Cy5 wave band fluorescence excitation chip and integrated detection system

Abstract

The invention relates to a Cy5 wave band fluorescence excitation chip and an integrated detection system. The fluorescence excitation chip comprises a multimode interference device, a transmission waveguide and a grating coupler, after exciting light is input into the fluorescence excitation chip, the exciting light is split by the multimode interference device, each light beam is transmitted to the grating coupler through the transmission waveguide, and the grating coupler vertically redirects the light to excite a sample on the fluorescence excitation chip. According to the Cy5-band fluorescence excitation chip, narrow-band excitation is achieved, interference of excitation light on emitted light is avoided, and a light path is simple and easy to miniaturize; a solution cavity of the micro-fluidic chip can realize multi-sample miniaturization, the sample volume is 17-20 microliters, and cross contamination of centrifugal tubes is avoided; quantification of fluorescence intensity and concentration can be realized through detection of a photoelectric detector. Therefore, the obtained integrated detection system has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of optoelectronic integration, and particularly relates to a Cy5 waveband fluorescent excitation chip and an integrated detection system. BACKGROUND

[0002] The biological sensing technology based on fluorescent labeling has been widely applied in the fields of pathogenic pathogen detection, food safety and environmental monitoring, etc. The fluorescent detection technology reflects a certain parameter index of the analyte through the properties of fluorescent intensity, excited state lifetime, spectral shape, etc., and has specific recognition ability to the detection molecules. At present, the high-pressure wide-mercury lamp is mostly used as the excitation fluorescent light source, the target light waveband is obtained through the light filtering module and the spatial light path structure, the emission light intensity is acquired, and then the concentration of the biological sample is calibrated. However, the existing detection system based on fluorescent labeling mainly has three problems: 1. The light source is large in size and difficult to be miniaturized and integrated; the excitation light is large in bandwidth, the background interference affects the determination of the emission light intensity, and the weak fluorescent signal is easily submerged; 2. The centrifugal tube of the sample reagent is easy to cause cross contamination and aerosol pollution; the volume of the detection sample is large; 3. The fluorescent signal detection mostly uses the light path structure of the fluorescence microscope, which is large in size and difficult to be miniaturized and integrated; and the qualitative determination through the visual field observation cannot achieve the quantitative determination. SUMMARY

[0003] In view of the defects of the prior art, the application aims to provide a Cy5 waveband fluorescent excitation chip and an integrated detection system. The fluorescent excitation chip is narrow-band excitation, which avoids the interference of the excitation light on the emission light, and the light path is simple and easy to be miniaturized.

[0004] The application provides a Cy5 waveband fluorescent excitation chip, which comprises a multimode interferometer, a transmission waveguide and a grating coupler. After the excitation light is input into the fluorescent excitation chip, the excitation light is split by the multimode interferometer, each beam of light is transmitted to the grating coupler through the transmission waveguide, and the grating coupler performs vertical redirection on the light to excite the sample on the fluorescent excitation chip.

[0005] Preferably, the center of the multimode interferometer is an interference region, and the two sides are connected with tapered waveguides. The number of the multimode interferometers can be multiple.

[0006] Preferably, the length of the interference region is 32-36 μm, and the width is 4-6 μm; the length of the tapered waveguide is 8-12 μm, and the width gradually changes from 0.9-1.3 μm to 420-480 nm.

[0007] Preferably, the grating coupler is a fan-shaped structure, and sequentially comprises a light guide region, a grating region and a reflection region. The number of the grating couplers can be multiple.

[0008] Preferably, the grating area is a fan ring, the inner radius is 20-30 mu m, the outer radius is 30-40 mu m, the period is 0.45-0.5 mu m, and the duty cycle is 0.4-0.6.

[0009] Preferably, the grating opening angle of the grating coupler is 34-38°, and the total length is 60-80 mu m.

[0010] Preferably, one end of the transmission waveguide is connected with the multimode interferometer, and the other end is connected with the grating coupler.

[0011] Preferably, the chip is divided into four layers from bottom to top, namely a silicon substrate, a silicon dioxide buried oxygen layer, a silicon nitride optical waveguide layer and a silicon dioxide protective layer.

[0012] The application provides an integrated detection system A, which comprises a Cy5 waveband fluorescent excitation chip, a biological microfluidic chip, a filter and a photoelectric detector from bottom to top, and the output voltage data of the photoelectric detector is collected by an oscilloscope.

[0013] Preferably, the Cy5 waveband fluorescent excitation chip is 1-3 cm long and 1-3 cm wide.

[0014] Preferably, a solution cavity is arranged in the biological microfluidic chip; the solution cavity is a circular through hole, the bottom of which is sealed by a PCR film, and the top of which is open for sample introduction.

[0015] Preferably, the filter is a Cy5 waveband filter, and the filter size is 10-13 mm in diameter, covering the photosensitive area of the photoelectric detector.

[0016] Preferably, the photoelectric detector adopts an avalanche photodiode (APD) to detect the excitation light.

[0017] The integrated detection system A takes Cy5 fluorescent dye as the application target, and provides a miniaturized integrated scheme for the currently commonly used fluorescent detection instruments such as polymerase chain reaction technology PCR and enzyme-linked immunoassay technology ELISA. The structure of the fluorescent excitation chip and the structure of the biological microfluidic chip can be customized with high freedom according to requirements.

[0018] The application also provides another integrated detection system B, which comprises a Cy5 waveband fluorescent excitation chip, a biological microfluidic chip and a fluorescence microscope. The fluorescence microscope is used for observation, a charge-coupled device (CCD) camera is used for collecting image information, and software is used for analyzing the fluorescence intensity.

[0019] The application also provides an application of the Cy5 waveband fluorescent excitation chip or the integrated detection system A / B in Cy5 fluorescein detection and virus detection.

[0020] Beneficial effects

[0021] The main advantages of the present application include: 1. Cy5 waveband fluorescence excitation chip narrowband excitation, avoiding the interference of excitation light on emission light, simple optical path and easy miniaturization; 2. Microfluidic chip solution cavity can realize miniaturization of multiple samples, sample volume 17 μL-20 μL, avoiding cross contamination of centrifugal tubes; 3. Photoelectric detector detection can realize quantitative fluorescence intensity and concentration. Therefore, the integrated detection system obtained has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a structural schematic diagram of the fluorescence excitation chip of the present application.

[0023] Figure 2 It is an enlarged view of the multimode interferometer (a) and the grating coupler (b) of the fluorescence excitation chip of the present application.

[0024] Figure 3 It is a structural diagram of the integrated detection system A of the present application.

[0025] Figure 4 It is the test result of different concentrations of Cy5 fluorescein of the integrated detection system A of the present application; wherein, (a) is a histogram of the test result of different concentrations of Cy5 fluorescein; (b) is a fitting curve of output voltage and concentration of Cy5 fluorescein solution.

[0026] Figure 5 It is the experimental result of the integrated detection system B of the present application under fluorescence microscope; wherein, (a) is a fluorescence image of different concentrations of Cy5 fluorescein solution; (b) is a histogram of fluorescence gray value of different concentrations of Cy5 fluorescein solution; (c) is a fitting curve of fluorescence gray value; the error bar shows the standard deviation of at least three independent experimental measurements.

[0027] Figure 6 It is the amplification curve of different plasmid concentrations under the PCR reaction system of the integrated detection system A of the present application.

[0028] Figure 7 It is the detection result of the integrated detection system A of the present application using the new coronavirus plasmid amplified under the PCR reaction system as the sample of different plasmid concentrations; wherein, (a) is a fluorescence image of the detection sample; (b) is the output voltage of the detection sample; the error bar shows the standard deviation of at least three independent experimental measurements. DETAILED DESCRIPTION

[0029] The application will be further described in connection with the following specific embodiments. It should be understood that these embodiments are only used to illustrate but not to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] Embodiment 1

[0031] The embodiment provides a Cy5 waveband fluorescence excitation chip, which comprises a multimode interferometer, a transmission waveguide and a grating coupler; after excitation light is input into the fluorescence excitation chip, the excitation light is split by the multimode interferometer, each beam of light is transmitted to the grating coupler through the transmission waveguide, and the grating coupler performs vertical redirection on the light to excite the sample on the fluorescence excitation chip.

[0032] Specifically, as shown in Figure 1 In the embodiment, three multimode interferometers and four grating couplers are used. The multimode interferometer can uniformly distribute one beam of light energy into two beams of outgoing light by using the mode coupling principle. The grating coupler performs vertical redirection on the planar transmission light to excite the sample on the fluorescence excitation chip.

[0033] Through optimization of the Cy5 waveband 645 nm target excitation wavelength, the structural parameters of the multimode interferometer and the grating coupler with the highest transmittance are obtained.

[0034] As shown in Figure 2 The center of the multimode interferometer is an interference region, and both sides are connected with tapered waveguides. The length of the interference region is 34 μm, and the width is 5 μm. The length of the tapered waveguide on both sides is 10 μm, and the width gradually changes from 1.1 μm to 450 nm. The single-end coupling efficiency is 49% at the 645 nm target wavelength.

[0035] As shown in Figure 2 The grating coupler is a fan-shaped structure, which sequentially comprises a light guide region, a grating region and a reflection region. The grating opening angle α of the grating coupler is 36°, and the total length L is 70 μm. The grating region is a fan ring, the inner radius is 25 μm, the outer radius is 35 μm, and the reflection region radius is 10 μm. The period of the grating region is 0.488 μm, and the duty cycle is 0.5. The middle recessed area of the grating can be completely removed by using electron beam exposure combined with reactive ion etching. The coupling efficiency is 15% at the 645 nm target wavelength.

[0036] The Cy5 waveband fluorescence excitation chip is divided into four layers, from bottom to top, which are a silicon substrate (500 μm), a silicon dioxide buried oxygen layer (2 μm), a silicon nitride light waveguide layer (0.22 μm) and a silicon dioxide protective layer (0.5 μm).

[0037] AsFigure 3 As shown, this embodiment also provides an integrated detection system A, including a Cy5 band fluorescence excitation chip, a bio-microfluidic chip, a filter, and a photodetector.

[0038] The Cy5-band fluorescence excitation chip is 2 cm long and 2 cm wide. The bio-microfluidic chip is laser-cut from a black acrylic plate measuring 8.5 mm long, 8.5 mm wide, and 2.85 mm thick, with a 3.5 mm diameter through-hole. A PCR sealing film is used to attach a semi-closed solution chamber to one side of the acrylic through-hole, into which the sample solution is placed. The solution chamber of the microfluidic chip is transparent, while other parts are opaque. A Cy5-band filter with a diameter of 12.7 mm is used, covering the photosensitive area of ​​the photodetector. The photodetector uses an APD to detect the excitation light.

[0039] This embodiment also provides another integrated detection system B, which includes a Cy5-band fluorescence excitation chip and a fluorescence microscope. The fluorescence microscope is used for observation, a CCD camera is used to acquire image information, and software is used to analyze the fluorescence intensity.

[0040] Using a pipette, 17–20 μL of prepared fluorescein solution (Cy5 fluorescein) was injected into each of the two sets of bio-microfluidic chips. One set was used for validation of integrated detection system A. The bio-microfluidic chip was placed on the fluorescence excitation chip, and the solution chamber of the bio-microfluidic chip was aligned with the photodetector. The excitation light carrying the fluorescence signal was filtered by a Cy5 filter to remove background stray light before being received by the photosensitive area of ​​the photodetector. The output voltage data of the photodetector was acquired using an oscilloscope. The other set was used for validation of integrated detection system B. It was observed using a fluorescence microscope (IX73, Olympus, Japan), and image information was acquired using a CCD camera. The fluorescence intensity was analyzed using ImageJ software, and the data will be further processed using Origin software.

[0041] The results of Cy5 luciferin testing under a fluorescence microscope are as follows: Figure 4 As shown. Figure 4 In (a), the concentrations of Cy5 fluorescein solutions corresponding to the fluorescence images are 0.5 μmol / L, 0.25 μmol / L, 0.125 μmol / L, 0.0625 μmol / L, and 0, respectively. The fluorescence intensity decreases with increasing Cy5 fluorescein dilution ratio. Figure 4 (b) The concentrations of Cy5 fluorescein solution from left to right are... Figure 4 (a) Correspondingly, the vertical axis in the figure represents the fluorescence grayscale value, which is determined by... Figure 4 The fluorescence image in (a) was extracted. Figure 4It can be seen that the fluorescence gray value decreases with the decrease of the concentration, which is consistent with the trend of the fluorescence image brightness. In the range of 0.0625 μmol / L to 0.5 μmol / L, the fitting curve of the fluorescence gray value and the Cy5 fluorescein solution concentration is as shown in Figure 4 (c). There is a good linear relationship between the fluorescence gray value and the calculated concentration of Cy5 fluorescein. The linear regression equation of the fluorescence gray value y and the concentration x is y = 89.73x + 26.27 (R2= 0.99203). The experimental results show that the concentration of the Cy5 fluorescein solution and the fluorescence intensity are linearly related under the same conditions.

[0042] The bio-microfluidic chip injected with the Cy5 fluorescein solution was placed in the integrated detection system A in turn, and the output signal was collected using an oscilloscope. The test results of different concentrations of Cy5 fluorescein are as shown in Figure 5 . Figure 5 The histogram of (a) shows that the concentrations of the Cy5 fluorescein solution from left to right are 0.5 μmol / L, 0.25 μmol / L, 0.125 μmol / L, 0.0625 μmol / L, and 0. The output voltage decreases with the increase of the dilution ratio of the Cy5 fluorescein solution. The error bar shows the standard deviation of the measurement results of at least three independent experiments. Compared with the control group DMSO, the output voltage of the Cy5 fluorescein solution with a concentration of 0.0625 μmol / L is significantly different, indicating that the integrated detection system A can realize the detection of the Cy5 fluorescein solution with a concentration of 0.0625 μmol / L. In the range of 0.0625 μmol / L to 0.5 μmol / L, the fitting curve of the output voltage and the Cy5 fluorescein solution concentration is as shown in Figure 5 (b). There is a good linear relationship between the output voltage and the calculated concentration of Cy5 fluorescein, which is compared with Figure 4 (c). Both of them have linear detection results, but the difference lies in the different slopes of the fitting curves, because the conversion ratio of the fluorescence intensity between them is not consistent. The photodetector is more sensitive to weak light signals and has a larger amplitude, so the slope is larger. The experimental results show that the fluorescence excitation and detection system realizes the functions of fluorescence excitation and collection and detection. The detection results are consistent with the detection results of the fluorescence microscope.

[0043] Example 2

[0044] To verify the performance of integrated detection systems A / B in practical applications, amplified novel coronavirus plasmids were used as detection samples. During the experiment, 17–20 μL of amplified reagent, unamplified reagent of the same concentration, and DEPC water (control group) were injected into each of the two sets of bio-microfluidic chips using a pipette. One set was tested using integrated detection system A. The bio-microfluidic chip was placed on a fluorescence excitation chip, and the solution chamber of the bio-microfluidic chip was aligned with the grating coupler. The excitation light carrying the fluorescence signal was filtered by a Cy5 filter to remove background stray light before being received by the photosensitive area of ​​the photodetector. The output voltage data of the photodetector was acquired using an oscilloscope. Detection was performed sequentially while maintaining constant detection conditions. The other set was used to verify integrated detection system B. Observation was performed using a fluorescence microscope (IX73, Olympus, Japan), and image information was acquired using a CCD camera.

[0045] An artificial plasmid containing the N gene from the novel coronavirus sequence, specific upstream and downstream primers, and a fluorescent probe were used. The excitation wavelength of the fluorescent probe was 650 nm, and the fluorescence emission wavelength was 670 nm, consistent with the operating wavelength of the fluorescence excitation and detection system. PCR amplification curves for four different plasmid concentration schemes are shown below. Figure 6 The data obtained using a photodetector can be found in [the image / document]. Figure 7 .Depend on Figure 6 , Figure 7 The results show that the PCR reaction amplification curve, the fluorescence image of integrated detection system B, and the output voltage of integrated detection system A can all clearly distinguish between negative and positive results. After amplification with different plasmid concentrations, the amplification curves eventually flattened and became similar, the fluorescence image brightness was almost the same, and the output voltage difference was not significant. The three detection results can mutually verify each other, proving that the proposed Cy5 band fluorescence excitation chip meets the requirements of PCR for fluorescence signal excitation. The constructed integrated detection system can accurately collect and detect fluorescence, possessing practical application capabilities and good application prospects.

Claims

1. A Cy5 waveband fluorescence excitation chip, characterized in that, The fluorescence excitation chip comprises a multimode interferometer, a transmission waveguide and a grating coupler; after excitation light is input into the fluorescence excitation chip, the excitation light is split by the multimode interferometer, each beam of light is transmitted to the grating coupler through the transmission waveguide, and the grating coupler performs vertical redirection on the light to excite the sample on the fluorescence excitation chip.

2. The Cy5 waveband fluorescence excitation chip of claim 1, wherein, The center of the multimode interferometer is an interference region, and both sides are connected with tapered waveguides.

3. The Cy5 waveband fluorescence excitation chip of claim 2, wherein, The length of the interference region is 32-36 μm, and the width is 4-6 μm; the length of the tapered waveguide is 8-12 μm, and the width gradually changes from 0.9-1.3 μm to 420-480 nm.

4. The Cy5 waveband fluorescence excitation chip of claim 1, wherein, The grating coupler is a fan-shaped structure, and sequentially comprises a light guide region, a grating region and a reflection region.

5. The Cy5 waveband fluorescence excitation chip of claim 4, wherein, The grating region is a fan ring, the inner radius is 20-30 μm, the outer radius is 30-40 μm, the period is 0.45-0.5 μm, and the duty cycle is 0.4-0.

6.

6. The Cy5 waveband fluorescence excitation chip of claim 1 or 4, wherein, The grating opening angle of the grating coupler is 34-38°, and the total length is 60-80 μm.

7. The Cy5 waveband fluorescence excitation chip of claim 1, wherein, The chip is divided into four layers, from bottom to top, which are a silicon substrate, a silicon dioxide buried oxygen layer, a silicon nitride optical waveguide layer and a silicon dioxide protective layer.

8. An integrated detection system A, characterized in that, The integrated detection system A comprises, from bottom to top, the Cy5 waveband fluorescence excitation chip of claim 1, a biological microfluidic chip, a filter and a photodetector.

9. An integrated detection system B, characterized in that The integrated detection system B comprises the Cy5 waveband fluorescence excitation chip of claim 1, a biological microfluidic chip and a fluorescence microscope.

10. Application of the Cy5 waveband fluorescence excitation chip of any one of claims 1-7 or the integrated detection system A / B of any one of claims 8-9 in Cy5 fluorescein detection and virus detection.

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