CN116337728B - Fluorescence detection device of monolithic integrated micro-flow cytometer - Google Patents
Fluorescence detection device of monolithic integrated micro-flow cytometer Download PDFInfo
- Publication number
- CN116337728B CN116337728B CN202310623997.5A CN202310623997A CN116337728B CN 116337728 B CN116337728 B CN 116337728B CN 202310623997 A CN202310623997 A CN 202310623997A CN 116337728 B CN116337728 B CN 116337728B
- Authority
- CN
- China
- Prior art keywords
- layer
- fluorescence
- electrode layer
- fluorescence detection
- bulk acoustic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02D—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN INFORMATION AND COMMUNICATION TECHNOLOGIES [ICT], I.E. INFORMATION AND COMMUNICATION TECHNOLOGIES AIMING AT THE REDUCTION OF THEIR OWN ENERGY USE
- Y02D30/00—Reducing energy consumption in communication networks
- Y02D30/70—Reducing energy consumption in communication networks in wireless communication networks
Abstract
The application discloses a fluorescence detection device of a monolithic integrated microfluidic cytometer, which comprises a confocal reflection fluorescence detection system, an ultrahigh frequency bulk acoustic resonator and a microfluidic chip, wherein the microfluidic chip is positioned above the ultrahigh frequency bulk acoustic resonator and comprises a sample inlet, a sample outlet and a micro-channel, and is used for controlling fluorescent particles to directionally pass through the micro-channel; the ultra-high frequency bulk acoustic wave resonator comprises a Bragg reflection layer and an electrode layer, wherein the electrode layer is deposited on part of the surface of the Bragg reflection layer, the other part of the surface is used as a laser irradiation area, the electrode layer is adjacent to the sample inlet, and the laser irradiation area is adjacent to the sample outlet; the confocal reflection fluorescence detection system is used for irradiating laser to the laser irradiation area and detecting fluorescence of fluorescent particles; the device can increase the maximum excitation quantity and emission intensity of fluorescent molecules, reduce noise interference and increase the detection intensity of fluorescent particles.
Description
Technical Field
The application belongs to the field of fluorescence detection, and particularly relates to a fluorescence detection device of a single-chip integrated micro-flow cytometer.
Background
One recent trend in flow cytometry is monolithic miniaturization and replacement of cumbersome components with smaller, cheaper counterparts. The microfluidic cytometer is a micro device which uses laser to excite a plurality of fluorescent samples, and collects fluorescence to a photoelectric detector through an objective lens or a waveguide to realize sample detection. The flow cytometry and microfluidic technologies are combined, and the flow cytometry and microfluidic device has the advantages of being small in size, low in cost and low in consumption. One of the most critical parameters of a microfluidic cytometer is the minimum detection limit of fluorescence, i.e. fluorescence sensitivity. At present, a plurality of methods can improve fluorescence detection performance, namely, excitation energy is improved, a high-power and low-noise laser is generally used for excitation, but the fluorescence quantity of a sample is certain, and the fluorescence saturation and even bleaching can be caused by the excessively high laser energy, meanwhile, background noise can be improved, and the fluorescence sensitivity can be reduced; secondly, increasing the fluorescence acceptance of the detector, typically using expensive high numerical aperture objectives and high sensitivity detectors, increases the design difficulty and cost of the microfluidic system. Thirdly, focusing the sample to be measured on the center and focal plane position of the laser spot is an essential method. The positioning ensures that the fluorescent sample is excited by consistent laser, thereby being beneficial to increasing the maximum excitation quantity and emission intensity of fluorescent molecules, reducing noise interference and increasing detection sensitivity. Various focusing methods such as inertial flow, sheath flow, electrophoresis, magnetophoresis, acoustophoresis and the like are currently available, but have certain limitations and can only be effective under specific conditions. Such as inertial flow, requires complex flow channel structures; sheath fluid is needed for sheath flow, reagent consumption can be increased, and electrophoresis can only control charged samples and the like. Fourth, the light path structure also can influence the fluorescence sensitivity of the system, and most flow cytometry adopts an orthogonal optical system, so that the fluorescence quantity at one angle can be collected, and the fluorescence sensitivity is lower. The confocal reflection light path structure can only detect fluorescence signals excited by particles in the optical detection area, so that the influence of stray light is reduced, and the signal-to-noise ratio can be improved. However, this structure has high selectivity to the substrate, and the transparent substrate has low reflectivity, and cannot effectively reflect fluorescence to the detector. The opaque substrate needs to have smooth surface and high reflectivity. Otherwise, more stray light and excitation light are reflected to the detector, greatly affecting the signal-to-noise ratio. In addition, with the development of optical thin film manufacturing technology, a reflective film, an interference film, etc. can be processed on a substrate, and fluorescence collection efficiency can be improved by utilizing the thin film reflection interference effect. However, the thin film processing requires a separate program or chip, lacks monolithic integration with a focusing structure, and is therefore complicated to operate, and is time-consuming and labor-consuming.
In a word, the current microfluidic cytometry adopts various focusing methods to improve fluorescence detection sensitivity, and the design and integration of an optical detection system are absent, so that the volume is large, and the signal to noise ratio is low. Therefore, a highly integrated miniaturized device with high fluorescence collection capacity, which is simple and portable, realizes high-sensitivity fluorescence detection and provides for the next accurate sorting, is needed. In particular point-of-care applications may benefit from an integrated optical detection device.
Disclosure of Invention
One recent trend in flow cytometry is monolithic miniaturization and replacement of cumbersome components with smaller, cheaper counterparts. The microfluidic cytometer is a micro device which uses laser to excite a plurality of fluorescent samples, and collects fluorescence to a photoelectric detector through an objective lens or a waveguide to realize sample detection. The flow cytometry and microfluidic technologies are combined, and the flow cytometry and microfluidic device has the advantages of being small in size, low in cost and low in consumption. One of the most critical parameters of a microfluidic cytometer is the minimum detection limit of fluorescence, i.e. fluorescence sensitivity. At present, a plurality of methods can improve fluorescence detection performance, namely, excitation energy is improved, a high-power and low-noise laser is generally used for excitation, but the fluorescence quantity of a sample is certain, and the fluorescence saturation and even bleaching can be caused by the excessively high laser energy, meanwhile, background noise can be improved, and the fluorescence sensitivity can be reduced; secondly, increasing the fluorescence acceptance of the detector, typically using expensive high numerical aperture objectives and high sensitivity detectors, increases the design difficulty and cost of the microfluidic system. Thirdly, focusing the sample to be measured on the center and focal plane position of the laser spot is an essential method. The positioning ensures that the fluorescent sample is excited by consistent laser, thereby being beneficial to increasing the maximum excitation quantity and emission intensity of fluorescent molecules, reducing noise interference and increasing detection sensitivity. Various focusing methods such as inertial flow, sheath flow, electrophoresis, magnetophoresis, acoustophoresis and the like are currently available, but have certain limitations and can only be effective under specific conditions. Such as inertial flow, requires complex flow channel structures; sheath fluid is needed for sheath flow, reagent consumption can be increased, and electrophoresis can only control charged samples and the like. Fourth, the light path structure also can influence the fluorescence sensitivity of the system, and most flow cytometry adopts an orthogonal optical system, so that the fluorescence quantity at one angle can be collected, and the fluorescence sensitivity is lower. The confocal reflection light path structure can only detect fluorescence signals excited by particles in the optical detection area, so that the influence of stray light is reduced, and the signal-to-noise ratio can be improved. However, this structure has high selectivity to the substrate, and the transparent substrate has low reflectivity, and cannot effectively reflect fluorescence to the detector. The opaque substrate needs to have smooth surface and high reflectivity. Otherwise, more stray light and excitation light are reflected to the detector, greatly affecting the signal-to-noise ratio. In addition, with the development of optical thin film manufacturing technology, a reflective film, an interference film, etc. can be processed on a substrate, and fluorescence collection efficiency can be improved by utilizing the thin film reflection interference effect. However, the thin film processing requires a separate program or chip, lacks monolithic integration with a focusing structure, and is therefore complicated to operate, and is time-consuming and labor-consuming.
In a word, the current microfluidic cytometry adopts various focusing methods to improve fluorescence detection sensitivity, and the design and integration of an optical detection system are absent, so that the volume is large, and the signal to noise ratio is low. Therefore, a highly integrated miniaturized device with high fluorescence collection capacity, which is simple and portable, realizes high-sensitivity fluorescence detection and provides for the next accurate sorting, is needed. In particular point-of-care applications may benefit from an integrated optical detection device.
The application provides a fluorescence detection device of a single-chip integrated micro-flow cytometer, which is characterized by comprising the following components: confocal reflection fluorescence detecting system, hyperfrequency bulk acoustic wave resonator and micro-fluidic chip, wherein:
the microfluidic chip is positioned above the ultrahigh frequency bulk acoustic resonator and comprises a sample inlet, a sample outlet and a micro-channel, and is used for controlling fluorescent particles to directionally pass through the micro-channel;
the ultra-high frequency bulk acoustic wave resonator comprises a Bragg reflection layer, an electrode layer and a substrate, wherein the electrode layer is deposited on part of the surface of the Bragg reflection layer, the other part of the surface of the Bragg reflection layer is used as an optical reflection area, the electrode layer is adjacent to the sample inlet, the optical reflection area is adjacent to the sample outlet, and the Bragg reflection layer is positioned on the surface of the substrate;
the confocal reflection fluorescence detection system is used for irradiating laser to the optical reflection area and detecting fluorescence reflected by fluorescent particles;
through the bulk acoustic wave provided by the ultra-high frequency bulk acoustic wave resonator, the fluorescent particles move along the edge of the electrode layer with the patterns, when the fluorescent particles reach the optical reflection area and are irradiated by laser, the fluorescent particles can excite fluorescent light waves, the fluorescent light waves interfere with each other under the action of the Bragg reflection layer, and the reflective fluorescence with the interference constructive is detected through the confocal reflective fluorescence detection system.
According to the application, bulk acoustic waves generated by the ultra-high frequency bulk acoustic wave resonator and transmitted to the interior of the micro-channel are utilized, and due to the acoustic flow effect, 3D vortex of fluid is caused, and a plurality of fluid vortices are connected in series along the edge of the electrode layer to form a plurality of 3D vortex tunnels. Therefore, when fluorescent particles pass through, the fluorescent particles can be subjected to sound radiation force and fluid vortex drag force and are rapidly captured in the 3D vortex tunnel, and a three-dimensional focusing effect is achieved. The fluorescent particles finally reach a tip or an inflection point of the electrode layer under the action of the fluid drag force, are separated one by one, pass through the laser spots in sequence, and are subjected to consistent and complete laser irradiation.
Furthermore, the micro flow channel of the micro flow control chip and the ultra-high frequency bulk acoustic wave resonator can have various stacking angles and positions, so that fluorescent particles in the micro flow channel can move along the boundary of at least one electrode layer. The micro flow channel of the micro flow control chip is arranged above one boundary of the ultra-high frequency bulk acoustic wave resonator and is used for controlling the orientation of fluorescent particles and only passing through the micro flow channel along one boundary, so that the phenomenon of crowding and collision caused by that a plurality of fluorescent particles reach the tip or inflection point of the electrode layer at the same time is avoided, and the effect that all the fluorescent particles are captured by vortex and released one by one at the same initial speed is ensured. The micro flow channel of the micro flow control chip is arranged above the boundaries of the ultra-high frequency bulk acoustic wave resonator and used for controlling the orientation of fluorescent particles and passing through the micro flow channel along the boundaries, so that the sample flux and the concentration are increased, and the time is saved and the blockage is avoided.
The laser incident light wave and the fluorescent light wave are reflected at different interfaces, and the Bragg reflection layer thickness is enough that the incident light wave does not interfere with each other, and the fluorescent light wave interferes with each other, so that the maximum fluorescence reflectivity of the interfaces can be realized, and the reflection of the laser at the interfaces is not enhanced.
Further, the electrode layer comprises a top electrode and a piezoelectric layer, patterning is carried out on the electrode layer through exposure and etching, and the laying area of the electrode layer is smaller than the area of the Bragg reflection layer. The longer electrode layer length can prolong the action time with fluorescent particles, improve the three-dimensional focusing effect, and the fluorescent particles can also keep consistency and move efficiently.
Further preferably, the pattern of the electrode layer is one or a combination of shapes of a circle, a semicircle, a shuttle, an ellipse, a triangle, a rectangle, and a pentagon.
Further, the topmost layer of the Bragg reflection layer is used as a bottom electrode of the electrode layer, and the piezoelectric layer is positioned on a partial area of the surface of the bottom electrode.
Further, laser emitted by the confocal reflection fluorescence detection system irradiates on the optical reflection area to form a laser spot, and the laser spot is positioned at the tip end of the patterned electrode layer or at the sharp angle, the right angle, the obtuse angle and the arc inflection point. The fluorescent particles passing through the 3D vortex tunnel can be subjected to uniform and complete laser irradiation one by one, so that the maximum excitation quantity and emission intensity of fluorescent molecules can be increased.
Further, the Bragg reflection layer is formed by alternately superposing a high refractive index layer and a low refractive index layer. The alternating stack of the high refractive index layer and the low refractive index layer comprises alternating stacks of a metal material and a nonmetal material, alternating stacks of a nonmetal material and a nonmetal material or alternating stacks of a metal material and a metal material;
the alternating stack of metallic material and non-metallic material includes alternating stack of molybdenum and aluminum nitride or alternating stack of molybdenum and silicon dioxide;
the alternating stacks of non-metallic material and non-metallic material include alternating stacks of aluminum nitride and silicon dioxide.
The alternating stack of metal material and metal material includes alternating stacks of tungsten and aluminum or alternating stacks of molybdenum and titanium.
The Bragg reflection layer is formed by alternately superposing a material with high acoustic impedance layer and higher optical refractive index and a material with low acoustic impedance layer and lower optical refractive index.
Further, the thickness of each of the Bragg reflection layers is 1/4 of the odd multiple of the wavelength of the sound wave and the wavelength of the fluorescent light. Meanwhile, interference constructive between the sound wave and the light wave is satisfied.
Further, the number of the Bragg reflection layers is 1-15, and most preferably 2-10. The number of Bragg reflection layers is set depending on the total reflection coefficient desired and the acoustic and optical wave reflection conditions that occur at each layer interface.
Further preferably, the Bragg reflection layer is formed by alternately superposing molybdenum/silicon dioxide, wherein the thickness of the molybdenum is 630-650nm, and the thickness of the silicon dioxide is 640-660nm;
or the Bragg reflection layer is formed by alternately superposing molybdenum/aluminum nitride, wherein the thickness of the molybdenum is 630-650nm, and the thickness of the aluminum nitride is 900-1100nm;
or the Bragg reflection layer is formed by alternately superposing molybdenum and titanium, wherein the thickness of the molybdenum is 630-650nm, and the thickness of the titanium is 610-630nm.
Further, the confocal reflection fluorescence detection system is located above or beside the microfluidic chip and comprises a light receiving device, a light source device, a detection device and a light modulation device:
wherein the light receiving device comprises an objective lens, a lens, an optical waveguide or an optical fiber;
the light source device comprises a laser, a light emitting diode or a mercury lamp;
the detection device comprises a photomultiplier, a photodetector or a photodiode;
the light modulation device comprises an attenuation sheet, a filter or a dichroic mirror.
Under the illumination of the bright field lamp, a laser spot is accurately placed at the position of an optical reflector adjacent to the electrode layer by adjusting a three-dimensional adjusting frame with an ultrahigh frequency resonator.
Compared with the prior art, the application has the beneficial effects that:
the present application monolithically integrates acoustic resonators and optical reflectors for the first time. The Bragg reflection layer structure and the laying range of the ultra-high frequency resonator are designed for the first time, so that the Bragg reflection layer structure and the laying range of the ultra-high frequency resonator not only meet the high reflection effect on bulk acoustic waves and ensure that acoustic wave energy is limited in a piezoelectric resonator stack to reach a high Q value, but also have the function of an optical medium mirror, so that light waves reflected on an interface interfere with each other, a series of reflected light wave bands are generated, the energy reflection of an excitation light wave band is restrained, and the reflectivity of light in a specific wave band and a fluorescent light wave band is increased. The ultrahigh frequency resonator has the functions of sound wave focusing and fluorescence reflection enhancement, so that the maximum excitation efficiency of a fluorescent sample is ensured, the fluorescent loss of the detector is reduced, and the optical detection sensitivity is greatly enhanced.
The application can obtain the fluorescence enhancement effect which is 4.6 times of the reflection of the silent wave focusing and the Bragg-free reflecting layer. The ultra-high frequency resonator, the micro-fluidic chip and the confocal reflection fluorescence detection system form a micro-flow cytometer together, and the micro-flow cytometer has the advantages of small volume, simplicity in operation and remarkable fluorescence enhancement effect.
Drawings
FIG. 1 is a schematic diagram of a fluorescence detection device of a monolithically integrated microfluidic cytometer according to an embodiment of the present application;
fig. 2 is a schematic structural diagram of an ultra-high frequency bulk acoustic wave resonator according to an embodiment of the present application;
FIG. 3 is a graph of reflectivity of a Bragg reflector layer illuminated with light of different wavelengths according to an embodiment of the present application, wherein FIG. 3a is a graph of reflectivity of an optical reflector illuminated with light of a fluorescent wavelength, and FIG. 3b is a graph of reflectivity of an optical reflector illuminated with light of a lasing wavelength;
fig. 4 is a schematic structural diagram of a microfluidic chip in a microfluidic chip according to an embodiment of the present application, which is located right above an ultrahigh frequency bulk acoustic resonator;
fig. 5 is a schematic structural diagram of a microfluidic chip in a microfluidic chip according to an embodiment of the present application, where the microfluidic chip is located above an ultrahigh frequency bulk acoustic resonator and is disposed at an angle;
fig. 6 is a graph of fluorescence signals detected when an ultra-high frequency bulk acoustic wave resonator is in operation and when the ultra-high frequency bulk acoustic wave resonator is not in operation in a fluorescence detection device of a monolithically integrated micro-flow cytometer according to an embodiment of the present application, wherein fig. 6a is a graph of fluorescence signals when the ultra-high frequency bulk acoustic wave resonator is in operation, and fig. 6b is a graph of fluorescence signals when the ultra-high frequency bulk acoustic wave resonator is not in operation.
The confocal reflection fluorescence detection system 100, a photomultiplier tube 110, an optical filter 120, a dichroic mirror 130, a laser 140, an attenuation sheet 150, an objective lens 160, a microfluidic chip 200, a sample inlet 210, a sample outlet 220, a microfluidic channel 230, an ultra-high frequency bulk acoustic wave resonator 300, an electrode layer 310, a top electrode 311, a piezoelectric layer 312, a Bragg reflection layer 320, a high refractive index layer 321, a low refractive index layer 322 and a substrate 330.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. The features of the following examples and embodiments may be combined with each other without any conflict.
Unless otherwise indicated, terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, it will be understood that terms defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense.
In order to collect more fluorescence and further realize higher fluorescence sensitivity detection, the application provides a fluorescence detection device of a monolithically integrated microfluidic cytometer, as shown in fig. 1, comprising: confocal reflection fluorescence detection system 100, microfluidic chip 200, and ultra-high frequency bulk acoustic resonator 300, wherein:
the confocal reflected fluorescence detection system 100 provided by the embodiment of the application comprises a photomultiplier 110, an optical filter 120, a dichroic mirror 130, a laser 140, an attenuation sheet 150 and an objective lens 160, wherein laser light is emitted through the laser 140, and is irradiated to an optical reflection area of a Bragg reflection layer 320 through the attenuation sheet 150, the dichroic mirror 130 and the objective lens 160 to form a laser spot, and fluorescent light waves emitted through the optical reflection area of the Bragg reflection layer are detected through paths of the objective lens 160, the dichroic mirror 130, the optical filter 120 and the photomultiplier 110.
The ultra-high frequency bulk acoustic resonator 300 provided in the embodiment of the present application is located below the confocal reflective fluorescence detection system 100, as shown in fig. 2, and includes an electrode layer 310, a bragg reflection layer 320, and a substrate 330.
The electrode layer 310 provided by the embodiment of the application comprises a top electrode 311 and a piezoelectric layer 312, wherein the electrode layer 310 is positioned in a partial area of the surface of the bragg reflection layer 320, i.e. the laying area of the electrode layer 310 is smaller than the area of the bragg reflection layer 320 and is adjacent to the sample inlet 210 of the microfluidic chip 200; the electrode layer 310 has an image, and the image has a shape of a line of symmetry, and preferably, the electrode layer 310 has a pattern of one or a combination of shapes selected from a circle, a semicircle, a shuttle, an ellipse, a triangle, a rectangle, and a pentagon.
The bragg reflection layer 320 provided in the embodiment of the present application includes a high refractive index layer 321 and a low refractive index layer 322 that are alternately stacked, and may be a stack of a metal material and a non-metal material, such as molybdenum and aluminum nitride that are alternately stacked, molybdenum and silicon dioxide that are alternately stacked, tungsten and silicon dioxide that are alternately stacked; it may also be a superposition of non-metallic materials and non-metallic materials, such as alternating superposed aluminum nitride and silicon dioxide; or a superposition of metallic materials and metallic materials, such as alternately superposed tungsten and aluminum, alternately superposed molybdenum and titanium.
Another partial region of the surface of the bragg reflector 320 is provided as an optical reflection region, and a laser spot formed at the optical reflection region is located at a tip or an acute, right, obtuse, or arc inflection point of the patterned electrode layer 310.
The microfluidic chip 200 provided in the embodiment of the application is located above the ultra-high frequency bulk acoustic wave resonator 300, and the contact positions are closely attached, as shown in fig. 4, the microfluidic chip 200 includes a sample inlet 210, a sample outlet 220 and a microfluidic channel 230, and the concentration of 10 μm fluorescent particles is 1×10 5 -1×10 8 Each ml, most preferably 1X 10 6 Fluorescent particles are injected into the microfluidic channel 230 from the sample inlet 210 using a syringe pump or peristaltic pump at a rate of 1-100. Mu.l/min, and most preferably 5-20. Mu.l/min.
In one embodiment, as shown in fig. 5, another way of placing the microfluidic chip 200 is provided in the embodiment of the present application, where the microfluidic chip is placed directly above the ultra-high frequency bulk acoustic wave resonator 300 and is placed at an angle, and the contact positions are closely attached. And starting the resonator, wherein the ultrahigh frequency resonator can form a 3D vortex tunnel. The fluorescent particles run straight along one boundary of the top electrode 311, reach one tip of the electrode layer under the action of fluid drag force, are separated one by one, pass through the laser spots one by one, and are subjected to uniform and complete laser irradiation.
The ultra-high frequency bulk acoustic wave resonator 300 is turned on with a power setting of 100-1000mW, and optimally 300-800mW. The uhf resonator 300 can generate a bulk acoustic wave that is exponentially attenuated toward the inside of the microchannel. The fluorescent particles move along the edge of the top electrode 311 with the patterned electrode layer, when the fluorescent particles reach the laser irradiation area and are irradiated by laser, the fluorescent particles can excite fluorescent light waves, the fluorescent light waves are interfered and constructive under the action of the optical reflection area, more fluorescent light is generated, the incident laser light waves are not interfered and coherent, noise caused by the laser light waves is reduced, and reflected fluorescent light with interference and constructive can be sensitively detected through the photomultiplier 110.
Example 1
The embodiment of the present application provides a confocal reflection fluorescence detection system 100, which adopts a 488nm laser 140, and uses a FITC fluorescence channel (525 nm) of a flow cytometer as an example to illustrate the design of an ultra-high frequency bulk acoustic wave resonator 300 provided by the embodiment of the present application.
As shown in fig. 2, the ultra-high frequency bulk acoustic wave resonator 300 provided by the embodiment of the present application sequentially includes, from bottom to top, a substrate 330, a bragg reflection layer 320, and an electrode layer 310: wherein, the material of the substrate 330 is silicon; the bragg reflection layer 320 is located above the substrate 330, and the bragg reflection layer 320 is formed by alternating molybdenum and silicon dioxide to form a 4-layer structure, wherein the molybdenum layer is used as the topmost layer of the bragg reflection layer 320 and also used as the bottom electrode layer 312 of the electrode layer 310, which is helpful for reducing thickness and saving materials; the electrode layer 310 includes a top electrode layer 311 and a piezoelectric layer 312, the piezoelectric layer 312 not entirely covering the Bragg reflection layer 320, leaving a distance of at least 30 μm long for the laser spot to be placed there (the diameter of the laser spot is about 20 μm). When the laser excites the fluorescent sample, the incident light wave (488 nm laser) and the fluorescent light wave (525 nm fluorescent) are reflected at different interfaces, and the Bragg reflection layer thickness meets the condition that the incident light wave does not interfere with each other and the fluorescent light wave interferes with each other, so that the maximum reflectivity of the fluorescent light of the interface can be realized, and the reflection of the laser at the interface is not enhanced.
The fabrication process of the Bragg reflection layer 320 provided by the implementation of the present application employs MEMS processing technology compatible with Complementary Metal Oxide Semiconductor (CMOS). The thickness of each layer of molybdenum and silicon dioxide is m times of 1/4 of the wavelength of the sound wave and the wavelength of the light wave, wherein m is an odd number. Because the acoustic wave speeds of molybdenum and silicon dioxide are different, the optical refractive indexes are different, and the minimum value of coherent constructive conditions of acoustic wave reflection and light wave reflection is simultaneously satisfied by taking the optical transparency and the layer number into consideration. Thus the thickness of molybdenum is 640nm and the thickness of silica is 650nm.
As shown in fig. 3a, the fluorescence light wave with a center wavelength of 525nm has a reflectivity of up to 83% at the 4 bragg reflection layer. As shown in fig. 3b, the reflectivity of the laser light wave with the center wavelength of 488nm at the 4 bragg reflection layers is only 38%. Compared with the reflectivity (R=38%) of the silicon wafer substrate without the Bragg reflection layer, the fluorescence light wave intensity is improved by 1.2 times.
The reflectivity (R) of the silicon substrate provided by the application can be calculated by the following formula:
R=（（n 1 - n 2 ）/（n 1 + n 2 ）） 2
r is reflectivity, n 1 Is air refractive index, n 2 Is silicon refractive index. The ultra-high frequency bulk acoustic wave resonator 300 provided by the embodiment of the application can realize the reflectivity of more than 80% in the light wavelength range of 515nm-550nm, has the bandwidth range of 35nm, and greatly expands the fluorescence detection range. Meanwhile, the excitation light reflectivity between 480nm and 500nm is kept below 50%, and the signal to noise ratio is greatly improved.
The ultra-high frequency bulk acoustic wave generated by the ultra-high frequency bulk acoustic wave resonator 300 provided by the embodiment of the application is totally reflected at the contact position of the upper electrode and air, and is reflected at the interface of the lower electrode and the Bragg reflection layer and at the interface in the Bragg reflection layer as much as possible. Therefore, sound waves with the opposite propagation direction to the original sound waves are generated, standing waves are further formed through superposition, resonance occurs, the resonance frequency is 2.45GHz, the quality factor Q is 11, and good acoustic performance is achieved.
As shown in fig. 4, the electrode layer 310 in the ultra-high frequency bulk acoustic resonator 300 according to the embodiment of the present application has a pattern formed by a triangle and a triangle with an arc, so that fluorescent particles can move to a tip along the edge of the pattern, a laser spot is disposed at an optical reflection area next to the tip of the electrode layer, and the size of the spot is set to be the size of the fluorescent sample or slightly larger than the fluorescent sample. The fluorescent particles are favorable to being irradiated by laser completely one by one, and precise focusing is realized.
The microfluidic chip 200 provided by the embodiment of the application is arranged right above the ultra-high frequency bulk acoustic wave resonator 300, and the contact positions are tightly attached to prepare fluorescent particles with the concentration of 1 multiplied by 10 and 10 micrometers 6 The micro flow channel was injected from the inlet with a syringe pump or peristaltic pump at a rate of 5. Mu.l/min. The resonator was turned on and the power was set at 600mW. The uhf resonator 300 can generate a bulk acoustic wave that is exponentially attenuated toward the inside of the microchannel. As the acoustic streaming effect causes 3D swirling of the fluid, multiple fluid swirls are connected in series along the edges of the electrode layers, forming two 3D vortex tunnels. Therefore, when fluorescent particles pass through, the fluorescent particles can be subjected to sound radiation force and fluid vortex drag force and are rapidly captured in the 3D vortex tunnel, and a three-dimensional focusing effect is achieved. The fluorescent particles finally reach one tip of the electrode layer under the action of the fluid drag force, are separated one by one, pass through the laser spots in sequence and are subjected to consistent and complete laser irradiation.
As shown in fig. 6a, when the ultra-high frequency bulk acoustic wave resonator 300 is turned on, the original peaks of the fluorescent particles detected by the photomultiplier tube 110 of the confocal reflection fluorescence detection system 100 are highly consistent, the average fluorescence intensity is 1.7V, and when the ultra-high frequency bulk acoustic wave resonator 300 is not turned on, the original peaks of the fluorescent particles detected by the photomultiplier tube 110 of the confocal reflection fluorescence detection system 100 are highly distributed, and the average fluorescence intensity value is 0.8V. At the optical reflector, the resonator focus increased the fluorescence intensity by a factor of 2.1 compared to unfocused. The above shows that the resonator has good 3D focusing effect, and the fluorescence detection intensity is improved.
The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily appreciate variations or alternatives within the scope of the present application. Therefore, the protection scope of the application is subject to the protection scope of the claims.
Claims (8)
1. A monolithically integrated microfluidic cytometer fluorescence detection device, comprising: confocal reflection fluorescence detecting system, hyperfrequency bulk acoustic wave resonator and micro-fluidic chip, wherein:
the microfluidic chip is positioned above the ultrahigh frequency bulk acoustic resonator and comprises a sample inlet, a sample outlet and a micro-channel, and is used for controlling fluorescent particles to directionally pass through the micro-channel;
the ultra-high frequency bulk acoustic wave resonator comprises a Bragg reflection layer, an electrode layer and a substrate, wherein the electrode layer is deposited on part of the surface of the Bragg reflection layer, the other part of the surface of the Bragg reflection layer is used as an optical reflection area, the electrode layer is adjacent to the sample inlet, the optical reflection area is adjacent to the sample outlet, and the Bragg reflection layer is positioned on the surface of the substrate;
the confocal reflection fluorescence detection system is used for irradiating laser to the optical reflection area and detecting fluorescence reflected by fluorescent particles;
the fluorescence particles move along the edge of the electrode layer with the patterns through the bulk acoustic wave provided by the ultra-high frequency bulk acoustic wave resonator, when reaching the optical reflection area and being irradiated by laser, the fluorescence particles can excite fluorescence waves, interference constructive interference occurs to the fluorescence waves under the action of the Bragg reflection layer, and reflected fluorescence with interference constructive interference is detected through the confocal reflection fluorescence detection system;
the Bragg reflection layer is formed by alternately superposing a high refractive index layer and a low refractive index layer;
the alternating stack of the high refractive index layer and the low refractive index layer comprises alternating stacks of a metal material and a nonmetal material, alternating stacks of a nonmetal material and a nonmetal material or alternating stacks of a metal material and a metal material;
the alternating stack of the metal material and the non-metal material comprises alternating stacks of tungsten and silicon dioxide, alternating stacks of molybdenum and aluminum nitride or alternating stacks of molybdenum and silicon dioxide;
the alternating stack of nonmetallic materials and nonmetallic materials includes alternating stacks of aluminum nitride and silicon dioxide;
the alternating stack of metal material and metal material includes alternating stacks of tungsten and aluminum or alternating stacks of molybdenum and titanium.
2. The fluorescence detection device of claim 1, wherein the electrode layer comprises a top electrode and a piezoelectric layer, the electrode layer is patterned by exposure and etching, and a laying area of the electrode layer is smaller than an area of the bragg reflection layer.
3. The apparatus of claim 2, wherein the pattern of electrode layers is one or a combination of shapes selected from the group consisting of circular, semi-circular, fusiform, oval, triangular, rectangular, and pentagonal.
4. The apparatus of claim 2, wherein the top-most layer of the bragg reflector layer is used as a bottom electrode of the electrode layer, and the piezoelectric layer is disposed on a partial region of the surface of the bottom electrode.
5. The fluorescence detection device of claim 1, wherein the laser light emitted by the confocal reflectance fluorescence detection system irradiates the optical reflection area to form a laser light spot, the laser light spot is located at a tip or a circular arc inflection point of the patterned electrode layer, and an angle of the tip is an acute angle, a right angle or an obtuse angle.
6. The microfluidic cytometer fluorescence detection device according to claim 1, wherein the microfluidic channel of the microfluidic chip and the ultra-high frequency bulk acoustic resonator can have a plurality of stacking angles, such that fluorescent particles in the microfluidic channel can move along at least one boundary of the electrode layer.
7. The fluorescence detection device of the monolithically integrated micro-flow cytometer according to claim 1, wherein the bragg reflection layer is composed of molybdenum/silicon dioxide alternately stacked, the thickness of the molybdenum is 630-650nm, and the thickness of the silicon dioxide is 640-660nm;
or the Bragg reflection layer is formed by alternately superposing molybdenum/aluminum nitride, wherein the thickness of the molybdenum is 630-650nm, and the thickness of the aluminum nitride is 900-1100nm;
or the Bragg reflection layer is formed by alternately superposing molybdenum and titanium, wherein the thickness of the molybdenum is 630-650nm, and the thickness of the titanium is 610-630nm.
8. The monolithically integrated microfluidic cytometer fluorescence detection device of claim 1, wherein the confocal reflectance fluorescence detection system is located above or beside the microfluidic chip, and comprises a light receiving device, a light source device, a detection device, and a light modulating device:
wherein the light receiving device comprises an objective lens, a lens, an optical waveguide or an optical fiber;
the light source device comprises a laser, a light emitting diode or a mercury lamp;
the detection device comprises a photomultiplier, a photodetector or a photodiode;
the light modulation device includes an attenuation sheet, a filter, and a dichroic mirror.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310623997.5A CN116337728B (en) | 2023-05-30 | 2023-05-30 | Fluorescence detection device of monolithic integrated micro-flow cytometer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310623997.5A CN116337728B (en) | 2023-05-30 | 2023-05-30 | Fluorescence detection device of monolithic integrated micro-flow cytometer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116337728A CN116337728A (en) | 2023-06-27 |
| CN116337728B true CN116337728B (en) | 2023-08-18 |
Family
ID=86891559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310623997.5A Active CN116337728B (en) | 2023-05-30 | 2023-05-30 | Fluorescence detection device of monolithic integrated micro-flow cytometer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116337728B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2148205A1 (en) * | 1994-05-02 | 1995-11-03 | Thomas M. Baer | Apparatus and method for volumetric capillary cytometry |
| CN101401722A (en) * | 2008-11-07 | 2009-04-08 | 上海奥通激光技术有限公司 | Multi-mode co-focusing imaging method and apparatus |
| CN104136922A (en) * | 2012-02-04 | 2014-11-05 | 细胞和分子平台中心（C-Camp） | Microfluidic flow analyzer and method thereof |
| CN105136763A (en) * | 2015-09-10 | 2015-12-09 | 大连海事大学 | Single microalgae cell activity dynamic monitoring novel method and device based on gas-liquid interface single cell capture and chlorophyll fluorescence characterization |
| CN105987870A (en) * | 2015-02-10 | 2016-10-05 | 博奥生物集团有限公司 | Flow cell sorting system and its focusing detection method and fluidic chip |
| CN107421934A (en) * | 2017-08-02 | 2017-12-01 | 重庆大学 | A kind of real-time detection chip system of novel portable bacterium and detection method |
| CN112076808A (en) * | 2019-06-13 | 2020-12-15 | 安行生物技术有限公司 | Method and equipment for controlling movement of particles in solution by using ultrahigh frequency sound wave |
| CN114112826A (en) * | 2021-11-25 | 2022-03-01 | 天津大学 | Acousto-optic interconnection microfluidic detection system and detection method for fluorescent particles |
| CN114471760A (en) * | 2022-02-10 | 2022-05-13 | 南通大学 | Microfluidic chip device based on magnetic field control fluorescence labeling cell sorting method and use method |
| CN115014478A (en) * | 2022-06-27 | 2022-09-06 | 昆明理工大学 | Solid-state assembly type micro mass sensor with all-metal structure and preparation method thereof |
| CN115041245A (en) * | 2022-08-12 | 2022-09-13 | 天津大学 | Method and device for capturing and separating particles based on ultrahigh frequency bulk acoustic wave acoustic current potential well |
-
2023
- 2023-05-30 CN CN202310623997.5A patent/CN116337728B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2148205A1 (en) * | 1994-05-02 | 1995-11-03 | Thomas M. Baer | Apparatus and method for volumetric capillary cytometry |
| CN101401722A (en) * | 2008-11-07 | 2009-04-08 | 上海奥通激光技术有限公司 | Multi-mode co-focusing imaging method and apparatus |
| CN104136922A (en) * | 2012-02-04 | 2014-11-05 | 细胞和分子平台中心（C-Camp） | Microfluidic flow analyzer and method thereof |
| CN105987870A (en) * | 2015-02-10 | 2016-10-05 | 博奥生物集团有限公司 | Flow cell sorting system and its focusing detection method and fluidic chip |
| CN105136763A (en) * | 2015-09-10 | 2015-12-09 | 大连海事大学 | Single microalgae cell activity dynamic monitoring novel method and device based on gas-liquid interface single cell capture and chlorophyll fluorescence characterization |
| CN107421934A (en) * | 2017-08-02 | 2017-12-01 | 重庆大学 | A kind of real-time detection chip system of novel portable bacterium and detection method |
| CN112076808A (en) * | 2019-06-13 | 2020-12-15 | 安行生物技术有限公司 | Method and equipment for controlling movement of particles in solution by using ultrahigh frequency sound wave |
| CN114112826A (en) * | 2021-11-25 | 2022-03-01 | 天津大学 | Acousto-optic interconnection microfluidic detection system and detection method for fluorescent particles |
| CN114471760A (en) * | 2022-02-10 | 2022-05-13 | 南通大学 | Microfluidic chip device based on magnetic field control fluorescence labeling cell sorting method and use method |
| CN115014478A (en) * | 2022-06-27 | 2022-09-06 | 昆明理工大学 | Solid-state assembly type micro mass sensor with all-metal structure and preparation method thereof |
| CN115041245A (en) * | 2022-08-12 | 2022-09-13 | 天津大学 | Method and device for capturing and separating particles based on ultrahigh frequency bulk acoustic wave acoustic current potential well |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116337728A (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7129809B2 (en) | Optical filters and spectrometers | |
| CA1126977A (en) | Ellipsoid-conic radiation collector and method | |
| JP2007171186A (en) | Sample detection system | |
| US20100019155A1 (en) | Luminescence sensors using sub-wavelength apertures or slits | |
| US8466435B2 (en) | Fine particle measuring device | |
| TW201728895A (en) | Optical sensing module | |
| JP4259762B2 (en) | Optical configuration for detecting light | |
| US11415514B2 (en) | Biochip device | |
| JP2009156659A (en) | Measuring apparatus and method | |
| CN111896511B (en) | Efficient fluorescence collection device and method for solid state spinning | |
| CN116337728B (en) | Fluorescence detection device of monolithic integrated micro-flow cytometer | |
| JP2005030830A (en) | Optical multiplexer/demultiplexer for fluorometric analysis, optical module for fluorometric analysis, fluorometric analysis apparatus, fluorescence/photothermal spectral analysis apparatus, and chip for fluorometric analysis | |
| CN116106797A (en) | Diamond NV color center magnetic detection device | |
| JP2014211326A (en) | Raman sensor | |
| JP4490281B2 (en) | Integrated light-emitting reader | |
| CN109752792B (en) | Fiber end optical antenna based on metal atom gas control and preparation method thereof | |
| JP2006250725A (en) | Flow cell unit, flow site meter and fluorescence detection method | |
| JPH0552895B2 (en) | ||
| KR101507879B1 (en) | Lighting apparatus using laser | |
| CN220367204U (en) | Microscopic light path coupling system for measuring deep ultraviolet photoluminescence spectrum | |
| CN113960009B (en) | Capillary fluorometer with low background signal | |
| CN216274200U (en) | Plasmon optical enhancement chip system | |
| CN215668052U (en) | Plasmon optical enhancement chip system | |
| WO2022168374A1 (en) | Emission optical system, emission device, and optical measurement device | |
| CN116879171A (en) | Microscopic light path coupling system and method for measuring deep ultraviolet photoluminescence spectrum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |