Microfluidic chip for processing exosomes and manufacturing method thereof

Abstract

The application discloses a micro-fluidic chip for processing exosomes and a manufacturing method thereof, and relates to the technical field of biological detection. Based on the structure of the micro-fluidic chip, when the nano-hole electrode layer and the nano-grating sensing electrode layer of the optical biosensing array unit are connected to a power supply, due to the different diameters of the detection channels, the two asymmetric gold nano-electrodes of each detection channel form a non-uniform electric field, the nanoparticles of the exosomes are polarized, dielectrophoresis force is generated on the nanoparticles, and the nanoparticles are driven to move and gather on the surface of the nano-grating sensing electrode layer at the bottom, so that the nanoparticles are effectively detected by the nano-grating sensing electrode layer, and the effect of efficiently and highly sensitively detecting the exosomes is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and in particular to a microfluidic chip for processing exosomes and a manufacturing method thereof. BACKGROUND

[0002] Microfluidic chip is a hot field of current micro total analysis system development. Microfluidic chip analysis takes chip as an operation platform, is based on analysis of electrochemical signals, relies on micro-electro-mechanical processing technology, takes micro-pipe network as a structural feature, and takes life science as a current main application object. The microfluidic technology aims to integrate the functions of the whole laboratory, including sampling, dilution, adding reagents, reaction, separation, detection, etc. on a microchip, and can be used multiple times. The microfluidic chip is mainly characterized in that the effective structure (channels, reaction chambers and other certain functional components) for containing fluid is at least micron level in one dimension. Due to the micron-level structure, the fluid shows and generates special performance different from that in macroscopic scale. At present, when detecting exosomes, the microfluidic chip cannot integrate the exosomes on the sensing surface at the bottom, resulting in low detection accuracy. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a microfluidic chip for processing exosomes and a manufacturing method thereof, which can improve the accuracy of exosome detection.

[0004] In one aspect, the present application provides a microfluidic chip for processing exosomes, comprising a photo-biosensor array unit, the photo-biosensor array unit comprising a nanohole electrode layer, a nanoring layer and a nanograting sensing electrode layer arranged in sequence.

[0005] The nanohole electrode layer comprises a plurality of first holes, and the nanoring layer comprises a plurality of second holes, the diameter of the first holes being greater than the diameter of the second holes.

[0006] The first holes of the nanohole electrode layer are aligned with the second holes of the nanoring layer to form a detection channel.

[0007] The nanohole electrode layer and the nanograting sensing electrode layer are used for connecting a power supply.

[0008] According to some embodiments of the present application, the nanograting sensing electrode layer comprises a glass substrate sublayer and a metal nanograting electrode sublayer, and the metal nanograting electrode sublayer is arranged between the nanoring layer and the glass substrate sublayer.

[0009] According to some embodiments of the present invention, the nanohole electrode layer is a gold nanohole electrode layer, the nanoring layer is a gold nanoring layer, and the metal nanograting electrode sublayer is a gold nanograting electrode sublayer.

[0010] According to some embodiments of the present invention, the microfluidic chip for exosome processing includes a base plate unit and a top plate unit, the optical biosensor array unit is disposed between the base plate unit and the top plate unit, and the nanograting sensing electrode layer of the optical biosensor array unit is disposed on the base plate unit;

[0011] The bottom plate unit is connected to the edge of the top plate unit to form the housing of the optical biosensor array unit.

[0012] According to some embodiments of the present invention, the base plate unit includes a separation region and a detection region, the separation region is provided with a micron column array, and the detection region is provided with the optical biosensor array unit;

[0013] One side of the top plate unit is provided with a detection inlet, and the other side of the top plate unit is provided with a particulate matter outlet and an exosome outlet.

[0014] According to some embodiments of the present invention, a microfluidic channel is provided on the side of the top plate unit facing the optical biosensor array unit.

[0015] On the other hand, embodiments of the present invention also provide a method for fabricating a microfluidic chip for exosome processing, comprising the following steps:

[0016] Photoresist was spin-coated onto the nano-grating sensing electrode layer;

[0017] A detection channel consisting of a first hole and a second hole is imprinted on photoresist using a three-dimensional nanopillar mold, and residual photoresist is removed. The three-dimensional nanopillar mold has multiple nanopillars, and each nanopillar is composed of two sub-pillars with different diameters.

[0018] Metal materials are deposited on the photoresist to form the detection channel to obtain a nanohole electrode layer, and an optical biosensor array unit is formed.

[0019] The optical biosensor array unit is packaged to obtain a microfluidic chip for exosome processing.

[0020] According to some embodiments of the present invention, the manufacturing method further includes the following steps:

[0021] Laying metal materials on a glass substrate;

[0022] Spin-coating photoresist onto a metal material;

[0023] A nanograting is imprinted on photoresist using a grating mold, and residual photoresist and metal materials are etched to form a nanograting sensing electrode layer.

[0024] According to some embodiments of the present invention, the process of encapsulating the optical biosensor array unit to obtain a microfluidic chip for exosome processing includes the following steps:

[0025] The base plate unit is molded using a micron column array mold so that the separation area of ​​the base plate unit is distributed with a micron column array;

[0026] The optical biosensor array unit is placed in the detection area of ​​the base plate unit;

[0027] The top plate unit is molded using a microfluidic channel mold, so that the top plate unit has multiple microfluidic channels distributed on it.

[0028] The top plate unit is bonded to the bottom plate unit, which is equipped with an optical biosensor array unit, to form a microfluidic chip.

[0029] According to some embodiments of the present invention, the process of encapsulating the optical biosensor array unit to obtain a microfluidic chip for exosome processing further includes the following steps:

[0030] The base plate unit is placed on a glass substrate with two electrode ports;

[0031] The nanograting sensing electrode layer is connected to one of the electrode ports via a first wire, and the nanohole electrode layer is connected to the other electrode port via a second wire.

[0032] The above-mentioned technical solution of the present invention has at least one of the following advantages or beneficial effects: After the nanohole electrode layer and the nanograting sensing electrode layer of the optical biosensing array unit are powered on, due to the different upper and lower diameters of the detection channels, the two asymmetric gold nanoelectrodes of each detection channel will form a non-uniform electric field, which will polarize the nanoparticles of exosomes, thereby generating a dielectric force on the nanoparticles and driving them to move and gather on the surface of the bottom nanograting sensing electrode layer, so that they can be effectively detected by the nanograting sensing electrode layer, achieving the effect of high efficiency and high sensitivity detection of exosomes. Attached Figure Description

[0033] Figure 1 This is an overall schematic diagram of a microfluidic chip for exosome processing provided in an embodiment of the present invention;

[0034] Figure 2 This is an overall schematic diagram of the optical biosensor array unit provided in an embodiment of the present invention;

[0035] Figure 3 This is a schematic diagram of a uniform electric field in a conventional channel provided in an embodiment of the present invention;

[0036] Figure 4 This is a schematic diagram of the non-uniform electric field of the detection channel provided in this embodiment of the invention;

[0037] Figure 5 This is a schematic diagram of the exosome separation process in the microfluidic chip provided in the embodiment of the present invention;

[0038] Figure 6 This is a schematic diagram of the exosome flow process provided in an embodiment of the present invention;

[0039] Figure 7 This is a schematic diagram illustrating the relationship between local surface plasmon resonance peaks and exosome concentrations provided in an embodiment of the present invention.

[0040] Figure 8 This is a schematic diagram of the microfluidic chip fabrication process provided in an embodiment of the present invention. Detailed Implementation

[0041] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar originals or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] In the description of this invention, it should be understood that the orientation descriptions, such as up, down, left, right, etc., are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0043] In the description of this invention, the use of terms such as "first," "second," etc., is merely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0044] This invention provides a microfluidic chip for exosome processing, referring to... Figure 1 The microfluidic chip for exosome processing in this embodiment of the invention includes an optical biosensor array unit 100.

[0045] Reference Figure 2The optical biosensor array unit includes a nanohole electrode layer 110, a nanoring layer 120, and a nanograting sensing electrode layer 130 arranged sequentially. The nanohole electrode layer includes multiple first pores, and the nanoring layer includes multiple second pores, with the diameter of the first pores being larger than the diameter of the second pores. The first pores of the nanohole electrode layer and the second pores of the nanoring layer are aligned to form a detection channel. The nanohole electrode layer and the nanograting sensing electrode layer can be used to connect a power source.

[0046] Specifically, the nanohole electrode layer, the nanoring layer, and the nanograting sensing electrode layer are arranged sequentially from top to bottom. The nanohole electrode layer can be a gold nanohole electrode layer, the nanoring layer can be a gold nanoring layer, and the metal nanograting electrode sublayer can be a gold nanograting electrode sublayer.

[0047] In related technologies, the detection channels of microfluidic chips, such as Figure 3 As shown, when the two uniform electrodes are energized, a uniform electric field is formed between them. This prevents the generation of dielectrophoretic forces on the exosomes, thus hindering the integration of the exosomes onto the bottom sensing surface and resulting in low detection accuracy. The detection channel in this embodiment of the invention is as follows: Figure 4 As shown, when the top nanohole electrode layer and the bottom nanograting sensing electrode layer of the optical biosensing array unit are energized, a non-uniform electric field is formed in the detection channel due to the two asymmetric gold nanoelectrodes. This polarizes the nanoparticles, generating a dielectrophoretic force on the nanoparticles and driving them to move onto the bottom nanograting sensing surface, thus achieving accurate detection of exosomes. The expression for the dielectrophoretic force of exosomes in the detection channel is as follows:

[0048]

[0049]

[0050]

[0051] Among them, F DEP ε is the dielectrophoretic force acting on the exosome, r is the effective radius of the exosome, and ε is the force applied by the electrophoresis. m F is the dielectric constant of the liquid. CM It is the Clausius-Mosotti factor, the magnitude of which determines the direction of the dielectrophoretic force. It is the root-mean-square gradient of the applied electric field, ε * It is the composite dielectric constant, ε p σ is the dielectric constant of the exosome, σ is the conductivity, and ω is the angular frequency of the applied electric field. When F... CM When the electric field strength is greater than 0, exosomes flow towards the direction of stronger electric field strength, a phenomenon known as positive dielectric electrophoresis. CMWhen the electric field strength is less than 0, exosomes flow towards the direction of weaker electric field strength, a phenomenon known as negative dielectric electrophoresis. By selecting an appropriate electric field frequency, exosomes can be driven to flow in the microfluidic system to the gold nanosensor array located at the bottom of the nanograting sensing electrode layer for detection.

[0052] In this embodiment, a non-uniform electric field can be formed by applying an AC voltage to the optical biosensor array unit to generate a dielectric force, thereby attracting exosomes in the fluid to flow to the underlying gold nanosensor array. Alternatively, multiple high-order local plasmon resonance modes, including Fano and Fabry-Perot resonances, can be generated by external light excitation. This can efficiently capture exosome particles in microfluidics while greatly improving the detection sensitivity of the optical biosensor array unit for exosome particles.

[0053] In some embodiments, please continue to refer to Figure 2 The nanograting sensing electrode layer includes a glass substrate sublayer 132 and a metal nanograting electrode sublayer 131, with the metal nanograting electrode sublayer disposed between the nanoring layer and the glass substrate sublayer.

[0054] In some embodiments, please continue to refer to Figure 1 The microfluidic chip for exosome processing includes a base plate unit 300 and a top plate unit 200. An optical biosensor array unit is disposed between the base plate unit and the top plate unit, and a nanograting sensing electrode layer of the optical biosensor array unit is disposed on the base plate unit. The edges of the base plate unit and the top plate unit are connected to form a shell of the optical biosensor array unit.

[0055] In some embodiments, please refer to Figure 1 and Figure 5 The base plate unit includes a separation area and a detection area. The separation area is provided with a micron column array 310, which is formed by multiple micron columns. The detection area is provided with an optical biosensor array unit 100. One side of the top plate unit is provided with a detection inlet 221, and the other side of the top plate unit is provided with a particulate matter outlet 222 and an exosome outlet 223.

[0056] In this embodiment, the fluid to be detected enters the microfluidic chip through the detection inlet. Since the volume and weight of large particles such as cells in the fluid are much higher than those of exosome nanoparticles, exosomes 510 and large particles such as cells 520 can be effectively separated when passing through the micron column array in the separation region. This allows the exosomes to flow towards the optical biosensor array unit in the detection region under the action of laminar flow and Stokes traction force in the microfluidic, while large particles such as cells flow out from the particle outlet of another channel. After the exosomes are detected, they flow out from the exosome outlet. Finally, the microfluidic chip can separate the exosomes from the fluid to be detected.

[0057] In some embodiments, please continue to refer to Figure 1 A microfluidic channel 210 is provided on the side of the top plate unit facing the optical biosensor array unit.

[0058] In this implementation, please refer to Figure 6 The flow process of exosomes within a microfluidic chip involves separation of the exosomes by a micron-pillar array and laminar flow. When an exosome encounters a microfluidic channel with a top-mounted three-dimensional nanograting structure, it experiences additional fluid shear force, altering its trajectory and allowing it to enter the pores of the underlying optical biosensor array unit. Simultaneously, the detection channel of the optical biosensor array unit consists of a first and a second pore with different diameters; the upper first pore is larger than the lower second pore. The detection channel generates a non-uniform electric field at a certain frequency, inducing dielectrophoretic force on the exosomes and driving them downwards to accumulate at the bottom of the optical biosensor array unit for detection. Once the exosomes fill the bottom and middle layers of the detection channel, the fluid velocity within the channel decreases, making it easier for new exosomes to flow into the next unfilled detection channel and be effectively detected. Further, referring to… Figure 7 As the concentration of the detected exosomes increases, the local surface plasmon resonance peaks shift significantly, thus enabling efficient and highly sensitive detection of exosomes.

[0059] In some embodiments, please continue to refer to Figure 1 The microfluidic chip in this embodiment of the invention also includes a chip glass substrate 400. The chip glass substrate is provided with two electrode ports, namely a first electrode port 410 and a second electrode port 420. The chip glass substrate is disposed below the base plate unit. The first electrode port is connected to the top electrode material of the nanohole electrode layer of the optical biosensing array unit through a wire, and the second electrode port is connected to the metal nanograting electrode sublayer of the nanograting sensing electrode layer through a wire.

[0060] On the other hand, embodiments of the present invention also provide a method for fabricating a microfluidic chip for exosome processing, comprising the following steps:

[0061] Photoresist was spin-coated onto the nano-grating sensing electrode layer;

[0062] A detection channel consisting of a first hole and a second hole is imprinted on photoresist using a three-dimensional nanopillar mold, and residual photoresist is removed. The three-dimensional nanopillar mold has multiple nanopillars, each of which consists of two sub-pillars with different diameters.

[0063] Metal materials are deposited on the photoresist to form the detection channel to obtain a nanohole electrode layer, and an optical biosensor array unit is formed.

[0064] The optical biosensor array unit is encapsulated to obtain a microfluidic chip for exosome processing.

[0065] In some embodiments, the manufacturing method of the present invention further includes the following steps:

[0066] A metallic material, such as gold or chromium, is deposited on a glass substrate.

[0067] Spin-coating photoresist onto a metal material;

[0068] A nanograting is imprinted on photoresist using a grating mold, and residual photoresist and metal materials are etched to form a nanograting sensing electrode layer.

[0069] In some embodiments, the step of encapsulating the optical biosensor array unit to obtain a microfluidic chip for exosome processing includes the following steps:

[0070] The base plate unit is molded using a micron column array mold so that the separation area of ​​the base plate unit is distributed with a micron column array;

[0071] The optical biosensor array unit is placed in the detection area of ​​the base plate unit;

[0072] The top plate unit is molded using a microfluidic channel mold, so that the top plate unit has multiple microfluidic channels distributed on it.

[0073] The top plate unit is bonded to the bottom plate unit, which is equipped with an optical biosensor array unit, to form a microfluidic chip.

[0074] In some embodiments, the step of encapsulating the optical biosensor array unit to obtain a microfluidic chip for exosome processing further includes the following steps:

[0075] The base plate unit is placed on a glass substrate with two electrode ports;

[0076] The nanograting sensing electrode layer is connected to one of the electrode ports via a first wire, and the nanohole electrode layer is connected to the other electrode port via a second wire.

[0077] For example, please refer to Figure 8 The fabrication process of the microfluidic chip in this embodiment of the invention is as follows:

[0078] S1. Depositing gold / chromium metallic material on a glass substrate;

[0079] S2. Spin-coating photoresist onto a metal material;

[0080] S3. Nanoimprinting is performed on photoresist by controlling temperature, pressure and ultraviolet exposure using a grating mold to imprint a nano grating.

[0081] S4. The residual photoresist is etched by reactive ion dry etching, and the gold / chromium film not covered by photoresist is removed by wet etching.

[0082] S5. Remove the photoresist to form a nano-grating sensing electrode layer;

[0083] S6. Spin-coat photoresist onto the nano-grating sensing electrode layer;

[0084] S7. A double-layered porous nanopillar structure (i.e., detection channel) is imprinted on photoresist using a three-dimensional nanopillar mold;

[0085] S8. Remove the photoresist residue, exposing the bottom gold nanograting under the double-layered hole-shaped nanopillars;

[0086] S9. A gold / chromium metal material is thermally evaporated and deposited on the photoresist forming the detection channel to form an optical biosensor array unit;

[0087] S10. The base plate unit is molded onto polydimethylsiloxane (PDMS) material using a micron column array mold;

[0088] S11. Place the base plate unit on a glass substrate with electrode ports;

[0089] S12. Place the optical biosensor array unit inside the base plate unit;

[0090] S13. A top plate unit with nano-microfluidic channels is molded onto polydimethylsiloxane (PDMS) material using a microfluidic channel mold;

[0091] S14. The top plate unit is placed upside down on the bottom plate unit, and the nano-grating sensing electrode layer and one of the electrode ports are connected through the first wire, and the nano-hole electrode layer and the other electrode port are connected through the second wire. The detection inlet, particulate matter outlet and exosome outlet are installed on the top plate unit.

[0092] In this embodiment, a three-dimensional multilayer gold nanoarray structure of an optical biosensor array unit is integrated into a single chip unit using nanoimprint lithography. This allows the three-dimensional multilayer gold nanoarray structure to not only generate multiple high-order plasmon resonance modes such as Fano and Fabry-Perot resonances, but also to act as gold nanoelectrodes to generate dielectrophoretic forces for the effective separation and capture of exosome particles in microfluidics, thereby significantly improving the detection sensitivity of exosomes. The fabrication process of the microfluidic chip is simple and controllable, making it suitable for large-area, large-scale mass production of optical biosensor chips based on three-dimensional multilayer gold nanoarrays.

[0093] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A microfluidic chip for exosome processing, characterized in that, The device includes an optical biosensing array unit, which comprises a nanohole electrode layer, a nanoring layer, and a nanograting sensing electrode layer arranged sequentially. The nanohole electrode layer is a gold nanohole electrode layer, the nanoring layer is a gold nanoring layer, and the metal nanograting electrode sublayer of the nanograting sensing electrode layer is a gold nanograting electrode sublayer. The nanohole electrode layer includes a plurality of first pores, and the nanoring layer includes a plurality of second pores, wherein the diameter of the first pores is larger than the diameter of the second pores; The first hole of the nanohole electrode layer is aligned with the second hole of the nanoring layer to form a detection channel; The nanohole electrode layer and the nanograting sensing electrode layer are used to connect to a power source; The nanograting sensing electrode layer includes a nanograting, which is exposed under the detection channel and is used to detect exosomes. The microfluidic chip for exosome processing includes a base plate unit and a top plate unit, the optical biosensor array unit is disposed between the base plate unit and the top plate unit, and the nanograting sensing electrode layer of the optical biosensor array unit is disposed on the base plate unit; The base plate unit includes a separation region and a detection region. The separation region is provided with a micron column array, and the detection region is provided with the optical biosensor array unit. The micron column array is used to separate exosomes to the detection region.

2. The microfluidic chip for exosome processing according to claim 1, characterized in that, The nanograting sensing electrode layer includes a glass substrate sublayer and a metal nanograting electrode sublayer, wherein the metal nanograting electrode sublayer is disposed between the nanoring layer and the glass substrate layer.

3. The microfluidic chip for exosome processing according to claim 1, characterized in that, The bottom plate unit is connected to the edge of the top plate unit to form the housing of the optical biosensor array unit.

4. The microfluidic chip for exosome processing according to claim 3, characterized in that, One side of the top plate unit is provided with a detection inlet, and the other side of the top plate unit is provided with a particulate matter outlet and an exosome outlet.

5. The microfluidic chip for exosome processing according to claim 3, characterized in that, A microfluidic channel is provided on the side of the top plate unit facing the optical biosensor array unit.

6. A method for fabricating a microfluidic chip for exosome processing as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Laying metal materials on a glass substrate; Spin-coating photoresist onto a metal material; A nanograting is imprinted on photoresist using a grating mold, and residual photoresist and metal materials are etched to form a nanograting sensing electrode layer. Photoresist was spin-coated onto the nano-grating sensing electrode layer; A detection channel consisting of a first hole and a second hole is imprinted on photoresist using a three-dimensional nanopillar mold, and residual photoresist is removed. The three-dimensional nanopillar mold has multiple nanopillars, and each nanopillar is composed of two sub-pillars with different diameters. Metal materials are deposited on the photoresist to form the detection channel to obtain a nanohole electrode layer, and an optical biosensor array unit is formed. The optical biosensor array unit is packaged to obtain a microfluidic chip for exosome processing. Specifically, this includes: molding a base plate unit using a micron column array mold so that the separation region of the base plate unit is distributed with a micron column array; and setting the optical biosensor array unit in the detection region of the base plate unit.

7. The method for fabricating a microfluidic chip for exosome processing according to claim 6, characterized in that, The process of encapsulating the optical biosensor array unit to obtain a microfluidic chip for exosome processing includes the following steps: The top plate unit is molded using a microfluidic channel mold, so that the top plate unit has multiple microfluidic channels distributed on it. The top plate unit is bonded to the bottom plate unit, which is equipped with an optical biosensor array unit, to form a microfluidic chip.

8. The method for fabricating a microfluidic chip for exosome processing according to claim 7, characterized in that, The process of encapsulating the optical biosensor array unit to obtain the exosome-processing microfluidic chip further includes the following steps: The base plate unit is placed on a glass substrate with two electrode ports; The nanograting sensing electrode layer is connected to one of the electrode ports via a first wire, and the nanohole electrode layer is connected to the other electrode port via a second wire.

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