Multi-component pollutant detection device based on multi-cavity fluorescent microsensor

Abstract

The utility model provides a multi-component pollutant detection device based on a multi-cavity fluorescent microsensor, which relates to the technical field of pollutant detection devices and comprises a fluorescent excitation module and an imaging module. The fluorescence excitation module comprises n (n > 1) excitation light sources and an optical filter used in cooperation with the n (n > 1) excitation light sources. The imaging module comprises an objective table, an objective lens, an optical filter unit and an image acquisition hole. By arranging the fluorescence excitation module and the imaging module, the excitation light source emits excitation light for exciting different fluorescent lights, so that the different fluorescent lights on different component pollutants are excited at the same time, the excited fluorescent lights are reflected to the optical filter unit through the objective lens, different optical filters are switched, and the different fluorescent lights are reflected to the terminal equipment through the image acquisition hole. The multi-component pollutants are analyzed without mutual interference, the specificity and the sensitivity are excellent, the detection efficiency is improved, and a new application technology is provided for pollutant detection in the fields of food safety and the like.

Description

TECHNICAL FIELD

[0001] The utility model relates to the technical field of pollutant detection device, concretely relates to a kind of multi-component pollutant detection device based on multi-chamber fluorescence microsensor. BACKGROUND

[0002] Current food pollution problem is complex and extensive, often involves including food source, food processing and storage multiple aspects.Polluted food types also include fruits and vegetables, grains, feed, meat and aquatic products etc..At the same time, the types of food pollutants are also very complex, involving pesticide residues, veterinary drug residues, mycotoxin pollution and heavy metal pollution etc..These small molecule pollutants cannot be ignored to food safety and human health, they can enter the body through transportation storage and production and processing etc.Links, reduce the immune function of livestock and poultry, and ultimately through food chain to human health bring serious threat.At present, with the rapid development of China's economy and the improvement of residents' living standards, the importance of food safety is further improved, and the state has clearly stipulated the residual limit of various pollutants in food.

[0003] The detection methods currently applied to small molecule pollutants mainly include enzyme-linked immunosorbent assay (ELISA), thin layer chromatography and high performance liquid chromatography etc.These methods are relatively mature, but also have some shortcomings such as high sample detection cost, large reagent demand and high professional requirements for detection personnel, and they are limited in application when facing multiple target detection in complex sample matrix.The corresponding detection device is also limited in the application of simultaneous detection of multiple target substances.Food pollutants often exist in the form of multiple components in food, food processing products or feed at the same time.Therefore, it is of important scientific significance and application value to develop a multi-component pollutant detection device based on multi-chamber fluorescence microsensor in the field of food safety. UTILITY MODEL CONTENT

[0004] (I) Technical problem solved

[0005] In view of the deficiencies of the prior art, the utility model provides a multi-component pollutant detection device based on multi-chamber fluorescence microsensor, which solves the technical problem that the detection device of the prior art can usually detect only a single pollutant.

[0006] (II) Technical scheme

[0007] To achieve the above purpose, the utility model realizes by the following technical scheme:

[0008] The multi-component pollutant detection device based on multi-chamber fluorescence microsensor comprises a fluorescence excitation module and an imaging module.

[0009] The fluorescence excitation module comprises n (n>1) excitation light sources and optical filters used in cooperation with the n (n>1) excitation light sources,

[0010] The optical filters are arranged on the excitation light sources, the excitation light emitted by the n excitation light sources cooperates with the transmission wavelength band of the corresponding optical filters, and the excitation light of different fluorescent excitation is respectively emitted;

[0011] The imaging module comprises a stage, an objective lens, an optical filter unit and an image acquisition hole,

[0012] The excitation light emitted by the excitation light source irradiates on the to-be-detected pollutants on the stage, the objective lens is fixed above the stage, the optical filter unit is composed of n optical filters which can transmit light of different colors, the optical filter unit is slidingly connected above the objective lens, and the image acquisition hole is located above the optical filter unit. The light reflected from the stage to the objective lens and then emitted to the optical filter unit is collected and then reflected to the terminal device.

[0013] Preferably, the detection device further comprises a light-proof shell, the light-proof shell comprises a top cover, an intermediate shell and a bottom plate, the top cover and the intermediate shell are mutually buckled to form a light-proof darkroom; the fluorescence excitation module and the imaging module are assembled on the light-proof shell; and the bottom plate and the intermediate shell are combined to fixedly encapsulate the inside of the intermediate shell.

[0014] Preferably, a sliding rail is arranged on the inner wall of the top cover, and the optical filter unit is slidingly arranged on the sliding rail.

[0015] Preferably, a handle extending to the outer end of the top cover is integrally connected to the end of the optical filter unit, and the handle is used to control the sliding of the optical filter unit.

[0016] Preferably, magnets are arranged at both ends of the stage in the length direction.

[0017] Preferably, the fluorescence excitation module further comprises a mounting bracket and an excitation light source control button, the excitation light source is fixed to the upper part of the mounting bracket and is at an angle of 45° with the device platform of the intermediate shell, and the excitation light source control button is used to control the opening and closing of the excitation light source.

[0018] Preferably, the imaging module further comprises a four-legged support, the four-legged support is erected above the fluorescence excitation module and the stage, and the objective lens is fixed at the center of the four-legged support.

[0019] Preferably, the detection device further comprises an incubation module, the incubation module comprises a heating sheet located on the device platform of the intermediate shell and a switch located on the side wall of the intermediate shell.

[0020] Preferably, the detection device further comprises a power module comprising a 12V lithium battery placed on the interior device platform of the intermediate housing and a battery control switch.

[0021] (III) Beneficial Effects

[0022] The utility model provides a kind of multi-component pollutant detection device based on multi-chamber fluorescence microsensor.Compared with prior art, it has the following beneficial effects:

[0023] By setting multiple excitation light sources in fluorescence excitation module, and configuring light filter that can transmit different light on excitation light source, excitation light source emits excitation light that excites different fluorescence, so that different fluorescence on different component pollutants is excited simultaneously, and the excited fluorescence is reflected to light filter unit by objective lens, different light filters are switched, different fluorescence is reflected to terminal equipment by image acquisition hole, so that multi-component pollutants are analyzed, and do not interfere with each other, specificity and sensitivity are excellent, and detection efficiency is improved, which provides new application technology for pollutant detection in food safety and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical scheme in the embodiments of the utility model or prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the utility model, and those skilled in the art can obtain other drawings according to these drawings without creating labor.

[0025] Figure 1 is the working principle diagram of the multi-chamber fluorescence microsensor detection method of embodiment 2 of the utility model;

[0026] Figure 2 is the overall structure schematic diagram of the portable microscopic imaging device of embodiment 1 of the utility model;

[0027] Figure 3 is the schematic diagram of the structure of each module of the portable microscopic imaging device of embodiment 1 of the utility model;

[0028] Figure 4 is the schematic diagram of the internal structure of the top cover of the portable microscopic imaging device of embodiment 1 of the utility model;

[0029] Figure 5 is the optical path schematic diagram of the portable microscopic imaging device of embodiment 1 of the utility model;

[0030] Figure 6 is the fluorescence imaging diagram of the multi-component target of the multi-chamber fluorescence microsensor detection of embodiment 2 of the utility model;

[0031] Figure 7is the detection stability data graph of the multi-chamber fluorescent micro-sensing system of the embodiment 3 of the present application;

[0032] Wherein, 1, light-tight shell; 11, top cover; 112, slide rail; 12, intermediate shell; 2, power module; 3, incubation module; 31, heating plate; 32, switch button; 4, fluorescence excitation module; 41, excitation light source; 42, filter; 43, mounting bracket; 44, control button; 5, imaging module; 51, stage; 511, magnet; 52, objective lens; 53, four-legged support; 54, filter unit; 541, handle; 55, image acquisition hole. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application is described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0034] The multi-component pollutant detection device based on the multi-chamber fluorescent micro-sensor provided by the embodiments of the present application solves the technical problem that the detection device of the prior art can usually only detect a single pollutant.

[0035] In order to better understand the above technical scheme, the above technical scheme will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0036] Embodiment 1: Construction of a microscopic imaging device

[0037] As shown in Figure 2 , Figure 3 , the microscopic imaging device includes a light-tight shell 1, a power module 2, an incubation module 3, a fluorescence excitation module 4, and an imaging module 5.

[0038] The light-tight shell 1 is composed of a top cover 11, an intermediate shell 12, and a bottom plate. The top cover 11 and the intermediate shell 12 are mutually buckled through a clamping groove to form a light-tight darkroom; the power module 2, the incubation module 3, the fluorescence excitation module 4, and the imaging module 5 are assembled on the light-tight shell 1; the bottom plate is combined with the intermediate shell 12 to fix and package the modules inside the intermediate shell 12. The combination of the top cover 11, the intermediate shell 12, and the bottom plate of the light-tight shell 1 has a cuboid structure with a length of 175 mm, a width of 170 mm, and a height of 150 mm.

[0039] The power module 2 includes a 12V lithium battery and a battery control switch placed on the equipment platform inside the intermediate shell 12.

[0040] The incubation module 3 includes heating fins on the equipment platform of the middle shell 12 and switches on the sidewall of the middle shell 12. The temperature of the heating fins is controlled by a heating controller inside the middle shell 12 to be constant at 37°C, and the switches are used to control the on-off of the heating controller.

[0041] In combination Figure 3 As shown, the fluorescence excitation module 4 includes excitation light sources 41, filters 42, mounting frames 43, and excitation light source 41 control buttons 44. There are three filters 42, each mounted on a corresponding excitation light source 41. The three excitation light sources 41 are fixed to the upper part of the three mounting frames 43 and are arranged at 45° to the equipment platform of the middle shell 12, and the three mounting frames 43 enclose the imaging module 5. The excitation light emitted by the excitation light source 41 cooperates with the transmission band of the filter 42 mounted on the excitation light source 41 to excite different fluorescent lights in different chambers of the microsensor.

[0042] The imaging module 5 includes a stage 51, an objective lens 52, a four-legged support 53, a filter unit 54, and an image acquisition hole 55.

[0043] The stage 51 is arranged on the equipment platform of the middle shell 12 and is located in the center of the three mounting frames 43 of the fluorescence excitation module 4, and is used to place a multi-chamber fluorescent microsensor. The excitation light emitted by the fluorescence excitation module 4 irradiates on the multi-chamber fluorescent microsensor. The stage 51 is provided with a magnet 511 at both ends in the length direction of the stage 51, which controls the magnetic particles in the chambers of the microsensor to maintain the balance in the direction of the microsensor.

[0044] In combination Figure 4 As shown, the four-legged support 53 is erected above the stage 51, and the objective lens 52 is fixed to the center of the four-legged support 53 and is located directly above the stage 51. The filter unit 54 is located directly above the objective lens 52 and is composed of three filters 42, which can respectively transmit red light, blue light and green light.

[0045] In combination Figure 4 As shown, the inner wall of the top cover 11 is provided with a sliding rail 112, and the filter unit 54 is slidingly arranged on the sliding rail 112. The end of the filter unit 54 is integrally connected with a handle 541 extending to the outer end of the top cover 11, and the handle 541 is used to control the sliding of the filter unit 54.

[0046] The image acquisition hole 55 is arranged on the top cover 11 and is located directly above the filter unit 54. The fluorescence beam passes through the image acquisition hole 55 to exit the microscopic imaging device.

[0047] Referring to Figure 5The use principle of the micro-imaging device in this embodiment is as follows: a plurality of groups of excitation light sources 41 emitting different excitation light are arranged to irradiate the multi-chamber fluorescent micro-sensor placed on the object table 51, different fluorescence in different types of pollutants in each chamber of the micro-sensor is excited at the same time, the excited fluorescence is reflected to the filter unit 54 through the objective lens 52, different filters 42 are switched to reflect different fluorescence to the terminal device through the image acquisition hole 55, so that different component pollutants are analyzed.

[0048] Embodiment 2: Construction of a multi-chamber fluorescent micro-sensor

[0049] 1. Selecting pollutants to be detected and their probe sequences

[0050] In this embodiment, aflatoxin B1 (PAT), aflatoxin B1 (AFB1) and ochratoxin A (OTA) commonly found in fruits and cereals are selected for detection. The probe sequences corresponding to the three target substances are as follows:

[0051] Table 1: Probe sequence table of PAT, AFB1 and OTA

[0052]

[0053] In Table 1, the hairpin sequence H1 is marked in bold and italic to indicate that the base is modified with a BHQ-1 group, and is marked in bold and underlined to indicate that the base is modified with a FAM group.

[0054] The synthesis and end modification of the aptamer strand and the complementary strand used in Embodiment 1 of the present application are synthesized by Beijing Lihe Huada Gene Technology Co., Ltd., and the experimental materials and reagents used are obtained from a regular and easily purchased manner.

[0055] The mixture of the aptamer sequence and the complementary primer strand of each mycotoxin and the H1 and H2 sequences are denatured at 95°C for 5 min, and then gradually cooled to room temperature to form double-stranded structures (dsDNA) of the aptamer and the complementary strand, and H1 and H2 form hairpin structures, respectively.

[0056] 2. Probe sequence modification polystyrene microparticles

[0057] The streptavidin-modified polystyrene microparticles were taken in a centrifuge tube, centrifuged at 10,000 rpm for 5 min, and the supernatant was removed, and then resuspended with a binding buffer (20 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 0.0005% Triton X-100). Three kinds of dsDNA were added to the resuspended polystyrene microparticles, incubated at 37°C for 30 min, and then the complex was washed with TEM buffer (10 mM Tris-HCl, 1 mM EDTA, 12.5 mM MgCl2) at pH 8.0 and resuspended for storage.

[0058] 3. Preparation of multi-chamber fluorescent microsensor

[0059] As shown in Figure 1 , red fluorescently labeled particles, blue fluorescently labeled particles, magnetic particles and functional polystyrene microparticles corresponding to PAT, AFB1 and OTA were uniformly mixed with sodium alginate solution to prepare the dispersed phase, which was stored in a syringe for standby; the above materials were sequentially passed through a microfluidic chip with six symmetrical channels, a microfluidic pump and an air flow control device to prepare micron-sized hydrogel microspheres. The obtained microspheres include six mutually separated and identical size chambers, and are sequentially loaded with red fluorescently labeled particles, blue fluorescently labeled particles, magnetic particles and three kinds of functional polystyrene microparticles. Among them, the chambers loaded with red fluorescently labeled particles and blue fluorescently labeled particles are used for labeling and positioning of the multi-chamber fluorescent microsensor, and the magnetic particles are used for magnetic auxiliary orientation of the multi-chamber fluorescent microsensor. The functional polystyrene microparticles corresponding to PAT, AFB1 and OTA are sequentially located in the three adjacent separated chambers beside the red labeled chamber.

[0060] 4. Simultaneous detection of three mycotoxins

[0061] The multi-chamber fluorescent microsensor and the hairpin sequences H1, H2 of the three toxins (PAT, AFB1, OTA) were added to the test solution containing the target, incubated on the 37℃ constant temperature heating piece of the microscopic imaging device for 45-50 min, and then washed. The incubation reaction occurs in the circular reaction tank formed by the bonding of the PDMS weir and the glass slide, and the incubation and washing operations are all completed in the circular reaction tank. Rapid imaging and fluorescence intensity analysis were performed by the microscopic imaging equipment. The direction of the microsensor chamber was maintained by controlling the magnetic particles in the chamber by the built-in magnet, and the positions and directions of the three detection chambers were determined by switching the three excitation light sources to achieve imaging of red and blue fluorescence. The fluorescence imaging of the detection results can be quickly transmitted to smart terminal devices such as mobile phones and computers, and the fluorescence intensity can be quickly processed and analyzed by processing software such as Image J.

[0062] As shown in Figure 6 , when the target exists, the multi-chamber fluorescent microsensor constructed by the present application can capture the target (PAT, AFB1, OTA) in the test solution through the fluorescence signal, and then obtain the fluorescence image of the target detection chamber through the microscopic imaging device.

[0063] Example 3: Stability of the multi-chamber fluorescent microsensor system

[0064] The multi-chamber fluorescent microsensor constructed by the present application can be stored at 4℃. To verify the relationship between the storage time and the detection ability, the multi-chamber fluorescent microsensor stored for 0-7 days was used for target detection, and the fluorescence signal intensity was compared. The detection results are shown inFigure 7 As shown in the results, the sensing system still has good detection ability within 6 days of preservation.

[0065] Compared with the prior art, the present application has the following beneficial effects:

[0066] The detection method of the present application loads functional polystyrene microparticles modified with different specific capture probes on the different chambers of the multi-chamber fluorescent microsensor, triggers aptamer melting and initiates chain hybridization of the hairpin sequence through target pollutant molecules, so as to produce a response fluorescent signal in each detection chamber. This detection method can realize simultaneous detection of multiple target components by a single fluorescent signal in a 3D structure with micron-level particle size, and does not interfere with each other, has excellent specificity and sensitivity, and improves the detection efficiency, providing a new application technology for pollutant detection in the field of food safety and the like.

[0067] It should be noted that, in this document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Elements denoted by the phrase "comprising a" do not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0068] The above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A multi-component pollutant detection device based on a multi-chamber fluorescent microsensor, characterized in that, Includes a fluorescence excitation module and an imaging module; The fluorescence excitation module includes n (n>1) excitation light sources and filters used in conjunction with the n (n>1) excitation light sources. The filter is disposed on the excitation light source, and the excitation light emitted by the n excitation light sources cooperates with the transmission band of the corresponding filter to emit excitation light that excites different fluorescence respectively; The imaging module includes a stage, objective lens, filter unit, and image acquisition port. The excitation light emitted by the excitation source illuminates the contaminant to be tested on the stage. The objective lens is fixed directly above the stage. The filter unit consists of n filters that can transmit different colors of light. The filter unit is slidably connected directly above the objective lens. The image acquisition hole is located directly above the filter unit. Light reflected from the stage to the objective lens and then emitted to the filter unit is collected and reflected to the terminal device.

2. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 1, characterized in that, It also includes a light-shielding shell, which includes a top cover and a middle shell, and the top cover and the middle shell are interlocked to form a light-shielding dark chamber; the power module, the incubation module, the fluorescence excitation module and the imaging module are assembled on the light-shielding shell.

3. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 2, characterized in that, The inner wall of the top cover is provided with a slide rail, and the filter unit is slidably mounted on the slide rail.

4. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 3, characterized in that, The filter unit is integrally connected to a handle extending to the outer end of the top cover, and the handle is used to control the sliding of the filter unit.

5. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 1, characterized in that, Magnets are provided at both ends of the stage along its length.

6. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 2, characterized in that, The fluorescence excitation module also includes a mounting bracket and an excitation light source control button. The excitation light source is fixed to the upper part of the mounting bracket and is at a 45° angle to the device platform of the intermediate housing. The excitation light source control button is used to control the excitation light source to turn on and off.

7. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 1, characterized in that, The imaging module also includes a quadruped bracket, which is mounted above the fluorescence excitation module and the stage, and the objective lens is fixed at the center of the quadruped bracket.

8. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 2, characterized in that, It also includes an incubation module, which includes a heating element located on the intermediate housing device platform and a switch located on the side wall of the intermediate housing.

9. The multi-component pollutant detection device based on a multi-chamber fluorescent microsensor as described in claim 2, characterized in that, It also includes a power module, which comprises a 12V lithium battery and a battery control switch placed on the equipment platform inside the intermediate housing.

Images