibody recognition for the sensor module, and then remove the sample solution to be detected;

[0120] Step 8: prepare a second antibody solution labeled with a fluorescent group, introduce the second antibody solution through the first injection line 411, the second injection line 421, the third injection line 431 and the fourth injection line 441, so that the second antibody solution fills the micro flow groove of the PDMS fluidic chip, provides the incubation environment required for antigen-antibody recognition for the sensor module, and then removes the second antibody solution;

[0121] Step 9: Turn on the excitation light source and obtain the image of the sample to be detected through the CMOS camera;

[0122] Step 10: Analyze and calculate the acquired dark background image, negative sample image and sample image to be detected through the signal processing module to obtain the fluorescence sensing characteristics of the sample to be detected on the biosensor chip 46.

[0123] It should be noted that modifying a suitable first antibody on the biosensor chip 46 in step 3 refers to attaching a layer of antibody molecules (self-assembled molecular layer) for capturing target analyte molecules on the nanostructured sensor array on the surface of the biosensor chip 46, such as gold particles, by physical or chemical means.

[0124] In this embodiment, a layer of first antibody molecules is modified on a solid surface (such as an ordinary glass slide or a surface of a precious metal nanostructure), and then the sample to be detected is added to provide the incubation environment required for antigen-antibody recognition. In this environment, the target analyte molecules will be captured by the first antibody molecules on the solid surface. In order to generate fluorescence and establish a biosensing association, it is necessary to remove the residual sample solution in the microcirculation groove, and then add a second antibody solution labeled with a fluorescent group to provide the incubation environment required for antigen-antibody recognition. In this environment, the second antibody as a fluorescent probe will specifically bind to the analyte molecules captured by the first antibody. After removing the excess second antibody solution, the number of fluorescent groups establishes a direct connection with the captured analyte molecules, and the fluorescence intensity has a positively correlated functional relationship with the number of fluorescent groups, thereby realizing fluorescent biosensor detection.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescence imaging sensor detection system, characterized in that: include: A dark box is provided with a containing cavity; An excitation light source module, disposed in the accommodating cavity, and used to provide an excitation light source; A sensor module is arranged on the top of the dark box, wherein a cover glass for bulk solution detection or a biosensor chip for solid surface detection is arranged in the sensor module, and the sensor module is connected with a sample delivery pipeline for providing a sample solution to be detected to the cover glass or the biosensor chip; An optical path adjustment module, the optical path adjustment module is connected to the bottom of the sensor module, and one side of the optical path adjustment module is connected to the excitation light source module, and is used to filter and steer the output light of the excitation light source, guide the output light of the excitation light source to the sample solution to be detected in the sensor module to make it emit fluorescence, and receive the fluorescence; An image acquisition module is disposed in the accommodating cavity, and the image acquisition module is disposed at the bottom of the optical path adjustment module, and is used to acquire a fluorescent image passing through the optical path adjustment module and convert it into a corresponding image data signal; The sensor module comprises: Fixture cover; The fixture bottom plate is provided with a receiving groove, a through hole for light transmission is opened at the bottom of the receiving groove, the through hole is covered with the cover glass or the biosensor chip, the receiving groove is connected with the sample delivery pipeline all around, and the fixture bottom plate is detachably connected to the fixture cover plate; A fluidic chip, the bottom surface of which is in contact with the top surface of the cover glass or the biosensor chip, and the fluidic chip is provided with a plurality of micro flow slots for storing a sample solution to be detected; The optical path adjustment module comprises: A mirror base is provided with a cavity, and a first light hole corresponding to the through hole is opened at the top of the mirror base, a second light hole corresponding to the image acquisition module is opened at the bottom of the mirror base, and a third light hole is opened on the side of the mirror base connected to the excitation light source module, wherein the first light hole, the second light hole and the through hole are coaxially arranged; A dichroic beam splitter prism is disposed in the cavity and is used to filter and steer the outgoing light of the excitation light source, and guide the outgoing light of the excitation light source in a direction perpendicular to the cover glass or the biosensor chip to the sample solution to be detected in the sensor module to make it emit fluorescence; A mirror base bracket, wherein the top end of the mirror base bracket is connected to the mirror base, and the bottom end of the mirror base bracket is connected to the bottom of the accommodating cavity.

2. The fluorescence imaging sensor detection system according to claim 1, characterized in that: The fluidic chip includes, from bottom to top: A flow channel layer, wherein the flow channel layer is provided with four micro flow grooves at intervals, the areas where the four micro flow grooves are located correspond to the through holes, and micro flow inlets and micro flow outlets are provided around the flow channel layer; The connecting layer is provided with four pairs of liquid-conducting grooves, one pair of the liquid-conducting grooves is correspondingly distributed at both ends of the micro-circulation groove, and each of the liquid-conducting grooves is respectively connected with the micro-circulation groove, the micro-flow inlet or the micro-flow outlet; The bonding layer covers the top of the connecting layer, and the flow channel layer, the connecting layer and the bonding layer are bonded and connected.

3. The fluorescence imaging sensing detection system according to claim 1, characterized in that: The mirror base bracket comprises: Base; A first connecting rod, wherein the first connecting rod is movably connected to the first connecting rod in a vertical direction, and a top end of the first connecting rod is connected to a transfer block; A second connecting rod, wherein the second connecting rod is arranged perpendicular to the first connecting rod, a first end of the second connecting rod is movably connected to the adapter block along a horizontal direction, and a second end of the second connecting rod is connected to the mirror seat.

4. The fluorescence imaging sensor detection system according to claim 1, characterized in that: The excitation light source module comprises the excitation light source, a converging lens, a pinhole diaphragm and a neutral density filter which are arranged in sequence; the centers of the light source, the converging lens, the pinhole diaphragm and the neutral density filter are arranged coaxially.

5. The fluorescence imaging sensing detection system according to claim 1, characterized in that: The excitation light source is used to provide narrow-band emission light with a wavelength range of 470nm-510nm, so as to excite fluorescence with a wavelength range of 500nm-560nm for the solution to be detected.

6. The fluorescence imaging sensing detection system according to claim 1, characterized in that: The image acquisition module comprises: A camera, wherein the camera is connected to the bottom of the accommodating cavity through a camera fixing member; A lens barrel is arranged on the lens of the camera, the top of the lens barrel is connected to the bottom of the lens holder, a relay lens is arranged in the lens barrel, and the relay lens, the lens of the camera and the second light hole are coaxially arranged; The fixing bracket is arranged between the lens base and the camera, the top end of the fixing bracket is connected to the lens base, and the bottom end of the fixing bracket is connected to the camera.

7. A fluorescence imaging sensing detection method, using the fluorescence imaging sensing detection system according to any one of claims 1 to 6, characterized in that: The following steps are involved: Determine the excitation light source parameters of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements; Place the cover glass in the receiving slot of the sensor module, make sure the excitation light source is turned off, and obtain a dark background image through the image acquisition module; preparing a negative sample solution containing a fluorescent probe, providing the negative sample solution with an incubation environment required for the fluorescent probe to recognize an analyte, and introducing the negative sample solution into a micro flow channel of the sensor module; Turn on the light source and obtain the image of the negative sample through the image acquisition module; Turn off the excitation light source, remove the negative sample solution in the micro flow-through slot, prepare a sample solution to be detected containing a fluorescent probe, provide the solution with an incubation environment required for the fluorescent probe to identify the analyte, and introduce the sample solution to be detected containing the fluorescent probe into the micro flow-through slot; Turn on the light source and obtain the image of the sample to be detected through the image acquisition module.

8. A fluorescence imaging sensing detection method, using the fluorescence imaging sensing detection system according to any one of claims 1 to 6, characterized in that: The following steps are involved: Determine the excitation light source parameters of the fluorescence imaging sensor detection system according to the characteristics of the sample to be detected and the experimental requirements; Modifying a suitable first antibody on the biosensor chip for identifying the analyte, placing the biosensor chip functionalized with the first antibody in a receiving slot of the sensor module, confirming that the excitation light source is in an off state, and obtaining a dark background image through an image acquisition module; preparing a negative sample solution, introducing the negative sample solution into the micro flow channel of the sensor module, providing the sensor module with an incubation environment required for antigen-antibody recognition, and then removing the negative sample solution; Prepare a second antibody solution containing a fluorescent group label, introduce the second antibody solution into the micro flow channel of the sensor module, provide the sensor module with an incubation environment required for antigen-antibody recognition, and then remove the second antibody solution; Turn on the light source and obtain the image of the negative sample through the image acquisition module; Turn off the excitation light source, clean the micro flow channel, prepare the sample solution to be detected, introduce the sample solution to be detected into the micro flow channel, provide the sensor module with the incubation environment required for antigen-antibody recognition, and then remove the sample solution to be detected; Prepare a second antibody solution containing a fluorescent group label, introduce the second antibody solution into the micro flow channel of the sensor module, provide the sensor module with an incubation environment required for antigen-antibody recognition, and then remove the second antibody solution; Turn on the light source and obtain the image of the sample to be detected through the image acquisition module.

Images