Alternating current modulated fluorescence microdevice for crisper detection

Abstract

The application discloses an alternating current modulation fluorescence micro device for CRISPR detection, and belongs to the field of biosensing and analysis instruments.The device comprises a circuit module, an optical detection module and a reaction unit; the main control board of the circuit module drives a PD detection plate and an LED excitation plate to perform excitation and collection of fluorescence signals; an excitation light source of the optical detection module is excited by the LED excitation plate to generate excitation light, which is filtered and corrected by an excitation filter and a convex lens, and then is reflected by a dichroic mirror into the reaction unit; a sample to be detected arranged on a microfluidic chip in the reaction unit is excited by the excitation light to generate emission light, which is transmitted through the dichroic mirror and an emission filter, and then the fluorescence signal is received by the PD detection plate, and the main control board is used for analysis and Bluetooth transmission.The application has the advantages of rapid detection and accurate quantification, and can realize multi-channel expansion, and is suitable for nucleic acid on-site rapid screening scenes based on CRISPR.

Description

Technical Field

[0001] This invention relates to the field of biosensing and analytical instruments, and more particularly to a highly integrated coaxial fluorescence detection platform, specifically to an AC-modulated fluorescence microdevice for CRISPR detection. Background Technology

[0002] With the continuous development of integrated circuits, microelectromechanical systems (MEMS), and precision optics, optical biosensors are evolving towards miniaturization, portability, and intelligence. The development of instant optical sensing detection devices in the diagnostic field largely benefits from the miniaturization and integration of these devices. Related research focuses on building easy-to-operate, portable optical detection systems capable of real-time analysis. The core objective of miniaturized portable optical sensor design is to achieve a compact and lightweight sensor design, improving portability and enhancing its application capabilities in various scenarios by reducing the overall system size and weight. Portable optical biosensors offer advantages such as cost-effectiveness, miniaturization, ease of use, and instant results, effectively meeting the application needs of personalized disease diagnosis, disease management, and telemedicine.

[0003] In recent years, CRISPR / Cas systems (such as Cas12 and Cas13) have demonstrated great potential in the rapid detection of nucleic acid molecules and proteins due to their highly specific target recognition and trans-cleavage capabilities. CRISPR detection methods based on fluorescence, with their high sensitivity and wide linear range, have become a core technological path for achieving rapid and accurate analysis of nucleic acids and proteins in the field of molecular diagnostics. However, the establishment of such detection systems is highly dependent on the accurate capture of the dynamic changes in fluorescence signals during the reaction process, which places extremely high demands on the optical transmission efficiency, signal acquisition stability, and environmental adaptability of the detection device. Existing CRISPR detection devices are bulky and cannot meet the needs of on-site rapid testing scenarios such as primary healthcare and home self-testing. Furthermore, existing detection devices often only focus on signal reading, neglecting the temperature sensitivity and cold chain dependence of enzymes and proteins in the CRISPR system, lacking a closed-loop hardware platform that integrates reagent room-temperature curing solutions (such as lyophilization) with high-precision reading circuitry. In addition, in portable and on-site rapid testing environments, complex ambient stray light and low-frequency noise within the circuitry often severely interfere with the extraction of weak fluorescence signals, causing the signal in the early stages of the CRISPR reaction to be masked by noise.

[0004] Therefore, although CRISPR systems offer a highly promising technological means for the field of biological detection, existing detection devices are limited by their large size, low optical path integration, and lack of design solutions for the stability of biological reagents. These limitations prevent current technologies from meeting the demands for highly sensitive reading and real-time analysis of CRISPR reaction signals in field settings. To address this key challenge and meet the aforementioned requirements for miniaturized and portable detection technologies, developing a miniature fluorescence detection device integrating a high-efficiency coaxial optical detection module, a high-precision fluorescence detection circuit based on AC modulation, and a lyophilized reaction unit with room-temperature stability is crucial for improving the efficiency of CRISPR rapid on-site detection, eliminating reliance on cold chains, and promoting the clinical adoption of point-of-care diagnostic technologies. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an AC-modulated fluorescence microdevice for CRISPR detection, aiming to solve the problems of the large size of existing fluorescence detection equipment and the inability to preserve biochemical reagents for long periods, which makes it difficult to popularize rapid on-site detection of biomarkers.

[0006] The objective of this invention is achieved through the following technical solution: an AC-modulated fluorescence microdevice for CRISPR detection, comprising: shell; The circuit module includes a main control board, a PD detection board, and an LED excitation board. The main control board drives the PD detection board and the LED excitation board to excite and collect fluorescence signals. An optical detection module includes an excitation light source, an excitation filter, a convex lens, a dichroic mirror, and an emission filter; the excitation light source is excited by an LED excitation plate to generate excitation light, and the excitation light, after being filtered and corrected by the excitation filter and the convex lens, is reflected by the dichroic mirror into the reaction unit; The reaction unit includes a sample cell and a microfluidic chip disposed within the sample cell; In this process, the sample to be tested in the microfluidic chip is excited by excitation light to generate emitted light, which passes through a dichroic mirror and an emission light filter, and the fluorescence signal is received by the PD detection board and analyzed by the main control board.

[0007] Furthermore, the microdevice includes a base, the reaction unit is disposed inside the base, the optical detection module is disposed above the base, and a through hole for light to pass through is opened above the base. The main control board and the PD detection board are disposed above the optical detection module, and the LED excitation board is disposed outside the optical detection module. The base is provided with a buckle, which connects to the outer shell to form a sealed environment.

[0008] Furthermore, the main control board outputs an AC square wave signal of a specific frequency to provide AC excitation to the LED excitation board, driving the LED excitation board to generate frequency-modulated excitation light, so that the excitation light is in a high-frequency pulse modulation state; the sample under test is excited to generate a weak fluorescence signal carrying the same frequency modulation characteristics. This signal is received by the photodetector of the PD detection board after passing through a dichroic mirror and an emission light filter and is converted into a weak photocurrent signal; the photocurrent signal is amplified by self-adjusting transimpedance and bandpass filtered, and then sampled at high frequency by the main control board; the main control board performs a fast Fourier transform operation on the sampled digital signal, converting the signal from the time domain to the frequency domain for analysis, and by extracting the amplitude at the modulation frequency feature point, eliminating the interference of ambient stray light and low-frequency noise of the circuit, finally outputting the target fluorescence intensity value reflecting the concentration of the analyte.

[0009] Furthermore, the main control board includes a microcontroller system, a digital-to-analog converter, a Bluetooth module, and a power management module, which are used to process and transmit fluorescence signals and provide power to the circuit module. The main control board, the PD detection board, and the LED excitation board are all electrically connected through a connecting board. The connecting board includes three inter-board connectors, which are respectively connected to the PD detection board, the LED excitation board, and the main control board.

[0010] Furthermore, the excitation light source is an LED point light source, which is placed at one focal length of the convex lens. The excitation filter is disposed between the LED point light source and the convex lens. The excitation filter filters out the spectral components of the excitation light that are not in the target wavelength range, and the convex lens calibrates the excitation light into parallel light.

[0011] Furthermore, the dichroic mirror is tilted so that after the excitation light is reflected by the dichroic mirror, it is located in the same coaxial optical path as the emitted light.

[0012] Furthermore, the microfluidic chip is provided with a fluid channel and a reaction chamber. A glass fiber membrane is provided at the bottom of the reaction chamber. The lyophilized reagent is placed on the glass fiber membrane and encapsulated by an encapsulation layer.

[0013] Furthermore, the lyophilized reagent comprises a trehalose protectant, target-designed crRNA, Cas enzyme, and a fluorescence quenching probe, and is prepared by a vacuum freeze-drying process.

[0014] Furthermore, the fluorescent quenching probe is a single-stranded nucleic acid with a fluorescent agent and a quencher modified at both ends, respectively, with the sequence 5'6-FAM-TTATT-BHQ1-3', an excitation wavelength of 494 nm, and an emission wavelength of 518 nm; wherein, the fluorescent agent contains a fluorescent group selected from one or more of fluorescein, rhodamine, or cyanine; and the quencher contains a quenching group selected from one of a dark quenching group or a fluorescent quenching group.

[0015] Furthermore, the center wavelength of the passband of the excitation filter is located within the excitation spectrum range of the fluorescent agent; the center wavelength of the passband of the emission filter is located within the emission spectrum range of the fluorescent agent.

[0016] The beneficial effects of this invention are as follows: This invention provides a miniature device and detection method suitable for CRISPR fluorescence detection. Through the design of the coaxial optical module, it is possible to achieve high-efficiency capture of dynamic fluorescence signals in CRISPR reaction in a very small volume. At the same time, the device integrates a high-gain signal processing circuit and a freeze-drying reaction unit. The operation process is extremely simple and does not require cold chain support, providing a brand-new hardware platform and technical path for portable CRISPR point-of-care detection.

[0017] Compared to traditional fluorescence detection equipment, the miniature fluorescence device for on-site CRISPR rapid detection of this invention features high integration, low cost, low background noise in the optical path, and independence from cold chain dependence. Its coaxial design and lyophilization integration method can be extended to the CRISPR detection of more nucleic acid and protein biomarkers, greatly improving the immediacy of detection. This completely frees CRISPR detection from the limitations of large-scale laboratory equipment, meeting the needs of primary healthcare and home testing, and providing an efficient and intelligent hardware solution for various infectious disease screenings and health management. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the AC-modulated fluorescence microdevice for CRISPR detection provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of a coaxial detection optical path based on a dichroic mirror provided in an embodiment of the present invention; Figure 3 This is a graph showing the relationship between the concentration of carboxyfluorescein and the peak fluorescence intensity provided in an embodiment of the present invention. Figure 4 This is a schematic diagram of the excitation spectrum of 6-carboxyfluorescein with a concentration range of 100 nM-1 mM provided in the embodiments of the present invention; Figure 5 This is a schematic diagram of the emission spectrum of 6-carboxyfluorescein with a concentration range of 0-10 μM provided in the embodiments of the present invention; Figure 6This is a schematic diagram of the transmission spectrum characterization results of dichroic mirrors at different tilt angles in the optical path design provided in the embodiments of the present invention; Figure 7 This is a schematic diagram of the waveform of the driving LED excitation light generated by the constructed fluorescence detection circuit provided in the embodiment of the present invention; Figure 8 This is a schematic diagram of the emitted light waveform generated by the fluorescence detection circuit constructed according to the embodiment of the present invention after being excited by a fluorescent agent; Figure 9 This is a schematic diagram of the output signal of the fluorescence detection circuit constructed according to the embodiment of the present invention after fast Fourier transform. Figure 10 This is a schematic diagram showing the frequency domain distribution of the fluorescence detection circuit constructed according to an embodiment of the present invention for detecting signals of different concentrations of fluorescent agents; Figure 11 This is a schematic diagram of the linear fitting between the detection results of different concentrations of fluorescent agents and the concentration of fluorescent agents provided in the embodiments of the present invention. Figure 12 This is a diagram showing the original output of the fluorescence detection system constructed according to the embodiment of the present invention for the CRISPR system, where ah represents the detection results read every 5 minutes from 0 to 35 minutes. Figure 13 This is a schematic diagram of the Fourier transform analysis of the measured output results of the fluorescence detection system constructed according to the embodiments of the present invention for the CRISPR system; Figure 14 This is a comparison chart of the experimental results of the constructed fluorescence detection system for the CRISPR system and the detection results of the fluorescence microplate reader provided in the embodiments of the present invention; Figure 15 This is a schematic diagram of the constructed microfluidic chip structure provided in the embodiments of the present invention; Figure 16 This is a comparison chart of the activity of CRISPR detection reagents before and after lyophilization with different amounts of trehalose provided in the embodiments of the present invention; Figure 17 This is a schematic diagram illustrating the activity retention rate of CRISPR detection reagents with different trehalose addition amounts provided in the embodiments of the present invention after lyophilization. Figure 18 This is a schematic diagram illustrating the stability of the CRISPR lyophilized reagent provided in this embodiment of the invention under low-temperature storage for the trans-cleavage activity of Cas12a. In the diagram: 1. Outer shell; 2. Main control board; 3. PD detection board; 4. LED excitation board; 5. Microfluidic chip; 6. Optical detection module; 7. Sample cell; 8. Light source; 9. Excitation filter; 10. Convex lens; 11. Excitation light; 12. Fluorescent sample to be tested; 13. Dichroic mirror; 14. Emitted light; 15. Emitted light filter; 16. Emitted light; 17. Emitted light filter; 18. Detailed Implementation

[0020] The embodiments, features, and aspects of this disclosure will be described in detail below with reference to the accompanying drawings, but this is not intended to limit the invention. All other embodiments obtained by those skilled in the art based on any extension of the embodiments of this invention, without inventive effort, are within the scope of protection of this invention. The same reference numerals in the drawings denote the same or similar functional elements. Although various aspects of the embodiments are shown in the drawings, they are not necessarily drawn to scale unless specifically indicated otherwise.

[0021] In addition, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art should understand that this disclosure can be implemented without certain specific details. Some methods and means well known to those skilled in the art are not described in detail in order to highlight the main points of this disclosure.

[0022] like Figure 1 As shown, this invention presents an AC-modulated fluorescence microdevice for CRISPR detection. The device is designed as a four-channel detection device, and the detection channels can be adjusted according to requirements. The shape of the detection device is designed based on the optical and circuit structure. The device adopts a layered assembly structure, including a shell 1, a main control board 2, a PD detection board 3, an LED excitation board 4, a microfluidic chip 5, a coaxial optical detection module 6, and a sample cell 7.

[0023] The outer shell 1 serves as an overall protective and structural support unit, providing physical isolation for the internal core components. The main control board 2 integrates the core control module through surface mount technology, enabling coordinated driving and signal acquisition of the LED excitation board 4 and the PD detection board 3. The LED excitation board 4 and the PD detection board 3 are responsible for the excitation and acquisition of fluorescence signals, respectively, and are electrically interconnected through an electronic connector. The microfluidic chip 5 works closely with the coaxial optical detection module 6, and the microfluidic chip 5 has a pre-set fluid channel and reaction chamber for sample loading and CRISPR reaction system construction. The coaxial optical detection module 6 conducts and acquires the fluorescence signal generated by the reaction sample. The sample cell 7 serves as a sample carrying unit, used to place the disposable and replaceable microfluidic chip 5.

[0024] like Figure 2The diagram shows a coaxial detection optical path based on a dichroic mirror designed according to the present invention. The optical path includes a light source 61, an excitation filter 62, a convex lens 63, an excitation light 64, a fluorescent sample 65, a dichroic mirror 66, an emission light 67, and an emission light filter 68. The light source 61 is an LED point light source, placed at one focal length of the convex lens 63, which converts the scattered light into parallel excitation light 64. The excitation filter 62 is located between the light source and the convex lens, used to filter out spectral components of non-target wavelengths, allowing only specific excitation wavelengths to pass through. After the convex lens 63 collimates the excitation light 64, it is reflected by the dichroic mirror 66 and focused into the fluorescent sample 65, achieving efficient excitation. The emission light 67 generated by the excitation of the fluorescent sample 65 passes through the dichroic mirror 66 and then undergoes secondary spectral selection by the emission light filter 68, effectively filtering out residual excitation light and stray light, and finally guiding the emission light 67 to the photodetector on the PD detection plate 3 for acquisition. The coaxial detection optical path design based on dichroic mirrors relies on precise optical element layout and angle matching to achieve coaxial propagation and efficient beam splitting of excitation and emission light, enabling high signal-to-noise ratio acquisition of weak fluorescence signals.

[0025] like Figure 3 As shown, this invention characterizes the transmission spectra of dichroic mirrors at different tilt angles. Based on the characterization results of the excitation and emission spectra of 6-carboxyfluorescein, the dichroic mirror needs to achieve spectral dispersion in the approximately 500 nm wavelength range. The incident angle of the light has a significant impact on the spectral dispersion band of the dichroic mirror. Therefore, the transmission spectra at different angles were characterized. As the angle between the dichroic mirror and the light plane gradually increases, the angle between the incident light and the normal gradually decreases, and the spectral dispersion band gradually redshifts. Therefore, the optical path design should ensure that the light rays incident on the dichroic mirror are parallel as much as possible. Meanwhile, the spectral dispersion characteristics at a 45° angle meet the design requirements; therefore, the preferred angle between the incident light and the dichroic mirror in the optical path design is 45°.

[0026] like Figure 4 As shown, this invention characterized the excitation spectrum of 6-carboxyfluorescein in the concentration range of 100 nM to 1 mM. Furthermore, the excitation spectrum of 6-carboxyfluorescein was characterized, and the results showed that the emission intensity reached its maximum under excitation light at a wavelength of approximately 490 nm, and increased with increasing fluorescein concentration; therefore, an excitation wavelength of 490 nm is preferred.

[0027] like Figure 5As shown, this invention characterized the emission spectra of 6-carboxyfluorescein in the concentration range of 100 nM to 1 mM. Similarly, the emission spectrum of 6-carboxyfluorescein was characterized, showing that its emission peak at around 520 nm continuously increases with increasing concentration. However, upon further increasing the concentration to 10 μM, a significant red shift occurs in the peak position. If the concentration is further increased to 1 mM, the overall fluorescence intensity decreases; therefore, the preferred emission wavelength is 520 nm.

[0028] like Figure 6 As shown, this invention characterizes the relationship between the concentration of 6-carboxyfluorescein and the peak fluorescence intensity. The emission intensity at wavelengths of 516 nm to 522 nm was selected to define the linear band between the 6-FAM fluorescein and its concentration. In the system, the fluorescence intensity exhibits a relatively obvious linear relationship with the concentration of the 6-FAM fluorescein in the range of 1 nM to 1 μM, preferably 500 nM.

[0029] like Figure 7 The image shows the emitted light wave generated by the fluorescence detection circuit of this invention. The square wave waveform generated by the microcontroller was detected using an oscilloscope, which corresponds to the waveform of the LED excitation light generated by the fluorescence detection circuit constructed in this embodiment. The square wave acquired by the oscilloscope within 1 second is clear and complete, with a frequency of 7Hz, meeting the LED excitation design requirements.

[0030] like Figure 8 The image shows the emission waveform generated by the fluorescence detection circuit of the present invention after excitation by a fluorescent agent. A 1 μM concentration of 6-carboxyfluorescein was selected, and an excitation signal with a frequency of 7 Hz was used for excitation. The signal information received by the PD was detected, and the collected fluorescence signal was a sine wave with relatively stable amplitude and frequency consistent with the excitation square wave.

[0031] like Figure 9 As shown, the output signal of the fluorescence detection circuit of the present invention is transformed by Fast Fourier Transform in the frequency domain. The fluorescence signal collected within a 10-second time interval is selected and subjected to Fast Fourier Transform (FFT). The center frequency of the signal falls at 7Hz, which is consistent with the excitation light. Therefore, the fluorescence intensity can be quantified by taking the amplitude at 7Hz.

[0032] like Figure 10 The figure shows the frequency domain distribution of the detection signals of the fluorescence detection circuit of the present invention for different concentrations of fluorescent agents. Based on the aforementioned spectral characterization results of 6-carboxyfluorescein, its linearity is good in the range of 1 nM-1 μM, which is also a common concentration range for its detection. A fast Fourier transform was performed on the fluorescence signals collected within a 10-second time interval to obtain its frequency domain distribution. As the concentration of the fluorescent agent increases, the amplitude at 7 Hz continuously rises.

[0033] like Figure 11 The diagram shows the linear fitting of the fluorescence detection circuit constructed in this invention for different concentrations of fluorescent agents with the fluorescent agent concentration. After completing three parallel measurements at different concentrations, FFT analysis was performed, and the peak value at 7Hz was taken. The voltage value obtained by converting the peak intensity was linearly fitted with the fluorescent agent concentration, and the coefficient of determination R0 was calculated. 2 It is 0.99.

[0034] like Figure 12 The figure shows the data from the detection using HPV16 viral nucleic acid in a plasmid vector as a positive target, incorporating it into the fluorescent detection device constructed in this invention using the CRISPR / Cas12a system. The detection results were read out every 5 minutes within the 0-35 minute timeframe, and the amplitude of the output waveform showed a significant increasing trend, consistent with the principle of positive samples activating Cas12a trans-cleavage.

[0035] like Figure 13 The figure shows the Fourier transform analysis of the measured output results. Performing Fourier transform analysis on the original output waveform reveals that the amplitude continuously increases at 7Hz.

[0036] like Figure 14 The image shows a comparison between the results of the fluorescence detection device and the results of the fluorescence microplate reader. A comparative analysis of the fluorescence signal values ​​and the results of the fluorescence microplate reader shows a good correlation between the two, with a coefficient of determination R0. 2 It is 0.98.

[0037] like Figure 15 The diagram shows a schematic of the microfluidic chip 5 constructed according to the present invention. The microfluidic chip 5 includes a fluid channel and a reaction chamber. A glass fiber membrane is located at the bottom of the reaction chamber, and the lyophilized reagent is placed on the glass fiber membrane and encapsulated by an encapsulation layer.

[0038] Preferably, the microfluidic chip 5 is constructed as follows: a polymer of PDMS preform and a crosslinking agent are mixed at a weight ratio of 10:1, then cast into a mold, and cured at 80°C for two hours to obtain PDMS. A glass fiber membrane of the corresponding size is placed in the cavity and fixed in the reaction chamber with PDMS adhesive. The PDMS channel layer and the encapsulation layer are bonded by plasma treatment. Subsequently, a fully functional microfluidic chip 5 is obtained through processes such as freeze drying. Its layering relationship is as follows: the bottom layer is a PDMS-based microfluidic channel layer, a glass fiber membrane is modified at the bottom of the reaction chamber, and after freeze drying, the freeze-drying reagent is attached to the glass fiber membrane. The top layer is a PDMS-based encapsulation layer.

[0039] like Figure 16The image shows a comparison of the activity of the CRISPR assay reagents provided by this invention before and after lyophilization with different amounts of trehalose. This invention optimizes the amount of trehalose added and evaluates the activity of the Cas enzyme before and after lyophilization to determine the optimal amount of trehalose. Without added trehalose, the Cas enzyme completely loses its trans-cleavage activity after lyophilization; when the trehalose addition is 4%, the activity of the Cas enzyme still shows a sharp decline after lyophilization; when the trehalose addition is above 8%, the activity of the Cas enzyme is largely preserved after lyophilization.

[0040] like Figure 17 The figure shows the Cas enzyme activity retention rate after freeze-drying with different concentrations of trehalose according to the present invention. When the trehalose content is 8%, the average activity retention rate after freeze-drying reaches 97.75%, while a higher trehalose content leads to a slight decrease in the activity retention rate. Therefore, the preferred trehalose content is 8%.

[0041] like Figure 18 The figure shows the stability of the CRISPR lyophilized reagent of this invention in terms of Cas12a trans-cleavage activity under low-temperature storage. The activity of the CRISPR lyophilized reagent did not change significantly during the first week. On the fourteenth and twenty-eighth days, its activity decreased slightly, but this decrease was not significant, demonstrating that the lyophilized reagent can achieve long-term stable storage under certain conditions.

[0042] This invention provides a miniature fluorescence device and detection method for rapid on-site CRISPR detection. By integrating AC-modulated excitation light and the system's optical path design, it solves the problems of large size and unsuitability for portable rapid detection scenarios in existing CRISPR fluorescence detection devices. The device integrates a coaxial optical system, a high-precision fluorescence detection circuit based on AC modulation, and a reaction unit pre-loaded with CRISPR lyophilized reagents. The coaxial optical system uses a dichroic mirror to ensure that the excitation and emission light are transmitted on the same physical axis, significantly improving the capture efficiency of the weak fluorescence signal released by the CRISPR system's trans-cleavage and effectively reducing the size of the optical module. The reaction unit integrates Cas enzyme, crRNA, and fluorescent probes into a microfluidic cavity based on lyophilization technology, achieving room-temperature stable storage of reagents and ready-to-use detection. This device is easily expandable, can be used for simultaneous detection of multiple targets, and has the advantages of fast detection speed and high quantitative accuracy. It is suitable for rapid on-site screening of nucleic acids based on CRISPR technology, providing a new technical solution for portable molecular diagnostics.

[0043] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made without creative effort should be included within the protection scope of the present invention.

Claims

1. An AC-modulated fluorescence microdevice for CRISPR detection, characterized in that, include: shell; The circuit module includes a main control board, a PD detection board, and an LED excitation board. The main control board drives the PD detection board and the LED excitation board to excite and collect fluorescence signals. An optical detection module includes an excitation light source, an excitation filter, a convex lens, a dichroic mirror, and an emission filter; the excitation light source is excited by an LED excitation plate to generate excitation light, and the excitation light is filtered and corrected by the excitation filter and the convex lens, and then reflected by the dichroic mirror into the reaction unit; The reaction unit includes a sample cell and a microfluidic chip disposed within the sample cell; In this process, the sample to be tested in the microfluidic chip is excited by excitation light to generate emitted light, which passes through a dichroic mirror and an emission light filter, and the fluorescence signal is received by the PD detection board and analyzed by the main control board.

2. The microdevice according to claim 1, characterized in that, The micro-device includes a base, a reaction unit disposed inside the base, an optical detection module disposed above the base, and a through hole for light to pass through on the top of the base. The main control board and PD detection board are disposed above the optical detection module, and the LED excitation board is disposed outside the optical detection module. The base is provided with a buckle, which connects to the outer shell to form a sealed environment.

3. The microdevice according to claim 1, characterized in that, The main control board outputs an AC square wave signal of a specific frequency to provide AC excitation to the LED excitation board, driving the LED excitation board to generate frequency-modulated excitation light.

4. The microdevice according to claim 1, characterized in that, The main control board includes a microcontroller system, a digital-to-analog converter, a Bluetooth module, and a power management module, which are used to process and transmit fluorescent signals and provide power to the circuit modules.

5. The microdevice according to claim 1, characterized in that, The excitation light source is an LED point light source, which is placed at one focal length of the convex lens. The excitation filter is disposed between the LED point light source and the convex lens. The excitation filter filters out spectral components of non-target wavelengths in the excitation light, and the convex lens calibrates the excitation light into parallel light.

6. The microdevice according to claim 5, characterized in that, The dichroic mirror is tilted so that the excitation light, after being reflected by the dichroic mirror, is located on the same coaxial optical path as the emitted light.

7. The microdevice according to claim 1, characterized in that, The microfluidic chip has a fluid channel and a reaction chamber. A glass fiber membrane is located at the bottom of the reaction chamber. The lyophilized reagent is placed on the glass fiber membrane and encapsulated by an encapsulation layer.

8. The microdevice according to claim 7, characterized in that, The lyophilized reagent contains a trehalose protectant, target-specific crRNA, Cas enzyme, and a fluorescence quenching probe, and is prepared by a vacuum freeze-drying process.

9. The microdevice according to claim 8, characterized in that, The fluorescent quenching probe is a single-stranded nucleic acid with a fluorescent agent and a quencher modified at both ends, respectively. The fluorescent agent contains a fluorescent group selected from one or more of fluorescein, rhodamine, or cyanine; the quencher contains a quenching group selected from one of dark quenching groups or fluorescent quenching groups.

10. The microdevice according to claim 9, characterized in that, The center wavelength of the passband of the excitation filter is located within the excitation spectrum range of the phosphor; the center wavelength of the passband of the emission filter is located within the emission spectrum range of the phosphor.

Images