Digital micro-fluidic chip, detection system and spectral information processing method

Abstract

The double-plate micro-fluidic chip comprises an upper polar plate, a lower polar plate, a detection chip, conductive gaskets and controllable liquid drops, the upper polar plate and the lower polar plate are oppositely arranged up and down at a preset interval, the two conductive gaskets are arranged between the upper polar plate and the lower polar plate, and the upper ends and the lower ends of the conductive gaskets are in contact with the upper polar plate and the lower polar plate respectively; a cavity is formed between the upper pole plate and the lower pole plate, the controllable liquid drop is arranged in the cavity between the upper pole plate and the lower pole plate, the upper part and the lower part of the controllable liquid drop are respectively contacted with the upper pole plate and the lower pole plate, the detection chip is arranged on the upper pole plate or the lower pole plate, and the detection chip extends into the cavity between the upper pole plate and the lower pole plate and can be contacted with the controllable liquid drop.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and in particular to a digital microfluidics chip, a detection system and a spectrum information processing method. Background Art

[0002] Digital microfluidics uses dielectric wetting phenomena and DC or AC voltage to change the contact angle of droplets, thereby moving, mixing, separating and other operations on microliter or even nanoliter droplets on a chip of several centimeters in size. This technology has many advantages such as small size, low reagent consumption, and automated operation. It can be widely used in biomedicine, chemical analysis, drug screening, biosensing, nucleic acid analysis and other detection. Compared with traditional microfluidics, digital microfluidics does not require the use of complex micro-pipes, micro-valves, micro-pumps, micro-structures and other micro-fabricated components to achieve the regulation of trace liquids, greatly reducing the size of the equipment and the complexity of preparation.

[0003] In order to realize the wide application of microfluidic technology in actual detection, a variety of detection and corresponding result readout methods are integrated on the basis of its flexible droplet operation, including paper-based detection, electrochemical detection and optical detection. At present, there are various types of biochemical sensing technologies based on digital microfluidic technology. However, how to realize the flexible manipulation of micro-droplets and the reading of detection results under the premise of fast, accurate and high-sensitivity detection results is one of the current development directions of detection technology. In view of this, the present invention is specially proposed. Summary of the invention

[0004] The present invention provides a digital microfluidic chip and a detection system and a spectral information processing method that integrate biochemical sensors and label-free spectral detection. By modifying different types of probes on the detection chip and filling multiple chips, specific detection can be achieved. When a transparent substrate is used, the light path can directly pass through the chip for in-situ detection, thereby avoiding damage and contamination to the biochemical chip during use, which affects the accuracy and sensitivity of the chip for detection.

[0005] In order to achieve the purpose of the present invention, the following technical solutions are adopted:

[0006] A double-plate microfluidic chip comprises an upper plate, a lower plate, a detection chip, a conductive pad and a controllable droplet, wherein: the upper plate and the lower plate are spaced apart from each other by a predetermined distance, two conductive pads are arranged between the upper plate and the lower plate, and the upper and lower ends of the conductive pads are respectively in contact with the upper plate and the lower plate; a cavity is formed between the upper and lower plates, the controllable droplet is arranged in the cavity between the upper plate and the lower plate, and the upper and lower parts thereof are respectively in contact with the upper plate and the lower plate, the detection chip is mounted on the upper plate or the lower plate, the detection chip extends into the cavity between the upper and lower plates, and can be in contact with the controllable droplet.

[0007] The double-plate microfluidic chip, wherein: the upper plate is provided with a mounting through hole, and the lower part of the detection chip passes through the mounting through hole into the cavity between the upper and lower plates.

[0008] The double-plate microfluidic chip, wherein: the lower electrode plate is provided with a mounting through hole, and the upper part of the detection chip passes through the mounting through hole into the cavity between the upper and lower electrode plates.

[0009] The double-plate microfluidic chip, wherein: the detection chip comprises a biochemical sensor chip, and the detection surface of the biochemical sensor chip extends into the cavity between the upper and lower plates.

[0010] The double-plate microfluidic chip, wherein: the detection chip includes a substrate, and the biochemical sensor chip is adhered to the substrate.

[0011] A digital microfluidic chip detection system comprises an optical path system, a spectrometer, a computer, a microfluidic chip control host and a control peripheral, wherein: the optical path system comprises a light source, the control peripheral is located on the optical path of the emitted light of the light source, the control peripheral is used to carry the double-plate microfluidic chip as claimed in any one of claims 1 to 5, the spectrometer is used to receive the reflected light of the double-plate microfluidic chip and send the spectral information to the computer, the microfluidic chip control host controls the control peripheral, and the computer is used to control the microfluidic chip control host.

[0012] A method for processing spectral information, wherein the spectral information is spectral information of a double-plate microfluidic chip detected by the detection system as described above, wherein the method comprises:

[0013] S1. Blank sensor chip detection: Place the microfluidic chip horizontally just below the optical path system, detect the reflected light R1(λ) formed by the thin film layer on the substrate surface of the blank sensor chip, and the reflected light R2(λ) formed by the substrate surface layer, and calculate the light intensity R formed by the interference of the two beams. base (λ);

[0014] S2. Incubation of the sensor chip for the detection probe: Place the microfluidic chip horizontally directly below the optical path system and detect the light intensity R formed by the interference of the reflected light formed on the probe surface and the light reflected from the substrate surface layer. capt (λ);

[0015] S3. Sensor chip detection for capturing detection targets: Place the microfluidic chip horizontally directly below the optical path system, control the manipulable droplets to move to the detection chip, contact and react with the probes of the sensor chip, and the probes capture the target molecules in the droplets. The light intensity R formed by the interference of the reflected light of the detection probes on the thin film on the surface of the detection chip after capturing the target molecules and the light reflected by the surface layer of the substrate dect (λ).

[0016] The spectral information processing method further comprises:

[0017] S4: After normalizing the spectra of the above steps S1-S3, time-frequency conversion is performed respectively to convert the spectra in the wavelength domain into the frequency domain;

[0018] S5: Identify the peak frequency in the frequency domain spectrum obtained in step S4 and the corresponding interference peak f peak , calculate the phase information at the peak;

[0019] S6: Calculate the phase difference of the probe and the data after capturing the target detection molecule relative to the blank chip at the peak frequency;

[0020] S7: Calculate the change in optical path length and dielectric layer thickness based on the phase difference calculated in step S6, and solve for the change in film layer thickness Δd caused by capturing target molecules. dect . BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of a double-plate microfluidic chip with an integrated sensor chip on the upper plate;

[0022] Figure 2 Schematic diagram of the structure of a double-plate microfluidic chip with an integrated sensor chip on the lower plate;

[0023] Figure 3 Schematic diagram of the structure of a double-plate digital microfluidic chip with an integrated sensor chip on the upper plate;

[0024] Figure 4 Schematic diagram of the structure of a double-plate digital microfluidic chip with an integrated sensor chip on the lower plate;

[0025] Figure 5 Schematic diagram of the structure of a double-plate digital microfluidic chip with multiple sensor chips integrated on the upper plate;

[0026] Figure 6 Schematic diagram of the structure of a double-plate digital microfluidic chip with an integrated sensor chip on the upper plate;

[0027] Figure 7 It is a schematic diagram of the setting methods of various sensor chips;

[0028] Figure 8 Schematic diagram of digital microfluidic chip detection system;

[0029] Fig. 9 A schematic diagram of the relevant steps of the method for calculating the concentration of the detected substance;

[0030] Fig.10 Schematic diagram of the droplet detection circuit structure. DETAILED DESCRIPTION

[0031] The following is combined with Figure 1-10 , the specific implementation methods of the present invention are described in detail. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The implementation methods are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0032] "One embodiment" or "some embodiments" described in this specification means that one or more embodiments of the present invention include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, in this specification, the terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized.

[0033] like Figure 1 As shown, the double-plate microfluidic chip of the upper plate integrated sensor chip includes an upper plate 1, a lower plate 2, a detection chip 3, a conductive gasket 4, and a controllable droplet 5. The upper plate 1 and the lower plate 2 are spaced apart at a predetermined distance, and the two conductive gaskets 4 are arranged between the upper plate 1 and the lower plate 2. Optionally, the area of ​​the lower plate 2 is larger than the area of ​​the upper plate 1, and the conductive gasket 4 is located at the edge of the upper plate 1. The upper and lower ends of the conductive gasket 4 are in contact with the upper plate 1 and the lower plate 2, respectively, and the two conductive gaskets 4 are arranged relative to each other at a predetermined distance, so that a cavity is formed between the upper and lower plates. The controllable droplet 5 is arranged in the cavity between the upper plate 1 and the lower plate 2, and its upper and lower parts are in contact with the upper plate 1 and the lower plate 2, respectively. The detection chip 3 is mounted on the upper surface of the upper plate 1, and the lower part of the detection chip 3 passes through the mounting through hole opened in the upper plate to enter the cavity between the upper and lower plates, and can contact the controllable droplet 5. The distance between the upper and lower plates can be controlled by the conductive pad 4. In addition, the conductive pad can be replaced by a conductive glue mixed with polystyrene balls of different sizes, and the distance between the chips can be controlled by the diameter of the balls. The controllable droplet 5 can be controlled by a program to move its position, so that the detection chip 3 can contact the control droplet 5 during use.

[0034] The upper plate 1 includes a substrate 11, an electrode layer 12 and a hydrophobic layer 13, wherein the electrode layer 12 is applied on the lower surface of the substrate 11, and the hydrophobic layer 13 is coated on the electrode layer 12, and the electrode layer 12 is arranged toward the lower plate 2; the lower plate 2 includes a substrate 21, an electrode layer 22, a dielectric layer 23 and a hydrophobic layer 24, wherein the electrode layer 22 is arranged on the upper surface of the substrate 21; the dielectric layer 23 covers the electrode layer 22, and the dielectric layer 23 completely covers the entire surface and side of the electrode layer 22; the hydrophobic layer 24 is coated on the dielectric layer 23, and the hydrophobic layer 24 is arranged toward the upper plate 1. Generally speaking, the upper plate 1 is used for grounding, and the lower plate 2 is used for connecting a controllable voltage source, and the controllable voltage range may be, for example, 30-150V.

[0035] The conductive gasket 4 is directly electrically connected to the lower electrode plate 2 , that is, the lower end of the conductive gasket 4 is electrically connected to the electrode layer 22 of the lower electrode plate 2 ; the upper end of the conductive gasket 4 is in contact with the hydrophobic layer 13 of the upper electrode plate 1 .

[0036] The detection chip 3 includes a biochemical sensor chip 31, an adhesive layer 32 and a substrate 33. The adhesive layer 32 is applied on the lower surface of the substrate 33. The biochemical sensor chip 31 is bonded to the substrate through the adhesive layer 32. The biochemical sensor chip 31 is inserted into the cavity between the upper and lower plates through the mounting through hole of the upper plate. During the use of the chip, the biochemical sensor chip 31 can contact the controllable droplet 5. The contact surface between the biochemical sensor chip 31 and the droplet 5 is called the detection surface.

[0037] In the above description, the substrate can be one of flat glass, paper, polyester film, and printed circuit board (PCB), preferably a transparent material. The electrode material can be one of metal, metal oxide and polysilicon. The material of the dielectric layer can be selected from polyparaxylene (Parylene), Si3N4, A12O3, etc. The hydrophobic material can use Teflon, CYTOP dispersion or FluoroPel. The biochemical sensor chip can use silicon-based materials, metal materials, non-metallic materials, compounds, polymer materials, etc. as the matrix, and its surface film layer can be formed by coating, growth, bonding or bonding methods. The target detection object of the biochemical sensor chip can be biological molecules, non-biological molecules, cells, tissues, viruses, microorganisms, bacteria, etc.

[0038] Figure 2The structure of the double-plate microfluidic chip with integrated sensor chip of the lower plate is shown. The main structure of the double-plate microfluidic chip with integrated sensor chip of the lower plate is the same as that of the double-plate microfluidic chip with integrated sensor chip of the upper plate, and the difference lies in the installation position of the detection chip 3. The lower plate 2 is provided with a mounting through hole, which passes through the substrate 21, the electrode layer 22, the dielectric layer 23 and the hydrophobic layer 24. The biochemical sensor chip 31 is inserted into the cavity between the upper and lower plates through the mounting through hole of the lower plate, and can contact with the controllable droplet 5 during the use of the chip. The contact surface between the biochemical sensor chip 31 and the droplet 5 is called the detection surface.

[0039] Figure 3 Another structure of a double-plate microfluidic chip with an upper plate integrated sensor chip is shown. Figure 1 The difference between the two-plate microfluidic chips shown is that Figure 1 The mounting through hole of the upper plate 1 of the double-plate microfluidic chip is facing an electrode 22 of the lower plate 2, and the detection chip 3 is also mounted at this position. Figure 3 The mounting through hole of the upper electrode plate 1 of the double-plate microfluidic chip faces the middle position of the two electrode plates 22 of the lower electrode plate 2, and the detection chip 3 is also mounted at this position.

[0040] Figure 4 Another structure of a double-plate microfluidic chip with an integrated sensor chip on the lower plate is shown. Figure 2 The difference between the two-plate microfluidic chips shown is that Figure 2 The mounting through hole of the lower electrode plate 2 of the double-plate microfluidic chip substantially coincides with the center of the square electrode (electrode plate 22), and the detection chip 3 is also mounted at this position. Figure 4 The mounting through hole of the lower electrode plate 2 of the double-plate microfluidic chip is located in the middle of the two square electrodes (electrode plates 22) of the lower electrode plate, and the detection chip 3 is also installed in this position.

[0041] Figure 5 Another structure of a double-plate microfluidic chip with an upper plate integrated sensor chip is shown. Figure 1 The difference between the two-plate microfluidic chips shown is that Figure 5 The detection chip 3 of the double-plate microfluidic chip includes two biochemical sensor chips 31, which are bonded to a substrate 33 via an adhesive layer 32 at a predetermined distance. The two biochemical chips 31 are respectively inserted into the cavity between the upper and lower electrodes through two mounting through holes opened in the upper electrode plate, and two manipulation droplets 5 can be controlled to contact the two biochemical sensor chips 31 respectively.

[0042] Figure 6 Another structure of a double-plate microfluidic chip with an upper plate integrated sensor chip is shown. Figure 6As shown, the double-plate microfluidic chip with an upper plate integrated sensor chip includes an upper plate 1, a lower plate 2, a detection chip 3, a conductive gasket 4, and a controllable droplet 5. The upper plate 1 and the lower plate 2 are spaced apart by a predetermined distance, and the two conductive gaskets 4 are arranged between the upper plate 1 and the lower plate 2. The upper and lower ends of the conductive gasket 4 are in contact with the upper plate 1 and the lower plate 2, respectively. The two conductive gaskets 4 are spaced apart by a predetermined distance and are arranged relative to each other, so that a cavity is formed between the upper and lower plates. The controllable droplet 5 is arranged in the cavity between the upper plate 1 and the lower plate 2, and its upper and lower parts are in contact with the upper plate 1 and the lower plate 2, respectively. The upper end of the detection chip 3 is embedded in the countersunk hole (blind hole) opened on the lower surface of the upper plate 1, which can save an additional fixed substrate, ensure a closed condition, and prevent the liquid between the plates from precipitating due to capillary action. The lower end of the detection chip 3 (biochemical sensor chip 31) protrudes from the lower surface of the upper electrode plate 1, extends into the cavity between the upper and lower electrodes, and can contact the controllable droplet 5. The distance between the upper and lower electrodes can be controlled by the conductive gasket 4. In addition, the conductive gasket can be replaced by a conductive glue mixed with polystyrene beads of different sizes, and the distance between the chips can be controlled by the diameter of the beads. The controllable droplet 5 can be moved by program control, and the biochemical sensor chip 31 of the detection chip 3 can contact the control droplet 5 during use.

[0043] like Figure 7As shown, the upper plate is in the form of a whole plate, and its electrode layer is also a whole layer structure. When the electrodes of the lower plate are distributed in an array, there are multiple ways to set the detection chip. Among them: one detection chip 331 can be set on the upper plate, or on the lower plate; multiple detection chips 332 can be set on the upper plate, or on the lower plate; multiple detection chips 333 of different shapes (including circular, rectangular, etc.) are set on the upper plate, or on the lower plate, wherein each detection chip 333 can be set at a position corresponding to a lower plate electrode, or at a position corresponding to multiple adjacent lower plate electrodes; multiple detection chips 334 are set at positions corresponding to multiple non-adjacent lower plate electrodes. The advantage of this setting method is that if the detection target is single, a single detection chip can be designed to be buried, or multiple chips can be buried to detect multiple samples, thereby improving the use efficiency of a single digital microfluidic chip; when there are multiple detection targets, multiple detection chips containing different probes can be buried, and the detection of multiple indicators of a single sample can be realized on a digital microfluidic chip. It is also possible to incubate different detection probes on the same detection chip and collect the detection results of the corresponding positions respectively. In addition, the size, shape and setting position of the detection chip can be changed as needed. For example, when the detection chip 31 is set at the position corresponding to the two adjacent lower plate electrodes, two different droplets are respectively controlled to contact the detection chip, then half of the detection chip 31 can detect sample 1, and the other half can detect sample 2. Therefore, a single chip can be used to detect multiple samples without being affected by crosstalk between samples. The step of burying multiple detection chips 31 can also be optimized to bury a single chip 31, simplifying the production process of the entire chip. Under the condition of long-term detection, since the droplets may evaporate, the detected sample is non-specifically adsorbed on the edge of the detection chip 31. Therefore, the use of a circular or smaller detection chip 31 can effectively prevent non-specific adsorption and improve the accuracy of subsequent sampling and detection.

[0044] like Figure 8 As shown, the digital microfluidic chip detection system includes an optical path system 51, an optical fiber 52, a spectrometer 53, a computer 54, a microfluidic chip control host 55, and a control peripheral 56.

[0045] The optical path system includes a light source 511 , a collimating lens 512 , a field lens 513 , a first aperture 514 , a beam splitter 515 , an objective lens 516 , an imaging lens 517 , and a second aperture 518 .

[0046] The collimating lens 512 is arranged at the lower part of the light source 511, and is used to convert the light beam emitted by the collimated light source 511 into a parallel light beam; the field lens 513 is arranged at the lower part of the collimating lens 512, and is used to make the light beam evenly irradiate the entire field of view of the sample; the first aperture 514 is arranged at the lower part of the field lens 513, and is used to control the size and shape of the light beam; the beam splitter 515 is used to split the light beam transmitted through the aperture 514 into two beams, wherein the emission direction of the first light beam is the same as the original light beam, and the light path where the light beam is located is called the first light path, and the emission direction of the second light beam is perpendicular to the emission direction of the first light beam, and the light path where the light beam is located is called the second light path; the objective lens 516 is arranged on the light path of the first light beam and is located at the lower part of the beam splitter, and is used to converge the light beam; the imaging lens 517 is arranged on a third light path opposite to the second light path, and is located on the outside of the beam splitter 515, and the second aperture 518 is arranged on the outside of the imaging lens 517, and is used to further control the size and shape of the light beam passing through and limit the size of the irradiation area.

[0047] The control peripheral 56 is arranged at the lower part of the objective lens 516. The control peripheral 56 carries the microfluidic chip as described above and has a moving part, which can control the chip to move freely in the three directions of XYZ to realize the detection and focusing of chips at different positions. The converging light beam transmitted by the objective lens 516 is irradiated on the microfluidic chip. The control peripheral 56 is electrically connected to the microfluidic chip control host 55, so that the position of the microfluidic chip is not restricted by the position of the control host 55 and can be freely moved; the control peripheral 56 is also provided with an electrical connection end (not shown in the figure), which is connected to the corresponding electrode of the microfluidic chip to control the movement, separation, mixing, etc. of the droplet 5; the converging light beam transmitted by the objective lens 516 is irradiated on the microfluidic chip, and the light beam is reflected by the beam splitter 515 after being reflected by the microfluidic chip, enters the third light path, and is received by the spectrometer 53 after passing through the imaging lens 517, the second aperture 518, and the optical fiber 52. The control peripheral 56 is electrically connected to the microfluidic chip control host 55, and the microfluidic chip control host 55 is used to control the operation of the microfluidic chip by applying a control signal to the control peripheral 56; the microfluidic chip control host 55 is connected to the computer 54 through a signal transmission line, and the computer 54 is used to control the microfluidic chip control host 55; the second aperture 518 is connected to the spectrometer 53 through the optical fiber 52, and the spectrometer 53 is used to analyze the optical signal and the spectrometer 53 is connected to the computer 54 through the signal transmission line, and the computer 54 is used to receive the signal detected by the spectrometer 53. That is, the reflected light of the microfluidic chip enters the spectrometer through the optical fiber transmission, and finally the spectral information detected by the spectrometer is transmitted to the computer through the data line for analysis. Since the spectral curve collected by the spectrometer before and after the reaction will produce a phase shift, the changes on the surface of the sensor (microfluidic chip) can be calculated according to the shift. At the same time, the control of the host can be realized through the program set in the computer, thereby realizing the control of the controllable droplets.

[0048] When the microfluidic chip is placed on the control peripheral 56, the sensor chip 31 needs to be set on the first optical path, and the biochemical sensor chip 31 is facing the light source 511. When it is set on the upper electrode plate 1, the lower electrode plate 2 is a transparent plate. When it is set on the lower electrode plate 2, the upper electrode plate 1 is a transparent plate, so that the light beam can pass through the lower electrode plate 2 or through the upper electrode plate 1 to irradiate the sensor chip 31.

[0049] like Fig. 9 As shown in the figure, the specific steps for processing the spectral information are as follows:

[0050] S1: Blank sensor chip detection

[0051] The integrated digital microfluidic chip is placed horizontally directly below the optical path system. Since there is a silicon dioxide film on the silicon layer substrate of the blank sensor chip 31, reflected light R1(λ) can be formed on the upper surface of the film, and reflected light R2(λ) can be formed on the surface of the substrate layer, where λ is the wavelength of the incident light. There is an optical path difference between the two beams of light, and the two beams of light interfere on the upper surface of the film. The light reflectivity formed by the interference of the two beams of light can be expressed as:

[0052] R base (λ)=R1(λ) 2 +R2(λ) 2 +2R1(λ)·R2(λ)·cos(4πn0d0 / λ) (1)

[0053] Among them, R base (λ) represents the reflectivity of the interference light reflected by the blank silicon wafer at wavelength λ, n0 is the refractive index of the oxide layer of the silicon wafer, and d0 is the thickness of the oxide layer.

[0054] S2: Sensor chip assay with incubation detection probes

[0055] The integrated digital microfluidic chip is placed horizontally just below the optical path system. Since the detection probes are incubated (also called fixed) on the film on the surface of the chip, the light incident on the sensor surface is reflected on the surface of the detection probes, scattered between the probe molecules, and then reflected by the substrate, which is equivalent to a change in the thickness of the film. At this time, the light formed by the interference of the two beams is:

[0056] R capt (λ)=R1(λ) 2 +R2(λ) 2 +2R1(λ)·R2(λ)·cos[2π(2n0d0+2n capt Δd capt ) / λ] (2)

[0058] Where n capt is the refractive index of the probe, Δdcapt is the relative change in the thickness of the oxide film layer caused by the probe.

[0059] S3: Sensor chip detection to capture the detection target

[0060] After the biosensor chip with fixed detection probes is embedded in the digital microfluidic chip, droplets to be detected are added into the cavity of the chip, and the droplets contain target molecules to be detected.

[0061] The droplet is driven to reach the sensor chip and move back and forth, so that the detection probe on the surface of the sensor chip binds to the target molecule. After the droplet is removed, the target molecule remains on the chip surface, thereby achieving the capture of the target molecule. Then optical detection is performed, and the light incident on the sensor surface is reflected on the surface of the target molecule, scattered between the target molecule and the detection probe, and then reflected by the substrate, which is equivalent to a further change in the thickness of the film. At this time, the light formed by the interference of the two beams is:

[0062] R dect (λ)=R1(λ) 2 +R2(λ) 2 +2R1(λ)·R2(λ)·cos[2π(2n0d0+2n capt Δd capt +2n dect Δd dect ) / λ] (3)

[0064] Where n dect is the refractive index of the target molecule, Δd dect is the relative change in film thickness due to the fixation of detection probes and the capture of target molecules.

[0065] S4: After normalizing the spectra of the above steps S1-S3, fast Fourier transform (FFT) is performed respectively to convert the spectra in the wavelength domain into the frequency domain.

[0066] F base (f) = FFT {R base (λ) / [U0(λ)·max(R base (λ))]} (4)

[0067] F capt (f) = FFT {R capt (λ) / [U0(λ)·max(R capt (λ))]} (5)

[0068] F dect (f) = FFT {R dect (λ) / [U0(λ)·max(R dect(λ))]} (6)

[0069] Where f is the frequency. base (f), F capt (f), F dect (f) The frequency domain signals of the fast Fourier transform of the interference spectra obtained after the cleaned bare (blank) chip, the incubated probe, and the detected target. U0(λ) represents the amplitude of the illumination light at wavelength λ.

[0070] S5: Identify the peak frequency in the frequency domain spectrum and the corresponding interference peak f peak , calculate the phase information at the peak.

[0071] Since the interference signal at the peak frequency is most sensitive to the optical thickness of the dielectric layer, the phase change is proportional to the thickness of the dielectric layer, so measuring the phase change at the peak frequency can more accurately detect the change in thickness. The peak frequency in the frequency domain spectrum and the corresponding interference peak f can be identified according to the following formula: peak , calculate the phase information at the peak:

[0072] φ base (f peak )=arg[F base (f peak )] (7)

[0073] φ capt (f peak )=arg[F capt (f peak )] (8)

[0074] φ dect (f peak )=arg[F dect (f peak )] (9)

[0075] In the formula, φ base (f peak ),φ capt (f peak ),φ dect (f peak ) are the phase information at the peak.

[0076] S6: Calculate the phase difference at the peak frequency of the probe and the data after capturing the target detection molecule relative to the blank chip.

[0077] Subtracting formula (7) from formula (8) yields:

[0078] Δφ1(f peak )=φ capt (f peak )-φbase (f peak ) (10)

[0079] Similarly, by subtracting formula (7) from formula (9), we can get:

[0080] Δφ2(f peak )=φ dect (f peak )-φ base (f peak ) (11)

[0081] In order to solve the sidelobe effect that may be caused by FFT transformation, the following is peak The nearby frequency components are averaged to reduce the effect of noise:

[0082]

[0083]

[0084] Where N is the number of frequency components within the selected frequency range, f min and f max are the upper and lower limits of the selected frequency range.

[0085] S7: Calculate the change in optical path length and dielectric layer thickness using the phase difference.

[0086] The length of the optical path is the physical thickness of the medium multiplied by its refractive index. Therefore, the change in the thickness of the dielectric layer can be calculated:

[0087]

[0088] Where ΔOP is the change in optical path length, and Δd is the change in dielectric layer thickness.

[0089] Relative to the blank silicon wafer, the optical path length changes after incubation of the probe and detection of the target are:

[0090]

[0091] In summary, according to formula (15) and formula (16), the change in film thickness Δd caused by capturing target molecules can be solved: dect .

[0092] According to the Δd dectThe concentration of target molecules in the controllable droplets can be obtained. This calculation method is an existing calculation method, and the present invention will not repeat it. In the experiment, it is ensured that there are enough detection probes fixed on the surface of the sensor chip. The higher the concentration of target detection molecules in the droplets, the more target molecules are captured by the detection probes, resulting in a greater change in the relative film thickness. Therefore, the target molecules in the droplets can be qualitatively or quantitatively detected by the sensor chip. In addition, the detection probes will bind to the corresponding target molecules, so specific detection of the target molecules can be achieved.

[0093] In order to achieve accurate control of droplet driving, the present invention also provides a droplet detection circuit and a droplet detection method. The method can be applied to the droplet driving control in step S3.

[0094] like Fig.10 As shown, the input voltage source V in The first terminal is connected to the sense resistor R test The first end of the sense resistor R test The second end is connected to the electrode layer 12 of the upper plate 1; the input voltage source V in The second end of is connected to the detection line through the first switch K1; the detection line is connected to the second electrode layer 22 of the lower plate 2 through the second switch K2, and the detection line is connected to the second electrode layer 22 of the lower plate 2 through the third switch K3. The voltage to be measured is the detection resistor R test The voltage V between the second end of and each electrode layer 22 of the lower electrode plate 2 out The voltage between the upper plate and each lower plate electrode layer can be detected in real time by the above circuit. Fig.10 The two V out1 and V out 2. Of course, the number of voltages to be measured is not limited thereto, and is the same as the number of electrode layers 22 of the lower electrode plate 2.

[0095] Since the dielectric constant of the droplet 5 is different from that of the surrounding air or material, when the droplet 5 moves between the upper and lower electrode plates, it will cause a change in capacitance. After the device is connected, the output voltage V out , the total capacitance C between the upper and lower plate electrodes in the current state can be calculated real-time , thereby tracking the position of the droplet in real time and detecting the current movement state of the droplet. The specific detection steps are as follows.

[0096] Assume the input signal is V in (t) = V m sin(ωt),capacitance C real-time The impedance under alternating current is Among them, ω=2πf is the angular frequency of the input signal. According to the voltage division, we can get:

[0097]

[0098] Therefore, C can be calculated real-time

[0099]

[0100] When there is no droplet in the gap between the plates corresponding to the lower plate single electrode, the measured total capacitance is equivalent to the dielectric layer 23 capacitance C dielectric Capacitance C of the air dielectric part air In series (the capacitance of the hydrophobic layer 24 is neglected here), assuming that the size of a single array electrode 22 of the lower plate is L1×L2, the total capacitance C total-air The expression is:

[0101]

[0102] Among them, ε0 and ε dielectric are the dielectric constants of air and dielectric layer 23, d air is the distance between the upper and lower plates filled with air, d dielectric is the thickness of the dielectric layer 23 .

[0103] When there is a droplet in the gap between the plates corresponding to the lower plate single electrode, the measured total capacitance is equivalent to the dielectric layer 23 capacitance C dielectric Capacitance C of the liquid dielectric part liquid In series, the total capacitance is C total-liquid The expression is:

[0104]

[0105] Among them, ε liquid is the dielectric constant of the droplet, d liquid is the distance between the upper and lower plates filled with liquid. liquid Usually greater than the dielectric constant ε0 of air, C total-liquid >C total-air , so the capacitance value measured is larger in the area covered by the droplet.

[0106] When the droplet moves between the plates, assuming that the droplet moves in the L1 direction, its position x determines the area covered by the droplet. Therefore, the capacitance of the gap between the two plates is the parallel capacitance formed by the part covered by the droplet and the part covered by the air:

[0107]

[0108] Among them, A liquid and A air are the areas covered by the droplet and air respectively, x is the coverage length of the droplet in the L1 direction, and L2 is the width of the array electrode. The capacitance of the dielectric layer 23 is A dielectric is the area of the corresponding dielectric layer. Since the dielectric layer 23 forms a series connection with the parallel capacitor, the total capacitance C total can be expressed as:

[0109]

[0110] The total capacitance changes with the position x. Therefore, by measuring the change in capacitance, the current position of the droplet 5 can be deduced, that is

[0111]

[0112] By monitoring the current total capacitance C total , solving the current position x of the droplet in real time, and establishing the curve of its relationship with time, the moving speed of the droplet can be calculated.

[0113] Assume that the size of the hole is L3×L4 (L3, L4 < L1, L2), and the area of the hole is A hole = L3×L4.

[0114] (1) When drilling a hole in the upper electrode plate, the effective area corresponding to the electrode decreases, resulting in a decrease in the total capacitance. When there is no droplet in the gap between the plates, the measured total capacitance is equivalent to the series connection of the dielectric layer 23 and the capacitance of part of the air medium. The expression of the total capacitance C t ′ otal-air is:

[0115]

[0116] When there is a droplet in the gap between the plates, the measured total capacitance is equivalent to the series connection of the dielectric layer 23 and the capacitance of part of the liquid medium. The expression of the total capacitance C t ′ otal-liquid is:

[0117]

[0118] (2) Similarly, when drilling a hole in the lower electrode plate, since the lower electrode plate contains the dielectric layer 23, the area corresponding to the dielectric layer will change when calculating the capacitance. The total capacitance corresponding to the electrode is:

[0119]

[0120] Therefore, when starting to monitor the capacitance C real-time corresponding to each electrode in real time, it is necessary to redefine the normal operation monitoring range of the capacitance at the drilling position. Similarly, when calculating the moving speed of the droplet, the influence of the hole needs to be considered and the formula needs to be corrected to ensure that the chip can operate normally and the speed can be detected. When multiple detection chips are filled on a microfluidic chip, by monitoring the speed of the droplet in real time, the consistency of the parallel experimental conditions can be effectively guaranteed.

[0121] The main advantages of the present invention are as follows:

[0122] (1) Compared with directly processing the microfluidic substrate, the method of this patent directly punches or digs holes in the substrate, which does not require the complex micro-nano processing method of the impedance chip, nor does it require the precious metal coating of SPR and Raman spectroscopy, and has little effect on the subsequent chip processing technology. Therefore, it can greatly simplify the chip preparation and processing steps and reduce the production cost. For bipolar plate microfluidic chips, this method can turn the detection surface of the biochemical sensor chip inward, avoiding the influence of external factors such as the environment.

[0123] (2) When the substrate opposite to the substrate where the chip is located is made of transparent material, the chip can be directly placed in the system, and the automated operation of the reaction and the real-time collection of the detection data can be realized. After adding the sample to be detected and the solution required for the detection to the digital microfluidic chip, the detection reaction and data collection can be performed using the system of this patent without the need for other additional experimental instruments, realizing the integrated operation from "sample in" to "result out".

[0124] (3) Existing fluorescent labeling measurement methods usually have complicated steps, and the following steps are required: adding samples for incubation, washing with cleaning solution, adding colorimetric substrate, washing with cleaning solution, and reading fluorescence results. The method of this patent omits the steps of adding fluorescent labels to substrates and the subsequent steps, and the detection speed is faster, the operation is simpler, and the consumption of detection solutions and the requirements for the professionalism of users are reduced. In addition, the label-free protein detection method does not require fluorescent or chemical labeling of proteins or other detection markers, and can avoid the effects caused by labeling, such as fluorescence quenching, enzyme inactivation, and other problems. In addition, label-free specific detection can be achieved under the condition that the corresponding capture probe is modified on the sensor surface.

[0125] The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A double-plate microfluidic chip, comprising an upper plate, a lower plate, a detection chip, a conductive gasket and a controllable droplet, characterized in that: The upper electrode plate and the lower electrode plate are arranged opposite to each other up and down at a predetermined distance, and two conductive gaskets are arranged between the upper electrode plate and the lower electrode plate, and the upper and lower ends of the conductive gasket are respectively in contact with the upper electrode plate and the lower electrode plate; a cavity is formed between the upper and lower electrode plates, and a controllable droplet is arranged in the cavity between the upper electrode plate and the lower electrode plate, and its upper part and lower part are respectively in contact with the upper electrode plate and the lower electrode plate; a detection chip is mounted on the upper electrode plate or the lower electrode plate, and the detection chip extends into the cavity between the upper and lower electrode plates and can contact the controllable droplet.

2. The double-plate microfluidic chip according to claim 1, characterized in that: The upper electrode plate is provided with a mounting through hole, and the lower part of the detection chip passes through the mounting through hole and enters into the cavity between the upper and lower electrode plates.

3. The double-plate microfluidic chip according to claim 1, characterized in that: The lower electrode plate is provided with a mounting through hole, and the upper part of the detection chip passes through the mounting through hole and enters into the cavity between the upper and lower electrode plates.

4. The double-plate microfluidic chip according to claim 2 or 3, characterized in that: The detection chip comprises a biochemical sensor chip, and the detection surface of the biochemical sensor chip extends into the cavity between the upper and lower electrode plates.

5. The double-plate microfluidic chip according to claim 4, characterized in that: The detection chip comprises a substrate, and the biochemical sensor chip is adhered to the substrate.

6. A digital microfluidic chip detection system, comprising an optical path system, a spectrometer, a computer, a microfluidic chip control host and control peripherals, characterized in that: The optical path system includes a light source, and the control peripheral is located on the optical path of the emitted light of the light source. The control peripheral is used to carry the double-plate microfluidic chip as described in any one of claims 1-5. The spectrometer is used to receive the reflected light of the double-plate microfluidic chip and send the spectral information to the computer. The microfluidic chip control host controls the control peripheral, and the computer is used to control the microfluidic chip control host.

7. A method for processing spectral information, wherein the spectral information is spectral information of a double-plate microfluidic chip detected by the detection system according to claim 6, characterized in that The method comprises: S1. Blank sensor chip detection: Place the microfluidic chip horizontally just below the optical path system, detect the reflected light R1(λ) formed by the thin film layer on the substrate surface of the blank sensor chip, and the reflected light R2(λ) formed by the substrate surface layer, and calculate the light intensity R formed by the interference of the two beams. base (λ); S2. Incubation of the sensor chip for the detection probe: Place the microfluidic chip horizontally directly below the optical path system and detect the light intensity R formed by the interference of the reflected light formed on the probe surface and the light reflected from the substrate surface layer. capt (λ); S3. Sensor chip detection for capturing detection targets: Place the microfluidic chip horizontally directly below the optical path system, control the manipulable droplets to move to the detection chip, contact and react with the probes of the sensor chip, and the probes capture the target molecules in the droplets. The light intensity R formed by the interference of the reflected light of the detection probes on the thin film on the surface of the detection chip after capturing the target molecules and the light reflected by the surface layer of the substrate dect (λ).

8. The spectral information processing method according to claim 7, characterized in that Also includes: S4: After normalizing the spectra of the above steps S1-S3, time-frequency conversion is performed respectively to convert the spectra in the wavelength domain into the frequency domain; S5: Identify the peak frequency in the frequency domain spectrum obtained in step S4 and the corresponding interference peak f peak , calculate the phase information at the peak; S6: Calculate the phase difference of the probe and the data after capturing the target detection molecule relative to the blank chip at the peak frequency; S7: Calculate the change in optical path length and dielectric layer thickness based on the phase difference calculated in step S6, and solve for the change in film layer thickness Δd caused by capturing target molecules. dect .

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