CN112657563A - Micro-fluidic liquid drop platform based on BRET bioluminescence technology - Google Patents
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Abstract
The invention discloses a BRET bioluminescence technology-based microfluidic droplet platform, which comprises a PDMS microfluidic chip; the PDMS microfluidic chip comprises a droplet generation module, a droplet mixing module and a fluorescence detection module. Compared with the traditional BRET bioluminescence detection technology by an enzyme-labeling instrument, the BRET bioluminescence detection method has the characteristics of high-throughput detection, rapid detection, low sample consumption and the like.
Description
Technical Field
The invention belongs to the technical field of microfluidics, and particularly relates to a microfluidic droplet platform based on a BRET bioluminescence technology.
Background
At present, the most widely applied optical analysis method in the field of biomedical analysis is mainly fluorescence analysis, and the method is mature in technology and wide in application. However, the existing fluorescence analysis technology often uses an external light source as excitation light, and has the problems that molecules with fluorescence properties in a biological sample generate autofluorescence, fluorescent molecules generate photobleaching due to high excitation energy of the external light source, and the external excitation light generates phototoxicity on photosensitive molecules.
Unlike fluorescence technology which relies on an external light source as excitation light, Bioluminescence Resonance Energy Transfer (BRET) is an Energy Transfer system in which luciferase, which catalyzes the luminescence of a substrate, serves as an Energy donor and a molecule having a fluorescent property serves as an Energy acceptor. The technology uses luciferase as a light source to excite an energy receptor to emit light, and avoids the problems of autofluorescence interference, phototoxicity, photobleaching and the like caused by external excitation light irradiation. The BRET-based detection system has the advantages of high sensitivity, good specificity, no need of expensive light sources for detection equipment and the like, and is widely concerned in the biomedical analysis fields of protein interaction detection, small molecule substance detection, disease marker detection (such as nucleic acids, proteins, polypeptides, carbohydrate disease markers and the like) and the like.
In the BRET technology, bioluminescence rapidly occurs and a fluorescence peak value is rapidly reached, but the conventional enzyme-linked immunosorbent assay method is utilized, the time from sample loading to fluorescence detection is in a neutral position, a sample loading device is matched to detect the fluorescence peak value, and the steps are complicated; the consumption of the microplate reader to the sample is large, and is usually hundreds of micro-upgrade; meanwhile, a sample in a detection system of the microplate reader is contacted with air, so that the external environment easily pollutes the sample to be detected, and the detection result is interfered; in addition, the fluorescence of BRET is detected by using a microplate reader, and the diffusion mass transfer of the pore plate serving as a reaction cell is slow.
Disclosure of Invention
In order to solve the problems in the background art, the present invention provides a microfluidic droplet platform based on BRET bioluminescence technology.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a BRET bioluminescence technology-based microfluidic droplet platform, which comprises a PDMS microfluidic chip; the PDMS microfluidic chip comprises a droplet generation module, a droplet mixing module and a fluorescence detection module which are sequentially arranged.
Furthermore, the microfluidic droplet platform comprises an upper layer and a lower layer, wherein the upper layer is a PDMS microfluidic chip, and the lower layer is a glass substrate.
Further, the droplet generation module is a flow focusing structure.
Further, the droplet mixing module is of a serpentine structure.
Further, the fluorescence signal in the fluorescence detection module is detected by a spatial optical detection system, and the spatial optical detection system and the microfluidic droplet platform are independent from each other.
Further, the spatial optical inspection system includes a microscope objective X20 (numerical aperture NA ═ 0.4), a dichroic mirror, a first photomultiplier tube equipped with a first filter for BRET energy donor emission light, and a second photomultiplier tube equipped with a second filter for BRET energy donor emission light.
Further, the fluorescence signal in the fluorescence detection module is collected by a microscope objective X20 (numerical aperture NA ═ 0.4).
Further, the fluorescence signal collected by the microscope objective lens X20 (numerical aperture NA ═ 0.4) separates the emitted light using a dichroic mirror, and separates the emitted light into two wavelengths.
Further, light of two wavelengths is sampled using a first photomultiplier tube equipped with a first filter for receptor emission of BRET energy and a second photomultiplier tube equipped with a second filter for receptor emission of BRET energy, respectively.
Further, the method for detecting BRET bioluminescence by the microfluidic droplet platform comprises the following steps: adding a BRET system, a luciferase substrate and oil into a droplet generation module respectively;
the BRET system, the luciferase substrate and the oil generate water-in-oil micro-droplets in the droplet generation module; then the 'water-in-oil' micro-droplets enter a droplet mixing module, and in the droplet mixing module, a substrate and an enzyme are combined into an energy receptor to provide energy in the BRET technology, so that the receptor emits fluorescence; and finally, the fluorescence signal is detected in a fluorescence detection module.
The invention has the beneficial effects that: the invention carries out fluorescence analysis based on BRET bioluminescence technology, and the technology utilizes luciferase as an endogenous excitation light source, so that the generated bioluminescence is very mild, and the problems of autofluorescence interference, phototoxicity, photobleaching and the like caused by external excitation light irradiation are effectively avoided. The BRET technology can be used for detecting protein interaction, detecting small molecular substances and detecting disease markers (such as nucleic acids, proteins, polypeptides, carbohydrate disease markers and the like), and has wide application.
The invention provides a BRET bioluminescence technology-based micro-fluidic droplet platform, which is characterized in that a micro-fluidic chip is manufactured through a micro-nano processing technology, a Flow Focus structure is designed in the micro-fluidic chip to prepare water-in-oil micro-droplets, the volume of the droplets is only picoliter (pL) grade, the consumption of a sample is greatly reduced, high-flux detection is realized, and in addition, the droplets are wrapped in an oil phase to avoid the contact of the sample and the external environment, so that the pollution possibility is greatly reduced; the micro-droplets are fully mixed with BRET system reactants through a micro-scale droplet mixing module, the droplet mixing module is designed into a winding snake-shaped channel, when the droplets pass through the structure, eddy current can be generated in the droplets and is continuously stirred, the components in the droplets can be fully mixed in a short time, the droplets are stretched and extruded through the width scaling of a flow channel, and the reaction efficiency in the droplets is improved again (the laminar flow effect in a micro-fluidic chip cannot realize the rapid and full mixing of BRET system reactants in the droplets); and finally, bioluminescence detection of the BRET system is realized through a space optical detection system with strong operability and the independence of the microfluidic chip is reserved, and the fluorescent detection module of the microfluidic chip determines the position with the strongest luminescence through detection of the luminescent signals of liquid drops at different positions, so that the fluorescent signal of the BRET system is quickly detected (the traditional microplate reader is matched with a sample injector for detecting the fluorescent peak value, the operation is complex, and the invention uses space optical detection and can quickly detect the fluorescent peak value after the luminescent position is determined). Compared with the traditional detection technical means of the microplate reader, the platform has the characteristics of rapid detection, high-flux detection, small sample consumption and the like.
Drawings
Fig. 1 is a schematic structural diagram of a microfluidic droplet platform based on BRET bioluminescence technology in example 1 of the present invention.
Fig. 2 is a schematic structural diagram of a microfluidic droplet platform based on BRET bioluminescence technology in example 2 of the present invention.
Fig. 3 is a schematic structural diagram of the microfluidic droplet platform based on BRET bioluminescence technology and a spatial optical detection system in example 2 of the present invention.
The system comprises a droplet generation module, a droplet mixing module, a fluorescence detection module, a microscope objective X20 (numerical aperture NA is 0.4), a dichroic mirror 5, a BRET energy supply receptor emission light first filter 6, a first photomultiplier 7, a BRET energy supply receptor emission light second filter 8, and a second photomultiplier 9.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. However, the following examples are illustrative and not restrictive, and any selection and modification may be made within the scope not affecting the technical effect of the present invention.
Example 1
A micro-fluidic liquid drop platform based on BRET bioluminescence technology comprises a PDMS micro-fluidic chip, wherein the structural schematic diagram of the PDMS micro-fluidic chip is shown in figure 1, and the PDMS micro-fluidic chip comprises a liquid drop generating module, a liquid drop mixing module and a fluorescence detection module which are sequentially arranged.
The method for detecting BRET bioluminescence by the microfluidic droplet platform comprises the following steps: adding a BRET system, a luciferase substrate and oil into a droplet generation module respectively;
the BRET system, the luciferase substrate and the oil generate water-in-oil micro-droplets in the droplet generation module; then the 'water-in-oil' micro-droplets enter a droplet mixing module, and in the droplet mixing module, a substrate and an enzyme are combined into an energy receptor to provide energy in the BRET technology, so that the receptor emits fluorescence; and finally, the fluorescence signal is detected in a fluorescence detection module.
Example 2
A micro-fluidic liquid drop platform based on BRET bioluminescence technology comprises a PDMS micro-fluidic chip, wherein the structural schematic diagram of the PDMS micro-fluidic chip is shown in figure 2, and the PDMS micro-fluidic chip comprises a liquid drop generating module 1, a liquid drop mixing module 2 and a fluorescence detection module 3 which are sequentially arranged. The droplet generation module 1 is a flow focusing structure. The generation of the liquid drop adopts a Flow Focus liquid drop generation structure, and the high-speed generation of the liquid drop can be realized. The droplet mixing module 2 has a serpentine structure. By designing the serpentine mixing structure which meanders, the components in the liquid drop are quickly and fully mixed. In the droplet mixing module 2, the size and number of mixing structures may be optimized according to reaction efficiency, reaction time, and the like. The fluorescence signal in the fluorescence detection module 3 is detected by a spatial optical detection system. The space optical detection system and the microfluidic droplet platform are mutually independent. A schematic structure diagram of a microfluidic droplet platform based on BRET bioluminescence technology and a spatial optical detection system used in combination is shown in fig. 3. The spatial optical detection system comprises a microscope objective X20 (numerical aperture NA ═ 0.4)4, a dichroic mirror 5, a first photomultiplier tube 7 equipped with a first filter 6 for BRET energy donor emission light and a second photomultiplier tube 9 equipped with a second filter 8 for BRET energy donor emission light. The fluorescence signal in the fluorescence detection module was collected by microscope objective X20 (numerical aperture NA ═ 0.4) 4. The fluorescence signal collected by the microscope objective lens X20 (numerical aperture NA ═ 0.4)4 separates the emitted light using the dichroic mirror 5, separating the emitted light into two wavelengths. The light of the two wavelengths is sampled by means of a first photomultiplier tube 7 equipped with a first filter 6 for the acceptor emission light and a second photomultiplier tube 9 equipped with a second filter 8 for the acceptor emission light.
The method for detecting BRET bioluminescence by the microfluidic droplet platform comprises the following steps: respectively enabling a BRET system, a luciferase substrate and oil to enter a liquid drop generation module 1 through three injection ports of the liquid drop generation module 1 (in the BRET bioluminescence technology, an energy donor is luciferase, and an energy acceptor is various fluorescent proteins, organic fluorescent molecules, quantum dots and the like); the BRET system, the luciferase substrate and the oil generate 'water-in-oil' micro-droplets in a droplet generation module 1 with a Flow Focus structure, and the droplets wrap the reaction system in the oil phase on one hand, so that the interference of the external environment on the reaction system is avoided; on the other hand, the volume of the liquid drop is very small and only has picoliter grade, compared with hundred-microliter grade required by the detection of the traditional microplate reader, the consumption of the sample is greatly reduced, and high-flux detection is realized;
then, the 'water-in-oil' micro liquid drops enter the liquid drop mixing module 2 with the zigzag structure, when the liquid drops enter the zigzag channel, vortex flow can be generated in the liquid drops, the liquid drops are continuously stirred, the components in the liquid drops are fully mixed in a short time, the liquid drops are stretched and extruded by the aid of the width scaling of the flow channels, and the reaction efficiency in the liquid drops is improved again. In the BRET technology in the liquid drop mixing module 2, the combination of a substrate and an enzyme provides energy for an energy receptor, so that the receptor emits fluorescence;
finally, the liquid drops after being fully mixed enter the fluorescence detection module 3, and the fluorescence signal is detected in the fluorescence detection module 3. The intensity of the emitted light from the donor and acceptor was collected and detected using a spatial optical detection system, and the energy transfer efficiency (BRET Ratio) was calculated as acceptor light intensity/donor light intensity.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A BRET bioluminescence technology-based microfluidic droplet platform is characterized in that the microfluidic droplet platform comprises a PDMS microfluidic chip; the PDMS microfluidic chip comprises a droplet generation module, a droplet mixing module and a fluorescence detection module which are sequentially arranged.
2. The BRET bioluminescence technology based microfluidic droplet platform of claim 1, wherein the microfluidic droplet platform comprises an upper layer and a lower layer, the upper layer is a PDMS microfluidic chip, and the lower layer is a glass substrate.
3. The BRET bioluminescence technology based microfluidic droplet platform of claim 1 or 2, wherein the droplet generation module is a flow focusing structure.
4. The BRET bioluminescence technology based microfluidic droplet platform of claim 1 or 2, wherein the droplet mixing module is a serpentine structure.
5. The BRET bioluminescence technology based microfluidic droplet platform of claim 1 or claim 2, wherein the fluorescence signal in the fluorescence detection module is detected by a spatial optical detection system that is independent of the microfluidic droplet platform.
6. The BRET bioluminescence technology based microfluidic droplet platform according to claim 5, wherein the spatial optical detection system comprises a microscope objective X20 (numerical aperture NA 0.4), a dichroic mirror, a first photomultiplier tube equipped with a first filter for BRET energy donor emission light, and a second photomultiplier tube equipped with a second filter for BRET energy donor emission light.
7. The BRET bioluminescence technology based microfluidic droplet platform of claim 6, wherein the fluorescence signal in the fluorescence detection module is collected by a microscope objective lens X20 (numerical aperture NA ═ 0.4).
8. The BRET bioluminescence technology based microfluidic droplet platform of claim 7, wherein the fluorescence signal collected by microscope objective X20 (numerical aperture NA ═ 0.4) uses a dichroic mirror to split the emission light into two wavelengths.
9. The BRET bioluminescence technology based microfluidic droplet platform of claim 8, wherein light of two wavelengths is sampled using a first photomultiplier tube equipped with a first filter for BRET energy acceptor emission and a second photomultiplier tube equipped with a second filter for BRET energy acceptor emission, respectively.
10. The BRET bioluminescence technology based microfluidic droplet platform of claim 1 or claim 2, wherein the method for detecting BRET bioluminescence by the microfluidic droplet platform is as follows: adding a BRET system, a luciferase substrate and oil into a droplet generation module respectively;
the BRET system, the luciferase substrate and the oil generate water-in-oil micro-droplets in the droplet generation module; then the 'water-in-oil' micro-droplets enter a droplet mixing module, and in the droplet mixing module, a substrate and an enzyme are combined into an energy receptor to provide energy in the BRET technology, so that the receptor emits fluorescence; and finally, the fluorescence signal is detected in a fluorescence detection module.
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| CN114917971A (en) * | 2022-05-19 | 2022-08-19 | 合肥工业大学 | Micro-fluidic chip for trace detection of hydrogen sulfide based on micro-droplets and detection method |
| CN116930142A (en) * | 2023-09-17 | 2023-10-24 | 苏州和迈精密仪器有限公司 | POCT homogeneous phase time resolution fluorescence detection device |
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