Plasmon metasurface chip biological image detection system integrated with digital microfluidics

Abstract

The invention discloses a plasmon metasurface chip biological image detection system integrated with digital microfluidics, and belongs to the field of microfluidics and metasurface biological image sensing. Comprising a digital micro-fluidic chip which is provided with a driving electrode array, a dielectric layer and a hydrophobic layer and is used for constructing a liquid path and controlling liquid drops; the plasmon metasurface chip is combined with the digital micro-fluidic chip to form a detection site, and biomolecules are detected by utilizing a plasmon resonance effect; the light source is used for exciting plasmon resonance; the CMOS image sensor is used for capturing an image at the detection site; and the control circuit is used for controlling the electrode of the digital micro-fluidic chip and realizing the construction of a liquid path and the control of liquid drops. A biomolecule solution to be detected can be accurately guided to a detection site of the plasmon metasurface chip, plasmon resonance is excited by using a light source, and an image is captured through the CMOS image sensor. And analyzing the image intensity change through image processing software to obtain a detection result of the biomolecules.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics and super-surface biological image sensing, and specifically relates to a plasmon super-surface chip biological image detection system integrated with digital microfluidics. Background Art

[0002] Microfluidics is a system used to control and manipulate small-scale liquids. It is a system that uses pressure, capillary force, and centrifugal force to manipulate small amounts of fluid (usually 10 -8 ~10 -12 L) is a technology for processing and transporting liquids. Compared with manual liquid processing, microfluidics can automatically transport samples, mix reactions, and detect a series of steps to achieve high-throughput processing. At the same time, it can also reduce the amount of samples and reagents used and increase the mixing rate between different reagents. These advantages make microfluidic systems have a huge contribution in the field of biological detection.

[0003] Digital microfluidics is a technology that manipulates microfluidics through the principle of dielectric electrowetting. Specifically, the dielectric electrowetting effect uses an electric field to change the wettability of a liquid on a solid surface: when a voltage is applied to the electrode, a potential difference is generated between two adjacent driving electrodes, and the contact angle between the droplet and the surface becomes smaller due to the dielectric wetting force and the dielectrophoretic force, generating a driving force pointing to the side with a higher potential, which manifests as a controllable movement of the droplet according to the order in which the electrodes are energized. Compared to typical microfluidics, digital microfluidics can customize the movement path of the droplet by digitally applying voltage to the electrodes, and is superior to traditional microfluidics in flexibility and portability; and digital microfluidic chips are small in size, fine in structure, and can react automatically, which is particularly suitable for many complex and cumbersome reactions. Therefore, digital microfluidic chips are widely used in various biochemical reactions and detections.

[0004] In recent years, surface plasmon technology has a wide range of application prospects in the instant detection of biomolecules, attracting the attention of a large number of researchers. Compared with traditional biomarker detection methods, biosensing based on plasmon metasurfaces can achieve label-free detection of biomolecules and has non-destructive detection capabilities, which means that there are fewer restrictions on operating conditions. As an improved version of plasmon metasurface sensing technology, image-based metasurface sensing technology can achieve multiplexing and simultaneously acquire multi-dimensional data, thereby providing more detailed information about the analyte. In addition, the detection sensitivity of image-based metasurface sensing technology is extremely high, and it is easy to observe the interaction between biomolecules. Therefore, the use of metasurface chips for image-sensing-based biodetection is more suitable for multi-indicator, large-scale instant detection.

[0005] In the process of using metasurface chips for image sensing-based biological detection, samples and biological reagents need to be added. Manual addition cannot accurately control the amount of reagents and samples, resulting in waste; at the same time, manual addition is prone to contamination of samples and reagents, thus affecting the test results. The use of traditional microfluidics requires the design of the liquid path required for biological detection of each indicator separately, which is not suitable for multi-indicator, large-scale instant detection, and it is impossible to temporarily change the detection indicators and modify the detection process, which is not conducive to the use of metasurface chips in instant detection. Summary of the invention

[0006] The purpose of the present invention is to provide a biological image detection system of a plasmon metasurface chip integrated with digital microfluidics to solve the above problems of the prior art. The system combines the automation and programmability of digital microfluidics with the label-free detection capability of the plasmon metasurface chip, and uses a portable metal oxide semiconductor (CMOS) image sensor to achieve miniaturized, high-throughput, and multi-index biological molecule detection.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions.

[0008] A biological image detection system of a plasmon metasurface chip integrated with digital microfluidics, comprising a digital microfluidics chip, a plasmon metasurface chip, a light source and a CMOS image sensor;

[0009] The digital microfluidic chip adopts a double-plate structure, consisting of a lower plate and an upper plate, and a running space for accommodating droplets is formed between the lower plate and the upper plate; the droplets to be processed and detected are located between the upper plate and the lower plate which are parallel and separated; the lower plate is provided with a driving electrode array, a dielectric layer covering the driving electrode array, and a first hydrophobic layer covering the dielectric layer in sequence from bottom to top, and the lower surface of the upper plate is provided with a second hydrophobic layer coated on the top plate; the driving electrode array is connected to a control circuit, and the liquid path required for detection is flexibly constructed through the control circuit;

[0010] The plasmon metasurface chip is combined with the lower electrode of the digital microfluidic chip. The plasmon metasurface chip is located on a single electrode to form a detection site for detecting biomolecules. The biomolecule solution to be detected can be controlled to move in and out by the digital microfluidic chip.

[0011] The light path formed by the light source and the CMOS image sensor passes through the detection site. The system captures the image at the detection site through the CMOS image sensor, and the image is used for subsequent analysis.

[0012] Furthermore, the running space between the upper electrode plate and the lower electrode plate is encapsulated with a filling medium for the movement of droplets.

[0013] Furthermore, the filling medium may be silicone oil or air.

[0014] Furthermore, the digital microfluidic chip can accommodate more droplets and construct more and more complex liquid circuits by increasing the number of electrodes.

[0015] Furthermore, the digital microfluidic chip has programmability of liquid circuits and can perform high-throughput or multi-index biomolecule detection at the same time.

[0016] Furthermore, the scheme is an imaging system designed based on a plasmon metasurface chip, and imaging is performed through a CMOS image sensor.

[0017] Furthermore, when the unlabeled biomolecules to be tested combine with the metal nanostructures on the metasurface chip, plasmon resonance will occur, thereby affecting the refractive index and causing a shift in the spectral absorption peak. This is specifically manifested as a change in the intensity of the image imaged by the CMOS image sensor, and the detection results can be obtained by analyzing the image through image processing software such as MATLAB.

[0018] Furthermore, the light intensity image acquired by the CMOS image sensor is processed using a MATLAB program to obtain the result of biomolecule detection.

[0019] Furthermore, multiple detection sites can be set on the digital microfluidic chip to achieve high-throughput multi-index detection, which requires the addition of additional optical path modules. Specifically, multiple detection sites are set on the digital microfluidic chip, each site corresponds to a plasmon metasurface detection unit, and by adding additional optical path modules, multiple biomolecular indicators can be detected simultaneously.

[0020] Furthermore, for intensity images obtained from multiple detection sites, regional images obtained by CMOS image sensor pixel units aligned with each detection site on the overall intensity image are selected as detection images of each detection point, and MATLAB program is used to process them respectively to obtain high-throughput or multi-index detection results.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] The present invention organically combines digital microfluidics, nano-optics and portable CMOS image sensors. First, the integrated digital microfluidic chip can automatically complete the mixing, reaction and chip cleaning of biological droplets, solve the problem of waste and pollution of samples and reagents in the traditional manual sampling scheme, reduce manual operation time, and simplify the operation steps; at the same time, the digital microfluidic chip has a programmable liquid path based on multiple electrodes, which solves the cumbersome process of customizing each liquid path when performing high-throughput multi-index biological molecule detection in the traditional microfluidic sampling scheme, and reduces the production cost of the overall system. Secondly, compared with other biological detection methods, the use of plasmon metasurface chips does not require labeling of the biological molecules to be tested, avoids damage to the biological molecules by the markers, simplifies the steps of the biological molecule reaction in the detection process, and reduces the time of the overall detection process. Thirdly, the plasmon intensity image used contains more effective information, realizing high-throughput multi-index detection. Finally, compared with the bulky and expensive spectrometer required by the traditional spectral-based plasmon detection scheme, the image-based detection scheme uses a portable CMOS image sensor to achieve miniaturization of the overall system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the overall framework of the biological image detection system of the plasmon metasurface chip integrated with digital microfluidics in Example 1 of the present invention;

[0024] Figure 2 It is a schematic diagram of a local structure in Example 1 of the present invention;

[0025] Figure 3 This is a schematic diagram of the overall framework of the system in Example 2 of the present invention;

[0026] Figure 4 It is a schematic diagram of a local structure in Example 2 of the present invention;

[0027] Figure 5 This is a schematic diagram of the overall framework of the system in Example 3 of the present invention;

[0028] Figure 6 This is a schematic diagram of the local structure in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. In the absence of conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0030] Example 1

[0031] Figure 1This is a schematic diagram of the overall framework of a plasmon metasurface chip biological image detection system integrated with digital microfluidics in a specific embodiment of the present invention. The detection part includes a light source 101, an optical path module 102, a digital microfluidic chip 103 combined with a plasmon metasurface chip, and a CMOS image sensor 105; the control part includes a digital microfluidic chip control module 104 and a host computer 106.

[0032] In this embodiment, the digital microfluidic chip control module is connected to the host computer, and data is transmitted to the control module through the host computer to control the movement of droplets on the digital microfluidic chip and construct a detection liquid circuit; the CMOS image sensor is also connected to the host computer, and the host computer controls the switch of the CMOS image sensor. The CMOS image sensor transmits the acquired image to the host computer, which is screened and processed by the MATLAB program in the host computer to finally obtain the detection result.

[0033] Figure 2 The schematic diagram of the local structure of the detection part of the digital microfluidic chip combined with the metasurface chip in this embodiment includes a collimated light source 201, a full width at half maximum (FWHM) filter 202, an upper electrode plate 203, a second hydrophobic layer 204, a droplet to be detected 205, a plasmon metasurface chip 206, a first hydrophobic layer 207, a driving electrode array 208, a dielectric layer 209, a lower electrode plate 210 and a CMOS image sensor 211. Among them, the broad arrow represents the light path, and the combination of the droplet to be detected and the plasmon metasurface chip is called a detection site.

[0034] In this embodiment, the system needs two images to obtain the results of the detection site, as follows: first, the plasmon metasurface chip is biofunctionalized to enable it to capture the biomolecules to be detected; then, a digital microfluidic chip is used to move a droplet of a buffer solution (such as a phosphate buffered saline solution) to the plasmon metasurface chip to obtain the first image as the original image; a droplet of the same buffer solution containing the biomolecule to be detected is moved to the plasmon metasurface chip to allow the biomolecule to be detected to bind to the metasurface, and the second image is obtained at this time as the detection image. By comparing the original image and the detection image and quantifying the difference, the detection results can be obtained qualitatively and quantitatively.

[0035] In this embodiment, the system adopts transmission imaging. Specifically, the light source of the system includes a collimated light source, and the collimated light source includes an LED array and a collimating lens module. The light path of the collimated light source passes through the FWHM filter and then transmits downward from the top of the digital microfluidic chip, passes through the droplets to be tested and the metasurface chip, and finally uses a CMOS image sensor to obtain a transmission intensity image under the digital microfluidic chip. The function of the FWHM filter is to allow the light passing through it to excite the plasmons on the metasurface chip. In order to ensure the intensity of transmission, the digital microfluidic chip uses transparent indium tin oxide (ITO) conductive glass as the substrate material for the upper and lower plates, and the plasmon metasurface chip uses glass as the substrate material.

[0036] In this embodiment, the collimated light source and the CMOS image sensor are located at the same center of a circle. The light spot range of the collimated light source passes through the detection site and completely covers the CMOS image sensor. The area of ​​the light spot is larger than the chip area of ​​the CMOS image sensor. While acquiring the plasmon transmission intensity image, it achieves full coverage of the CMOS photosensitive array, thereby ensuring the quality of imaging.

[0037] Example 2

[0038] Figure 3 The overall framework diagram of the biological image detection system of the plasmon metasurface chip integrated with digital microfluidics in another specific embodiment of the present invention is shown. The system has multiple detection points. The detection part includes a light source 301, an optical path module 302, a digital microfluidic chip 303 combined with a plasmon metasurface chip, an additional optical path module 305 and a CMOS image sensor 307, and the control part includes a digital microfluidic chip control module 304 and a host computer 306. Different from Example 1, the transmission range of this embodiment is larger, and the transmitted light needs to be focused by an additional optical path module before it can be acquired by the CMOS image sensor.

[0039] Figure 4 The schematic diagram of the local structure of the detection part of the digital microfluidic chip combined with the metasurface chip in this embodiment includes a collimated light source 401, a FWHM filter 402, an upper plate 403, a second hydrophobic layer 404, a plurality of droplets to be tested 405, a plurality of plasmon metasurface chips 406, a first hydrophobic layer 407, a driving electrode array 408, a dielectric layer 409, a lower plate 410, a focusing lens 411 and a CMOS image sensor 412. The optical path is marked in the schematic diagram, and the combination of the droplets to be tested and the plasmon metasurface chip is called a detection site. This embodiment is the same as Example 1, and two images are required to obtain the results of the detection site.

[0040] This embodiment is the same as Embodiment 1. The system adopts transmission imaging. The digital microfluidic chip adopts transparent indium tin oxide (ITO) conductive glass as the substrate material of the upper and lower plates. The plasmon metasurface chip adopts glass as the base material. The collimated light source and the CMOS image sensor are located at the same center. The difference is that since there are multiple detection points in this embodiment, the light source should cover all detection points. However, large-area CMOS image sensors are relatively expensive. Therefore, in order to reduce costs, an additional focusing lens is selected to focus the transmitted light on a small-area CMOS image sensor to obtain a plasmon transmission intensity map related to multiple detection points.

[0041] In this embodiment, the overall transmission intensity image obtained by the host computer needs to be segmented and screened to obtain the transmission intensity image of each detection site, and the pixel area with valid detection information can be selected on the image according to the positional relationship between the optical path and the detection site. At the same time, since the transmission intensity of the detection site is significantly different from other locations, machine learning technology can be used to assist in the identification and judgment of the pixel area corresponding to the detection site.

[0042] Example 3

[0043] Figure 5 This is a schematic diagram of the overall framework of a biological image detection system of a plasmon metasurface chip integrated with digital microfluidics in another specific embodiment of the present invention, wherein the detection part includes a light source 501, an optical path module 504, a digital microfluidic chip 505 combined with a plasmon metasurface chip, and a CMOS image sensor 502, and the control part includes a digital microfluidic chip control module 503 and a host computer 506. Different from embodiments 1 and 2, this embodiment adopts reflective imaging.

[0044] Figure 6 The schematic diagram of the local structure of the detection part of the digital microfluidic chip combined with the metasurface chip in this embodiment includes a collimated light source 602, a FWHM filter 603, an upper plate 604, a second hydrophobic layer 605, a droplet to be detected 606, a plasmon metasurface chip 607, a first hydrophobic layer 608, a dielectric layer 609, a drive electrode array 610, a lower plate 611 and a CMOS image sensor 601. The incident light path and the reflected light path are marked in the schematic diagram. The combination of the droplet to be detected and the plasmon metasurface chip is called a detection site. As in Example 1, this embodiment requires two images to obtain the results of the detection site.

[0045] Since this embodiment adopts reflective imaging, there is no need to select transparent materials to manufacture digital microfluidic chips and plasmon metasurface chips, which is slightly different from the previous embodiment. The digital microfluidic chip in this embodiment can be manufactured using PCB technology, and the electrode is a double-layer metal material with copper on the bottom and gold on the top, and the plasmon metasurface can be further processed (such as nanoimprinting) directly on the gold on the electrode. When the dielectric layer and hydrophobic layer of the digital microfluidic lower plate are made, the metasurface part is masked to protect its biological detection characteristics; a position can also be reserved on the digital microfluidic chip to embed and combine the plasmon metasurface chip with silicon as the substrate.

[0046] In this embodiment, the image obtained by the CMOS image sensor is a plasmon reflection intensity image. The image obtained by the CMOS image sensor should be as complete as possible, which requires strict requirements on the positional relationship between the light source, the detection site and the CMOS image sensor, such as the incident angle and reflection angle formed by the incident light path, the reflected light path and the normal of the plasmon metasurface chip should be equal. The spot range of the collimated light source should cover the detection site, and the reflected spot area should also be larger than the CMOS image sensor chip area to ensure the quality of the result.

[0047] Through the system of the present invention, the biomolecule solution to be detected can be accurately guided to the detection site of the plasmon metasurface chip, the plasmon resonance can be excited by the light source, and the image can be captured by the CMOS image sensor. The detection result of the biomolecule can be obtained by analyzing the image intensity change through the image processing software.

[0048] In addition, the system of the present invention also has high-throughput detection capabilities. By designing multiple electrodes and plasmon metasurface areas, it can detect multiple biological molecules at the same time, greatly improving detection efficiency and accuracy.

[0049] The digital microfluidic chip of the present invention can be a double-plate digital microfluidic chip. The lower plate can use ITO glass as the substrate of the driving electrode, and the driving electrode array is made by photolithography. The dielectric layer can use polyethylene tetramethyl terephthalate to cover the driving electrode array to prevent short circuits between electrodes. The first hydrophobic layer can use polytetrafluoroethylene coating to increase the contact angle between the droplet and the surface, so that the surface presents hydrophobicity. The upper plate can use transparent ITO glass, and the lower surface is coated with a second hydrophobic layer, and the material can be the same as the first hydrophobic layer.

[0050] Plasmon metasurface chips can use gold nano-hole structure arrays as plasmon metasurfaces, which are made on transparent silicon dioxide SiO2 or sapphire Al2O3 substrates through electron beam etching technology. The diameter of each gold nano-hole structure can be 100-200nm, and the array period can be 300-600nm. The plasmon metasurface chip is aligned and bonded to the specific electrode position of the lower plate of the digital microfluidic chip through a precision positioning device.

[0051] Light source and CMOS image sensor: LED light source is selected as the excitation light source with a wavelength of 635nm. After passing through a 10nm FWHM filter, it can excite the plasmon metasurface chip. The CMOS image sensor uses a high-sensitivity, high-resolution model, and focuses on the detection site through the lens system to ensure a clear image.

[0052] During detection, the biomolecule solution to be tested (such as protein, exosomes, etc.) and various reagent solutions involved in biological reactions can be loaded on specific electrodes of the digital microfluidic chip. By controlling the activation of the electrodes, various reagent solutions are moved to pre-treat the biomolecule solution to be tested, and the plasmon metasurface chip is biofunctionalized, and then the treated biomolecule solution to be tested is moved to the detection site of the plasmon metasurface chip. The light source is activated, and the image at the detection site is captured by the CMOS image sensor. The image is processed using the MATLAB program, and the image intensity changes are analyzed to obtain the detection results.

[0053] The biological image detection system of the present invention has the advantages of simple operation, high detection sensitivity, strong high-throughput detection capability, etc. It is suitable for biomedical research, clinical diagnosis, drug screening and other fields, and provides a new technical means for the rapid and accurate detection of biological molecules.

[0054] Although the contents of the present application are specifically displayed and described in conjunction with the preferred embodiments, those skilled in the art should understand that various changes made to the present application in form and detail without departing from the spirit and scope of the present application as defined by the appended claims and without making any creative effort are within the scope of protection of the present application.

Claims

1. A biological image detection system of a plasmon metasurface chip integrated with digital microfluidics, characterized in that: Includes digital microfluidic chips, plasmon metasurface chips, light sources and CMOS image sensors; The digital microfluidic chip adopts a double-plate structure, including a lower plate and an upper plate, and a running space for accommodating droplets to be tested is formed between the lower plate and the upper plate; the droplets to be processed and tested are located between the upper plate and the lower plate which are parallel and separated; the lower plate is provided with a driving electrode array, a dielectric layer covering the driving electrode array, and a first hydrophobic layer covering the dielectric layer in sequence from bottom to top, and the lower surface of the upper plate is provided with a second hydrophobic layer coated on the top plate; the driving electrode array is connected to a control circuit, and the liquid path required for detection is flexibly constructed through the control circuit; The plasmon metasurface chip is combined with the lower electrode of the digital microfluidic chip, and the plasmon metasurface chip is located on a single electrode to form a detection site for detecting biomolecules, and the biomolecule solution to be detected is controlled to move in and out by the digital microfluidic chip; The light source is used to excite plasmon resonance on the plasmon metasurface chip; the CMOS image sensor is used to capture the image at the detection site; the light path formed by the light source and the CMOS image sensor passes through the detection site, the system is imaged in the CMOS image sensor, and the image is used for subsequent analysis.

2. A biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: The running space between the upper plate and the lower plate is encapsulated with a filling medium for the movement of droplets.

3. A biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 2, characterized in that: The filling medium is silicone oil or air.

4. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: The driving electrode array of the digital microfluidic chip is designed with multiple electrodes to accommodate more droplets, build a complex fluid network, and achieve high-throughput detection.

5. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: The plasmon supersurface chip includes a gold nanostructure array, and the size, shape and arrangement of the gold nanostructure are adjusted to adapt to the detection of different biological molecules.

6. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: The wavelength of the light source matches the resonance wavelength of the plasmon metasurface chip to excite plasmon resonance.

7. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: Multiple detection sites are set on the digital microfluidic chip, each site corresponds to a plasmon metasurface detection unit, and by adding additional optical path modules, multiple biomolecular indicators can be detected simultaneously.

8. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: It also includes image processing software for analyzing images captured by the CMOS image sensor, extracting image intensity changes caused by plasmon resonance, and obtaining detection results.

9. A biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 8, characterized in that: The image processing software is a MATLAB program, which can process images of multiple detection sites in batches to achieve high-throughput, multi-index biomolecule detection.

10. The biological image detection system of a plasmon metasurface chip integrated with digital microfluidics as claimed in claim 1, characterized in that: The digital microfluidic chip has programmability of liquid circuits and can perform high-throughput or multi-index biomolecule detection at the same time.

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