CN111060482B - Detection equipment based on microspheres and microporous plates and use method thereof - Google Patents
Detection equipment based on microspheres and microporous plates and use method thereof Download PDFInfo
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Abstract
The invention provides a detection device based on microspheres and a microporous plate and a use method thereof. The detection device comprises a sample processing part and a detection part; the detection part comprises a multicolor fluorescence excitation module, a front scattering imaging module, a fluorescence emission filtering module and an alternating electric field control module; the front scattering imaging module is used for placing a microporous plate chip and imaging micropores of the microporous plate chip, and the alternating electric field control module is used for controlling the movement of microspheres in the microporous plate chip. The detection equipment provided by the invention applies an electric field on the microporous plate chip to push the microspheres into the micropores of the microporous plate chip, and utilizes the front scattering light imaging module to image, so that the reaction state of each micropore in the microporous plate chip is more clearly obtained, and the accurate qualitative and quantitative determination of the target molecules to be detected is realized.
Description
Technical Field
The invention belongs to the technical field of biological detection, particularly relates to detection equipment for molecular diagnosis, and particularly relates to detection equipment based on microspheres and a microporous plate.
Background
Molecular diagnosis is to utilize molecular biology technology and method to study the existence, structure or expression regulation and control change of human endogenous (i.e. organism gene) or exogenous (e.g. virus, bacteria, etc.) biological molecules and molecular systems, and provide information and decision basis for prevention, prediction, diagnosis, treatment and outcome of diseases. Nucleic acid amplification technology has the widest application range in the field of molecular diagnostics.
Compared with the traditional clinical diagnosis method, the molecular diagnosis can make up for certain defects of the traditional clinical diagnosis method, for example, the molecular diagnosis can directly reveal the existence of pathogens, can objectively reflect the infection and activity conditions of the pathogens in a human body, and can be used as an effective monitoring means in clinical treatment. In addition, the molecular diagnosis can be used for detecting pathogens which are difficult to detect by the conventional detection method, for example, the problem of the window period from infection to antibody generation in the enzyme immunoassay technology can be overcome.
The current commercial molecular diagnostics are mainly based on PCR amplification techniques combined with different detection techniques, and in particular typically include: a small amount of bacterial or viral DNA molecules are selectively and massively copied or the DNA obtained by reverse transcription of viral RNA molecules is selectively and massively copied, and a specific nucleic acid sequence is copied and modified by a designed primer so as to carry out detection. Common detection modes include methods such as second-generation gene sequencing, real-time fluorescence detection during amplification, capillary electrophoresis (first-generation sequencing), flow-type fluorescence detection, gene hybridization chip and the like. Methods for implementing molecular diagnostics using PCR amplification techniques have several problems, such as: cross contamination is easy to occur, the existing clinical molecular test also needs highly skilled operators and has high requirements on fields, wherein the PCR clinical diagnosis also needs to carry out sample treatment, amplification and detection in a separated room, the investment is high in the early stage of pollution prevention, and the sample is difficult to clean and the multi-index detection is difficult to carry out after the pollution.
The microfluidic chip (microfluidic chip) completes multiple steps including nucleic acid extraction, reverse transcription, amplification, detection and the like in a closed micro volume. The micro-fluidic chip has the characteristics of controllable liquid flow, extremely less consumption of samples and reagents, ten-fold or hundred-fold improvement of analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter time, and can realize the whole processes of pretreatment and analysis of the samples on line. All reactants are closed systems after the sample is added, and pollution is avoided.
However, the microfluidic chip has a small surface area and is divided into many micropores, and droplets or microspheres are easily affected by the acting force such as surface tension when entering the micropores, and cannot enter the micropores, thereby affecting the accuracy of the result. Meanwhile, the traditional microfluidic chip detection equipment based on PCR amplification generally does not have an imaging system, is only responsible for converting the number and the intensity of detected fluorescent signals into data, realizes the qualitative and quantitative determination of fluorescent molecules or detection samples to be detected, and an experimenter cannot visually observe a detection result, so that the experimental result has uncertainty.
The manipulation of target substances at microscopic dimensions and real-time observation are very widely used, for example, the manipulation of micro-sized microspheres coupled with antibodies or nucleic acids can be used for the study of intermolecular forces on these coupled molecules.
The conventional control method is mainly suitable for a scanning probe microscope, magnetic tweezers or optical tweezers, and the scanning probe microscope is used for directly adhering the microspheres at the probe needle tip and moving the microspheres through the piezoelectric ceramic moving needle tip with multiple degrees of freedom. Magnetic tweezers are used to directly manipulate microspheres containing magnetic materials by changing the strength and direction of the magnetic field. Optical tweezers move microspheres by making an optical energy potential well in a medium, wherein the optical energy potential well can capture the microspheres, and the force generated by the manipulation of the microspheres is generally in the range of fN-nN. The scanning probe method and the optical tweezers method can only operate single microspheres or a small number of microspheres generally, and the flux is extremely low. Magnetic tweezers can be operated by hundreds of parallel microspheres, but flux is also low, requirements are difficult to meet, and parallel operation of each microsphere is difficult to realize due to uneven magnetic field strength.
Therefore, there is an urgent need to develop a detection device capable of rapidly and effectively transferring microspheres into a microplate chip, and simultaneously, enabling a researcher to more intuitively and accurately obtain experimental results, so as to meet the research requirements in the technical field of biological detection.
Disclosure of Invention
In view of the problems in the prior art, the invention provides detection equipment based on microspheres and a microporous plate, the detection equipment is easy to control the microspheres in a medium, good in repeated stability, large in generated force, flexible and variable in direction of force, capable of more conveniently controlling the particles in a solution to be detected and transferring the particles to micropores of a microporous plate chip, and meanwhile, the equipment can also directly image the microporous plate chip to be detected to obtain images of the microporous plate chip under different filtering wavelengths, so that the detection result is clearer and more visual. In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a detection device based on microspheres and a microplate, the detection device comprising a sample processing portion and a detection portion;
the detection part comprises a multicolor fluorescence excitation module, a front scattering imaging module, a fluorescence emission filtering module and an alternating electric field control module; the front scattering imaging module is used for placing a microporous plate chip and imaging micropores of the microporous plate chip, and the alternating electric field control module is used for controlling the movement of microspheres in the microporous plate chip.
In the invention, the working principle of the detection equipment is as follows: the micro-porous plate chip containing the microspheres is placed in the front scattering imaging module, the alternating electric field control module applies an electric field on the micro-porous plate chip, the difference between the dielectric constant of the microspheres and a solution is very large, and the difference between the dielectric constant of a material forming a micro-porous structure and the dielectric constant of a liquid environment is relatively large, so that a non-uniform electric field with very large change is generated, particularly in the vicinity of micro-pores, the microspheres are pushed into the micro-pores of the micro-porous plate chip by applying dielectrophoresis force generated by applying the alternating electric field, the size of the dielectrophoresis force is in direct proportion to the volume of the microspheres and the change degree of the size of the electric field, and is a function of the frequency of the applied electric field, and the direction of the force can be changed by 180 degrees under different frequencies.
Under specific frequency, the microsphere is pushed into the micropore by dielectrophoresis force, so that the electric field structure in the micropore is changed, the second microsphere is difficult to obtain the same force and is pushed into the pore again, and the single microsphere detection of a single micropore is ensured. Meanwhile, the multi-color fluorescence excitation module is used for exciting fluorescence with different wavelengths to carry out near field excitation, namely, the excitation light of a far field is transmitted in a medium under a specific angle, so that light at the bottom of the micropore cannot be refracted to enter, near field light is generated, the fluorescence generated after the fluorescence microsphere is excited is transmitted and scattered to all directions, finally, a front scattered light imaging module is used for photographing, and images under different filtering wavelengths can be obtained by matching with the fluorescence emission filtering module. After the detection is finished, the alternating electric field control module can adjust the direction of the electric field frequency changing force to push the microspheres out of the micropores, so that the repeated use of the microporous plate chip is realized.
The mode for detecting the microscopic manipulation is usually fluorescence or white light microscope observation, for the highly parallel manipulation, the data quantity required to be obtained is large, the large-area detection is difficult to realize by a common coaxial excitation fluorescence microscope, and the invention combines the front scattering technology to carry out the large-area highly parallel detection while improving the manipulation parallelism. The detection mode mainly depends on near-field light to excite the fluorescent microspheres, and emitted fluorescent signals are received by a far field of a detector, so that the high-parallelism manipulation and the simultaneous detection of the manipulations are realized, and data are obtained.
The detection part of the device provided by the invention is integrated with a multicolor fluorescence excitation module, a front scattering imaging module, a fluorescence emission filtering module and an alternating electric field control module. The multicolor fluorescence excitation module is used for exciting the multiple fluorescence coding microspheres and fluorescent substances released by substrate molecules under the action of a catalyst; obtaining a low-background fluorescence photo of the microsphere-introduced microporous plate by a front scattering imaging module; the fluorescence emission filtering module is used for distinguishing the coding microspheres and the micropore brightness change (the concentration and the change of fluorescent substances released by substrate molecules through the action of a catalyst); the magnetic absorption module and the alternating electric field control module are used for helping the microspheres to be led into or led out of the micropores. The sample processing part and the detection part in the detection equipment based on the microspheres and the microporous plate can exist independently.
To realize a large area (mm) 2 –cm 2 ) The patent combines two technologies of dielectrophoretic force and evanescent wave to build the equipment platform. The reason for using evanescent waves for detection is that high sensitivity is required to observe microscopically manipulated microspheres in real time, while in large area detection it is difficult to maintain high lateral resolution and sensitivity, and using evanescent waves allows for large area microscopic resolution with low numerical aperture while maintaining high longitudinal resolution and sensitivity, since only the total intensity of the optical signal needs to be collected. The device comprises two core components:
1. alternating electric field control module (high robustness large area high parallel dielectrophoresis force control)
When a dielectric material (mainly micron-sized microspheres and cells are described in the present invention, taking microspheres as an example) is exposed in a non-uniform electric field medium (taking an aqueous solution as an example), an electric field polarizes the microspheres and molecules in the aqueous solution around the microspheres to achieve different polarization strengths, and the microspheres or the molecules around the microspheres move to a high electric field strength to reduce free energy generated by the system and an external electric field, which is called dielectrophoresis, and the generated force is dielectrophoresis force. The dielectrophoretic force is proportional to the volume of the microspheres and to the rate of change of the electric field strength, a particular phenomenon being the positive and negative forces calculated under a specific system, as shown in figure 1, since the permittivity is a function of the frequency of the applied electric field, which produces forces in diametrically opposite directions at different frequencies.
In a specific system, that is, the microspheres are fixed in size and the solution is fixed, the maximum value of the force can be changed by enhancing the change speed of the electric field, the common method is to micro-machine and encapsulate electrodes with complex shapes, and the electrodes are generally in a network structure, and in the structure, alternating current is applied between different electrodes to generate high field intensity gradient, but the method has high cost and complex process, and is difficult to realize stable and high-repeatability large-area implementation. In the invention, the high field strength gradient is generated by using the dielectric constant difference between dielectric materials, such as a common high polymer material structure processed by a photoetching method in two flat electrodes. The flat electrodes are provided with uniform electric field, i.e. there is no field intensity gradient, taking fig. 2 as an example, a-d represents that the flat electrodes apply electric field to the microspheres under different states, a '-d' represents the electric field gradient around the microspheres under the state, a larger electric field gradient can be generated by simply arranging high molecular material, a micropore is arranged between the two flat electrodes, the electric field can be seen to generate larger distortion around the micropore, thereby generating non-uniform electric field
This phenomenon is also applicable to only adding microspheres, when the microspheres are close to an electrode, because the direction of the electric field is perpendicular to the plane of the electrode, the field intensity measured at the upper and lower sides of the microspheres is no longer symmetrical, and a large field intensity contrast is generated, so that the dielectrophoresis force is generated. In the micropores, only the microspheres near the micropores can be pushed into and out of the micropores by force due to the strong dielectrophoresis force near the micropores, and after one microsphere enters the micropores with the size close to that of the microsphere, the field intensity change is greatly reduced, and because the dielectrophoresis force is reduced, a second microsphere is difficult to enter the micropores, so that the system repeatability of the method is ensured, and one micropore corresponds to one microsphere.
2. Front scattering imaging module (Large area front scattering detection system)
When the microspheres move longitudinally in the microwells, the device incorporates evanescent field based front scattering optics to accurately detect this longitudinal movement. As shown in fig. 3, when a light propagates at a specific angle, a total reflection phenomenon occurs in the propagating medium, and an evanescent field is generated at an interface where the total reflection occurs, and the near-field light is characterized in that the field intensity decreases exponentially with the increase of the distance from the interface, and the light intensity changes greatly. By using the system, after the microspheres are placed in the micropores, when the microspheres are close to the interface, namely the microspheres are at the bottom of the micropores, stronger scattered light can be generated and can be transmitted towards all directions, and when the microspheres are gradually far away from the interface, namely the microspheres move away from the interface, the scattered light intensity index is decreased progressively. The low-power objective lens is used in the equipment system, the scattered far-field light is received by the low-numerical aperture resolution objective lens and the scanning of a low-resolution CCD/CMOS or large-area scanner, each microsphere only needs one pixel to detect the change of the intensity, and the longitudinal movement of each microsphere can be detected with high sensitivity, so that the microspheres manipulated by the dielectrophoresis force can be monitored in real time. It is also possible to use fluorescent microspheres with a fluorescent excitation light system, the excitation light forming an evanescent field, observing the fluorescent emission light signal generated by longitudinal movement of the microspheres near the interface.
As a preferred technical scheme of the invention, the microspheres are magnetic microspheres with fluorescence codes.
The dielectric material generates opposite forces at two ends of the dipole due to the polarization of the electric field in the medium, when the field intensities at the two ends of the dipole are different, the dielectric material is stressed integrally, the force is in direct proportion to the volume of a stressed object and the change difference of the field intensities at the two ends of the dipole, and the force is a function of the frequency of the applied alternating electric field. In one common system, microspheres are placed in a solution medium and microstructures are placed in the solution, the difference between the dielectric constants of the materials of the microstructures and the medium is large, so that distortion of an electric field is generated near the microstructures, and the microspheres are stressed and move in the nonuniform field.
As a preferred technical solution of the present invention, the detection portion further includes a magnetic module, and the magnetic module is configured to move the magnetic microspheres in the microplate chip out of the microplate chip.
Preferably, the detection part further comprises a liquid path control module, and the liquid path control module is used for sealing the surface of the microplate chip.
As a preferred technical scheme of the invention, the sample processing part comprises a sample extraction module, a liquid transfer module, a mixing module and a magnetic suction module; the sample extraction module is used for extracting target molecules in a sample to be detected, the liquid transfer module is used for transferring the extracted target molecules to the mixing module, the mixing module is used for mixing the target molecules with a solution containing magnetic microspheres, and the magnetic attraction module is used for separating the microspheres combined with nucleic acid in the mixing module.
In the invention, the sample extraction module of the non-amplification nucleic acid molecule diagnosis equipment is used for cracking a biological sample, extracting nucleic acid molecules (DNA or RNA) released into a solution by adopting an automatic magnetic particle method or a disposable silica gel column mode, capturing the nucleic acid molecules on magnetic microspheres in a non-specific way, carrying out magnetic separation on the microspheres, washing and eluting the nucleic acid molecules on the surfaces of the microspheres by using water to obtain a pure nucleic acid solution; the liquid transfer module transfers the microspheres connected with the probes, the sample solution to be detected, the detection probes, the catalyst base solution and other reagents to the mixing module; the microspheres connected with the molecules to be detected in the mixing module are hybridized with the detection molecules (sequences); the magnetic module can wash the microspheres for capturing target molecules to be detected and detection molecules through enrichment.
Preferably, the sample processing part further comprises a liquid pre-storing module for storing a solution used when processing the sample.
Preferably, the sample processing part further comprises a liquid path cleaning module, and the liquid path cleaning module is used for cleaning the detection device.
The liquid prestoring module of the sample processing part is used for storing reaction solutions such as buffer solution and the like; the liquid path cleaning module cleans the liquid path to prevent cross contamination.
As a preferred technical solution of the present invention, the detection device further includes an automatic control module, and the automatic control module is configured to control all modules in the detection device.
Preferably, the detection device further comprises an image recognition and analysis module, wherein the image recognition and analysis module is used for recognizing the brightness of each micropore in the microplate chip and analyzing the reaction state in the microplate chip.
In the invention, all modules of the non-amplification detection equipment are automatically controlled by a bottom layer program, an image recognition analysis module is integrated in the equipment, a system automatically recognizes the brightness in each micropore and obtains the distribution of the micropore brightness, and whether a reaction occurs in each micropore is automatically judged. If the microspheres with the fluorescent codes are used, each reaction can correspond to the fluorescent number of the microsphere, the device can obtain the dynamic detection range of the analog signal through brightness analysis, and the number of the reactions of the microspheres with the same number is used as the dynamic detection range of the digital signal.
In a second aspect, the present invention also provides a method for using the detection apparatus according to the first aspect, the method comprising:
transferring the processed microspheres to a microporous plate chip, and transferring the microspheres to the bottom of the microporous plate chip by using an alternating electric field control module; the fluorescence is excited by using a multicolor fluorescence excitation module, the fluorescence irradiates on the microporous plate chip, the fluorescence emission filtering module distinguishes the brightness of the microspheres and the micropores, and the front scattering imaging module collects and images the fluorescence signal of each micropore on the microporous plate chip.
As a preferred embodiment of the present invention, the step of treating the microspheres with a sample treatment section comprises: adding a sample to be detected into a sample extraction module of the sample processing part, extracting target molecules in the sample to be detected by using a gel chromatography or an automatic magnetic particle method, transferring the target molecules to a mixing module by using a liquid transfer module, mixing the target molecules with a solution containing microspheres, and separating the microspheres combined with the target molecules by using a magnetic absorption module of the sample processing part.
Preferably, the step of performing oil phase sealing on the surface of the microplate chip by using the liquid path control module is further included after the magnetic microspheres are transferred to the bottom of the microplate chip.
Preferably, the use method further comprises the step of cleaning the detection device by liquid path cleaning after the imaging is completed.
As a preferable technical solution of the present invention, the using method includes:
(1) Adding a sample to be detected into the sample extraction module, extracting target molecules in the sample to be detected, transferring the sample to a mixing module by using the liquid transfer module, mixing the sample with a solution containing microspheres, and separating the microspheres combined with the target molecules by using the magnetic absorption module of the sample processing part;
(2) Transferring the magnetic microspheres to a microporous plate chip, transferring the magnetic microspheres to the microporous plate chip by using the detection part of the detection equipment sample, and transferring the magnetic microspheres to the bottom of the microporous plate chip by using an alternating electric field control module;
(3) The method comprises the steps of exciting fluorescence by using a multicolor fluorescence excitation module, irradiating the fluorescence on a microporous plate chip, distinguishing the brightness of microspheres and micropores by using a fluorescence emission filtering module, collecting and imaging a fluorescence signal of each micropore on the microporous plate chip by using a front scattering imaging module, and obtaining the reaction state of each micropore on a microporous plate chip by using an image recognition analysis module.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) According to the detection equipment based on the microspheres and the microporous plate, the alternating electric field control module is used for applying the non-uniform alternating electric field to control the microspheres, so that the microspheres can more effectively enter micropores of a microporous plate chip, signals of the microspheres cannot be captured due to the fact that the microspheres are gathered on the surface of the microporous plate chip are prevented, and the accuracy of a detection result is ensured;
(2) The detection equipment is provided with a front scattering imaging module for capturing the fluorescent signal of the microplate chip and converting the fluorescent signal into an image, so that the reaction state of each micropore in the microplate chip can be more clearly obtained, and the detection result is more visual;
(3) The detection device provided by the invention integrates the functions of sample treatment, sample detection and sample analysis, simplifies the operation steps of molecular diagnosis, reduces the cross contamination between reagents and the external environment in the molecular diagnosis, and ensures that the operation of the molecular diagnosis is simpler and more convenient and the result is more accurate.
Drawings
Figure 1 is a graph of dielectrophoretic force as a function of frequency of the electric field.
FIG. 2 is a schematic diagram of the electric field gradient around the microspheres in different states between the plate electrodes.
Fig. 3 is a schematic diagram of the working principle of the front scattering imaging module in the detection apparatus based on the microsphere and the microplate provided by the invention.
FIG. 4 is a schematic diagram of a detection portion of a detection apparatus based on microspheres and a microplate provided by the invention.
FIG. 5 is a photograph of a microplate containing fluorescently encoded microspheres obtained using the detection apparatus provided by the present invention in example 1.
FIG. 6 (a) shows a microplate chip (scale 100 μm) used in example 2; FIG. 6 (b) shows a microplate chip (scale 200 μm) used in example 2.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.
Example 1
The embodiment provides detection equipment based on microspheres and a microporous plate, which is used for detecting whether a sample to be detected contains influenza A virus and influenza B virus. The detection equipment comprises the following components in percentage by weight:
(1) A sample processing part:
1. a sample extraction module: extracting all nucleic acids of the culture solution of the influenza A virus and the influenza B virus by a magnetic particle method, and finally eluting in water. The method comprises the following specific steps: to 200uL of the virus culture medium were added 130uL of lysis solution (50mM Tris, pH 8.0,4M guanidine hydrochloride, 1mM EDTA,1% Triton-X100), 10uL proteinase K (20 mg/mL), and water bath at 55 ℃ for 10 minutes; adding 150uL of isopropanol into each sample, mixing, blowing and beating uniformly, and adding 500ug of silica magnetic beads with hydroxyl on the surface; standing for five minutes, adsorbing the magnetic beads on a magnetic frame, removing supernatant by using a pipette gun, washing the magnetic beads by using 70% ethanol, and eluting by using 50uL water.
2. A liquid transfer module: transferring the sample extract to a mixing module using a movable liquid transfer module;
3. a mixing module: adding 1mg/mL fluorescent microsphere 1 coupled with oligonucleotide probe and fluorescent microsphere 2, mixing in equal volume to obtain 0.2mL of solution, adding 0.2mL of hybridization buffer (50 mM sodium citrate, pH 7.2, 750mM NaCl) and 0.2mL of nucleic acid probe coupled with biotin, wherein the nucleic acid probe can specifically bind with influenza A virus nucleic acid molecules or influenza B virus nucleic acid molecules, uniformly mixing for 5 minutes, then carrying out magnetic attraction and washing, adding 0.2mL of streptavidin-beta-galactosidase conjugate connected on the nucleic acid probe to serve as detection molecules, uniformly mixing for 5 minutes, then carrying out magnetic attraction and washing, and dispersing magnetic beads into 100 mu M dihydrofluorescein-di-beta-D-galactopyranoside solution.
The preparation method of the magnetic microsphere containing the fluorescent code comprises the following steps:
styrene monomer, polymethyl methacrylate, a cross-linking agent (divinylbenzene) and acrylic oligomer are mixed in chloroform to be used as a high molecular solution, 4.5mL of the high molecular solution is added with 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles, and then the high molecular solution is placed in two reactors with 300mL of deionized water and sodium dodecyl sulfate to obtain emulsion.
Ultrasonic generators (50uJ, 100uJ) and ultrasonic transmitters are respectively arranged on two sides of a container for bearing the emulsionThe sensor (4 MHz,6dB bandwidth, 4.2MHz, focusing length 10.5 cm) controls the ultrasonic wave to the Fe in the microsphere emulsion in a closed loop manner through the real-time feedback of the sensor 3 O 4 The magnetic nano particles are modulated, the magnetic particles absorb ultrasonic wave bands and keep the polarity orientation to be induced by a field of external ultrasonic waves according to the principle of a magnetic resonance imaging device, and in order to keep the modulation efficiency of induction, the sensor passes through Fe 3 O 4 Specific absorption spectrum of magnetic nano-particles can timely modulate energy of ultrasonic pulse so as to controllably modulate Fe 3 O 4 The orientation of the magnetic nano particles is adopted to obtain liquid drops with uniformly distributed functional particles and consistent dipole directions;
uniformly dispersing the solution into droplets of 10 mu m, adding an initiator azobisisobutyronitrile into the solution, heating to perform polymerization crosslinking reaction, slowly dissolving chloroform in each droplet in water after 24 hours, volatilizing, polymerizing and crosslinking monomers to finally form the fluorescent coding microspheres;
two parts of fluorescent coding microspheres are taken, the microsphere 1 is marked by a complementary single-stranded DNA molecule (capture molecule 1) of an influenza A virus nucleic acid molecule, and the microsphere 2 is marked by a complementary single-stranded DNA molecule (capture molecule 2) of an influenza B virus nucleic acid molecule. The method comprises the following specific steps:
taking microsphere 1 as an example, 1mg of microsphere 1 is dispersed in 1mL of PBS buffer, 5mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 5mg of Sulfo-NHS (N-hydroxythiosuccinimide) are added, mixed uniformly and kept stirred for 10 minutes, then influenza A virus nucleic acid complementary single-stranded DNA molecules with amino groups at the 3' end are added, then 10% BSA is added as a blocking agent, stirring is carried out for 30 minutes, then microsphere 1 is washed by magnetic separation, finally the microspheres are dispersed in a hybridization buffer (50 mM sodium citrate, pH 7.2, 750mM NaCl) to obtain fluorescent microsphere 1 labeled with capture molecule 1, and the preparation method of fluorescent microsphere 2 labeled with capture molecule 2 is the same as the above.
4. Module is inhaled to magnetism: the composite microspheres are magnetic microspheres, so that the magnetic microspheres can be adsorbed and gathered by using the magnetic adsorption module.
(2) The detection part, the relation among the modules of the sample processing part is shown in figure 4:
1. alternating electric field control module (electric tweezers system): the composite microspheres are gathered and transferred to the surface of a microporous plate chip, 10MHz alternating current is applied by using an alternating electric field control module, the composite microspheres are guided into micropores by using electric tweezers, and galactosidase on the surfaces of the composite microspheres can catalyze reaction substrates to react, so that the concentration of the galactosidase in each micropore is increased by multiple orders of magnitude due to the small volume of the micropore, and the luminescence of a single micropore can be sufficiently catalyzed;
2. multi-color fluorescence excitation module: after reacting for 2 minutes, exciting the light with wavelength of 488nm by using a multicolor fluorescence excitation module;
3. a fluorescence emission filtering module (the fluorescence emission filtering module is represented by taking an emission light filter as a main component in fig. 4) and a front scattering imaging module (the front scattering imaging module is represented by taking a low-magnification lens and a CCD/CMOS/scanner as main components in fig. 4): filtering with filter 1 (512 nm transmission, 20nm bandwidth) and filter 2 (570 nm transmission, 30nm bandwidth) of fluorescence emission filtering module; the fluorescence signal on the micro-porous plate chip is received by the front scattering imaging module and transferred to the charge coupled element for imaging; then, the light was excited again with 532nm wavelength light, and the image was taken through filter 3 (615 nm transmission, 30nm bandwidth).
FIG. 6 is a photograph of a microplate containing fluorescence-encoded microspheres 1 obtained by the amplified nucleic acid molecule diagnostic apparatus when the filter 1 is excited by light with a wavelength of 488nm for taking a picture. As can be seen from FIG. 5, the fluorescent microspheres 1 capturing the influenza A virus nucleic acids uniformly fall into the microwells, each microwell has only one microsphere and is limited in the microwell, and the labeled enzyme on the microspheres amplifies the fluorescent signal and is detected by the front scattering imaging module.
Example 2
The embodiment provides a detection device based on microspheres and a microporous plate, which is used for detecting whether a sample to be detected contains influenza A virus and influenza B virus. The detection equipment comprises the following components in percentage by weight:
(1) Sample processing section
The working principle of the sample extraction module, the liquid transfer module, the mixing module and the magnetic suction module of the device in the embodiment is the same as that of the device in the embodiment 1;
the difference is that the sample processing part also comprises a liquid pre-storing module and a liquid path cleaning module, after the detection is finished, the liquid path cleaning module can add ethanol into each module of the device for pretreatment, and cleaning is carried out by flowing in cleaning liquid.
(2) Detecting part
The working principle of the alternating electric field control module, the multicolor fluorescence excitation module, the fluorescence emission filtering module and the front scattering imaging module is the same as that of the embodiment 1;
the detection part also comprises a liquid path control module, and after the microspheres enter the micropores, the liquid path control module adds silicone oil on the surface of the microporous plate chip to seal the micropores;
meanwhile, the detection part also comprises a magnetic absorption module, most of magnetic microspheres in the microporous plate chip can be moved out of the microporous plate chip by the magnetic absorption module, a cleaning solution flows in after the ethanol pretreatment is carried out by the liquid path cleaning module, and the alternating electric field control module applies 10kHz alternating current to more thoroughly remove the magnetic microspheres in the micropores so as to realize the recycling of the microporous plate chip.
(3) An automatic control module: controlling all modules in the detection device.
(4) The image recognition analysis module: the device also comprises an image recognition and analysis module, the system automatically recognizes the brightness in each micropore and obtains the distribution of the micropore brightness, and as a result, as shown in fig. 6 (a) and 6 (b), the system automatically judges whether a reaction occurs in each micropore, and the existence of brightness in the micropore means that the reaction occurs.
Example 3
In the embodiment, the fluorescent microspheres are manipulated in an area of 3mm multiplied by 3mm by using an alternating electric field control module and a front scattering imaging module in the equipment and are monitored in real time.
Processing 4 μm diameter and 4 μm high micro-hole on the surface plated with gold electrode by photoetching method to form 6mm × 6mm chip, and assembling metal electrode on the opposite surface to form micro-fluidic flow channel. The two electrodes are connected to the positive and negative terminals of a high-frequency voltage signal generator. The bottom of the micropore is irradiated by 532nm laser with an incident angle of 75 degrees, and filtered light of 580nm is detected by a 4-time objective lens with NA0.1 and a 500-ten-thousand-pixel CMOS chip. A3 μm rhodamine 6 dyed microsphere dispersion was flowed into the channel and applied with a voltage of 100kHz 10V pp to push the microspheres into the microwells close to the bottom interface of the microwells, the CMOS detector received a signal of high brightness for each microsphere, and when applied with a voltage of 1kHz 10Vpp, the brightness of the microspheres was greatly reduced and pushed out of the microwells.
Example 4
The embodiment utilizes the alternating electric field control module and the front scattering imaging module in the equipment to perform 100mm 2 The area of (a) was used to manipulate the cells and monitored in real time.
And processing micropores with the diameter of 20 microns and the height of 20 microns on the surface plated with the gold electrode by using a photoetching method to form a chip with the diameter of 10mm multiplied by 10mm, and assembling a metal electrode on the opposite surface of the chip to finally form the microfluidic flow channel. The two electrodes are connected to the positive and negative terminals of a high-frequency voltage signal generator. White light at an incident angle of 75 degrees at the bottom of the microwell was illuminated and detected with a conventional single lens reflex camera lens in conjunction with a scanner. A T cell dispersion solution was flowed into the channel, and a voltage of 100kHz 10Vpp was applied to push single cells into the wells close to the bottom interface of the wells, and a light intensity signal scattered by each cell was received by the detector, and when a voltage of 1kHz 10Vpp was applied, the brightness of the cells decreased greatly and pushed out of the wells.
In summary, according to the detection apparatus based on the microsphere and the microplate provided by the invention, the electric field is applied to the microplate chip to push the microsphere into the micropores of the microplate chip, and the front scattering light imaging module is used for imaging, so that the reaction state of each micropore in the microplate chip is more clearly obtained, and thus, the accurate qualitative and quantitative determination of the target molecule to be detected is realized.
The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (15)
1. The detection equipment based on the microspheres and the microporous plate is characterized by comprising a sample processing part and a detection part;
the detection part comprises a multicolor fluorescence excitation module, a front scattering imaging module, a fluorescence emission filtering module and an alternating electric field control module;
the multicolor fluorescence excitation module is used for exciting the multiple fluorescence coding microspheres and fluorescent substances released by substrate molecules under the action of a catalyst;
the front scattering imaging module is used for placing a microporous plate chip and imaging micropores of the microporous plate chip;
the fluorescence emission filtering module is used for distinguishing the coded microspheres and micropore brightness changes;
the alternating electric field control module is used for controlling the movement of the microspheres in the microporous plate chip, the microspheres are transferred to the bottom of the microporous plate chip by using the alternating electric field control module, and the sizes of the micropores are close to that of the microspheres, so that only one microsphere can enter each micropore.
2. The detection apparatus of claim 1, wherein the microspheres are fluorescently encoded magnetic microspheres.
3. The detection apparatus according to claim 2, wherein the detection portion further comprises a magnetic attraction module for moving the magnetic microspheres in the microplate chip out of the microplate chip.
4. The detection apparatus of claim 1, wherein the detection portion further comprises a liquid path control module for sealing the microplate chip surface.
5. The testing device of claim 2, wherein the sample processing portion comprises a sample extraction module, a liquid transfer module, a mixing module, and a magnetic attraction module;
the sample extraction module is used for extracting target molecules in a sample to be detected, the liquid transfer module is used for transferring the extracted target molecules to the mixing module, the mixing module is used for mixing the target molecules with a solution containing magnetic microspheres, and the magnetic attraction module is used for separating the microspheres combined with the target molecules in the mixing module.
6. The detection apparatus according to claim 5, wherein the target molecule is a nucleic acid molecule.
7. The testing device of claim 5, wherein the sample processing portion further comprises a liquid pre-storage module for storing a solution used when processing the sample.
8. The testing device of claim 5, wherein the sample processing portion further comprises a fluid path cleaning module for cleaning the testing device.
9. The detection apparatus according to claim 1, further comprising an automatic control module for controlling all modules in the detection apparatus.
10. The detection apparatus according to claim 1, further comprising an image recognition and analysis module for recognizing brightness of each microwell in the microplate chip and analyzing a reaction state in the microplate chip.
11. The method of using a microsphere and microplate based detection apparatus according to any one of claims 1-10, wherein the method of use comprises:
transferring the processed microspheres to a microporous plate chip, and transferring the microspheres to the bottom of the microporous plate chip by using an alternating electric field control module;
the fluorescence is excited by using a multicolor fluorescence excitation module, the fluorescence irradiates on the microporous plate chip, the fluorescence emission filtering module distinguishes the brightness of the microspheres and the micropores, and the front scattering imaging module collects and images the fluorescence signal of each micropore on the microporous plate chip.
12. The method of use of claim 11, wherein the step of treating the microspheres with a sample treatment moiety comprises:
adding a sample to be detected into a sample extraction module of the sample processing part, extracting target molecules in the sample to be detected by using a gel chromatography or an automatic magnetic particle method, transferring the target molecules to a mixing module by using a liquid transfer module, mixing the target molecules with a solution containing microspheres, and separating the microspheres combined with the target molecules by using a magnetic absorption module of the sample processing part.
13. The use method of claim 11, further comprising the step of performing oil phase blocking on the surface of the microplate chip by using a liquid path control module after transferring the microspheres to the bottom of the microplate chip.
14. The method of use of claim 11, further comprising the step of cleaning the detection device with a fluid path cleaning module after imaging is complete.
15. The method of use according to claim 11, comprising:
(1) Adding a sample to be detected into the sample extraction module, extracting target molecules in the sample to be detected, transferring the sample to a mixing module by using a liquid transfer module, mixing the sample with a solution containing microspheres, and separating the microspheres combined with the target molecules by using a magnetic absorption module of the sample processing part;
(2) Transferring the magnetic microspheres to a microporous plate chip, transferring the magnetic microspheres to the microporous plate chip by using a sample detection part of the detection equipment, and transferring the magnetic microspheres to the bottom of the microporous plate chip by using an alternating electric field control module;
(3) The method comprises the steps of exciting fluorescence by using a multicolor fluorescence excitation module, irradiating the fluorescence on a microporous plate chip, distinguishing the brightness of microspheres and micropores by using a fluorescence emission filtering module, collecting and imaging a fluorescence signal of each micropore on the microporous plate chip by using a front scattering imaging module, and obtaining the reaction state of each micropore on the microporous plate chip by using an image recognition analysis module.
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