Miniaturized portable fluorescence microplate reader for instant fluorescence detection

Abstract

The invention relates to the technical field of biological detection, in particular to a miniaturized portable fluorescence microplate reader for instant fluorescence detection, and aims to provide a fluorescence detection scheme which is portable, high in sensitivity and quick in response. The detector integrates an MCU microcontroller, a voltage conversion module, a light source driving module, a motor driving module, a photoelectric detection module, an AD sampling module, an automatic temperature control module based on a PID algorithm, a signal processing system and a data processing system. High-sensitivity and high-resolution detection of a fluorescent sample can be realized, the concentration of the sample can be detected in real time, and the change process of the concentration of the sample can be presented through a kinetic curve. Through the optimization of the structure, optics and electronic circuits, the instrument is more miniaturized and has portability. Due to the advantages of portability, high sensitivity, quick response and the like, the kit has wide application prospects in multiple fields of basic research of nucleic acid, gene expression, sequence analysis, transgenic detection and the like.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and particularly to a miniaturized portable fluorescence microplate reader for instant fluorescence detection. Background Art

[0002] In recent years, as a core tool in the fields of scientific research and industrial analysis, fluorescence detection instruments have shown remarkable speed and breadth in technological innovation and application expansion globally. In China, although the commercialization process of fluorescence detection technology started relatively late, thanks to policy support, capital investment, and the unremitting efforts of scientific research personnel, this technology has rapidly emerged in many key fields such as biomedicine, environmental protection, and food safety, and its application scope and depth are increasing at an unprecedented rate. Meanwhile, on the international stage, fluorescence detection technology has already entered the mature development stage. Many well-known enterprises and top research institutions, relying on their profound technical accumulation and innovation capabilities, continuously promote product iteration and upgrade, improving performance stability and diversity. Especially in the high-end market, their products dominate with excellent technical advantages. The development direction of fluorescence detection technology is becoming increasingly clear, that is, to pursue higher sensitivity to capture weaker signals, stronger selectivity to distinguish target substances in complex environments, and faster detection speed to meet the growing demand for instant detection. With the continuous emergence of new fluorescence probes and detection methods, the application potential of fluorescence detection technology has been further explored, providing strong support for scientific research and technological innovation. In the domestic and international markets, the demand for high-precision and high-efficiency fluorescence detection instruments continues to grow, especially in fields related to social well-being and sustainable development such as healthcare, life sciences, and environmental monitoring, where the application demand is particularly urgent and extensive.

[0003] However, although traditional desktop fluorescence detection devices are powerful, they are unable to meet the modern detection requirements of rapid response and on-site instant measurement. These devices are often bulky, occupying a large amount of space, and have complex operations, requiring professional personnel to be trained professionally to use them proficiently. More importantly, their portability is extremely poor and they cannot adapt to complex and changeable detection environments such as the wild, emergency rescue, and remote areas. In addition, there are also certain limitations in the sensitivity and detection speed of traditional devices, making it difficult to meet the high requirements for detection performance in scenarios such as rapid screening in medical diagnosis, real-time monitoring in environmental monitoring, and rapid detection in food safety. With the acceleration of globalization, the spread of diseases is accelerating, environmental pollution problems are becoming increasingly severe, and the public's attention to health and safety is continuously increasing. The market's demand for a portable fluorescence detection technology that can respond quickly, is easy to operate, and has high sensitivity is becoming more and more urgent.

[0004] To meet these requirements, the development of a miniaturized portable fluorescence microplate reader becomes particularly important. The design of this instrument aims to overcome the limitations of traditional devices and provide a solution that can quickly respond to market demands and technological advancements. It not only represents the transformation of fluorescence detection technology from the laboratory to the field but also brings innovative solutions to the fields of global health, environment, and food safety. The portability and ease of use of this instrument enable it to be used by non-professionals in various environments, thus broadening the application scenarios of fluorescence detection technology.

[0005] The innovation of the new portable fluorescence microplate reader lies in its highly integrated optical system, electronic control technology, and software algorithms. The combination of these technologies not only improves the sensitivity and speed of detection but also simplifies the operation process for users. By using advanced photodetectors and optimized light source technology, the new device can detect lower concentrations of fluorescent markers, and the intelligent algorithm based on a microcontroller can automatically adjust the detection parameters to adapt to different sample characteristics. The development of the portable fluorescence microplate reader can not only provide rapid on-site detection capabilities but also provide reliable detection results for professionals or non-professionals without the support of a professional laboratory. Summary of the Invention

[0006] The present invention aims to provide a rapid, simple, and low-cost fluorescence detection solution with high sensitivity and high selectivity to meet diverse application requirements. The instrument adopts cutting-edge optical and electronic technologies to achieve efficient detection within a short time. The user interface is intuitive and easy to use, simplifying the operation process. It is applicable to a variety of application scenarios, including but not limited to biomedical detection, environmental monitoring, and material analysis.

[0007] To achieve this goal, the miniaturized portable fluorescence microplate reader designed by the present invention integrates multiple functional modules to form a rapid and simple analysis system, including a sample cell, a control main board, an optical path system, a signal acquisition board, a sensor module, a drive control board, a temperature control board, a signal processing system, and a data processing system, etc.

[0008] Specifically, the instrument is equipped with eight sample cells, each of which is adapted to a 0.2 mL centrifuge tube. The detection range of the instrument is 0.1 nM - 100 nM, and it has a high detection rate and accuracy.

[0009] Specifically, the control main board includes a power interface, an RS232 serial asynchronous communication interface, a signal acquisition interface, a microcontroller, a light source, a motor controller, etc. Different modules of the entire system are powered through the power interface and the voltage conversion module. Communication between the upper computer and the lower computer is achieved through the RS232 communication protocol and the communication interface, such as the issuance of control instructions and the upload of collected data. The microcontroller is the core device of the control main board, and the STM32F103C8T6 chip is selected. It can read the information fed back by all sensors through the GPIO port and control each drive module of the system, enabling them to cooperate with each other to complete the instructions issued by the upper computer.

[0010] Specifically, the communication interface can be connected to a portable device, such as a laptop, a tablet computer, or a mobile phone, through a serial-to-USB cable to receive data, enabling fast data transmission and processing. The instrument can be compatible with most mainstream portable devices on the market without the need for additional adapters or converters. Users can receive and analyze the data sent from the fluorescence microplate reader through dedicated software or applications on the portable device. This provides a convenient and fast data reception and processing solution for users, greatly expanding the application scope and usage scenarios of the fluorescence microplate reader.

[0011] Specifically, the detection modes of the instrument are two types: endpoint detection and kinetic detection. The endpoint detection mode only detects once at the time set by the user and is suitable for rapid screening and preliminary judgment of sample characteristics. In this mode, the instrument collects data at specific time points and provides immediate analysis results, which is suitable for occasions that do not require continuous monitoring or have low requirements for time resolution. The kinetic detection mode performs multiple detections at the detection duration and detection interval set by the user, and can continuously track and record the fluorescence changes of the sample over a period of time. This mode is suitable for research that requires observing the reaction process, analyzing dynamic changes, or collecting time series data. By setting an appropriate detection interval, users can obtain data with high time resolution, thereby deeply analyzing the dynamic behavior or reaction kinetics of the sample.

[0012] Specifically, the optical path system consists of an excitation light LED, a lens, a filter, a dichroic mirror, and an optical fiber. The light generated by the excitation light LED is filtered by the filter and reflected by the dichroic mirror, and then irradiated onto the sample through the lens and the optical fiber. After the sample is irradiated by the excitation light, the molecules in it absorb light energy and transition from the ground state to the excited state. The molecules in the excited state return to the ground state through radiative transition, and during this process, light of a certain wavelength is emitted. The emitted light of the sample reaches the dichroic mirror through the same optical fiber and lens. The light of this wavelength is transmitted by the dichroic mirror to the lens for convergence, and then filtered by the filter and detected at the photodiode.

[0013] Specifically, the signal acquisition board integrates signal acquisition and signal processing. When the light source excites the sample, emitted light is generated, and the photodiode can convert the emitted light signal of the sample into a current signal. Since this current signal is too weak and contains a lot of noise and is not easy to detect, it needs to pass through a transimpedance amplifier circuit with a high input impedance and low noise characteristics first to convert the current signal into a voltage signal and amplify it. The processed voltage signal then passes through a first-order high-order band-pass filter circuit to reduce the noise in the signal. The voltage signal after noise reduction processing also needs to pass through a first-order differential amplifier circuit, which not only increases the intensity of the signal but also effectively suppresses noise. Finally, after the signal is buffered by a voltage follower, it can be sampled by the acquisition circuit composed of the AD7682 chip and its peripheral circuits.

[0014] Specifically, the signal acquisition board takes electromagnetic compatibility and signal integrity into consideration in its design, and adopts reasonable wiring and shielding technologies to reduce external interference and signal attenuation. In addition, the signal acquisition board also adopts the temperature compensation method to suppress the possible influence brought by temperature drift.

[0015] Specifically, the sensor module includes a DS18B20 temperature sensor and a position sensor. The temperature sensor can monitor the subtle temperature changes inside the instrument cavity in real time and transmit them to the MCU. The MCU automatically adjusts the temperature inside the cavity according to the temperature fed back by the DS18B20 and the PID algorithm. The position sensor is used to align the position of the sample cell. When the sample cell reaches the corresponding position, the position sensor feeds back a high-level signal to the MCU, and then the stepper motor stops rotating.

[0016] Specifically, the drive control board includes light source drive control and stepper motor drive control. The drive control board is connected to the GPIO port of the main control board, and outputs PWM pulse signals through the GPIO port to control the brightness of the light source and the angle and direction of the rotation of the stepper motor, so as to achieve the required effect. The stepper motor is mainly used to drive the sample cell in and out of the chamber and move the sample to a specified position for detection.

[0017] Specifically, the temperature control board consists of a thermoelectric cooler, a fan and corresponding drive circuits. After the MCU receives the temperature fed back by the DS18B20, it can calculate the difference between the current temperature and the user-set temperature. After the calculated temperature difference is processed by an algorithm, the output value is used to control the heating and cooling of the thermoelectric cooler and the operating speed of the fan. When the temperature difference is large, the MCU controls the thermoelectric cooler and the fan to increase the power output; when the temperature difference is small, the power of the thermoelectric cooler and the fan is reduced.

[0018] Specifically, after the thermoelectric cooler is connected to the corresponding drive circuit, it is then connected to the main control board. The main control board controls the thermoelectric cooler through two red and black wires. When the red wire outputs a high level and the black wire outputs a low level, the thermoelectric cooler is in the heating mode; when the red wire outputs a low level and the black wire outputs a high level, the thermoelectric cooler is in the cooling mode.

[0019] Specifically, the signal processing system first processes the collected signals such as amplification, noise reduction, filtering, and voltage stabilization by the hardware circuit to ensure the quality and stability of the signals; then the software system processes the collected voltage values such as selection and calculation of the average value.

[0020] Specifically, the data processing system analyzes and interprets the data processed by the signal processing system by the upper computer software, generates a microbial growth curve, and provides relevant statistical and analysis results.

[0021] In summary, a miniaturized portable fluorescence microplate reader and method for instant fluorescence detection according to the present invention provides a new type of portable, highly sensitive, and easy-to-operate fluorescence microplate reader for the fields of biomedical detection, environmental monitoring, material analysis, etc. by integrating multiple functional modules and adopting advanced control algorithms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. The drawings are only used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0023] Figure 1 is a system block diagram of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0024] Figure 2 is a signal processing flow chart of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0025] Figure 3 is a schematic optical path diagram of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0026] Figure 4 is an internal structure diagram of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0027] Figure 5 is a motor movement and optical fiber alignment diagram of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0028] Figure 6 It is the structural diagram of the excitation light and photoelectric detection module of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0029] Figure 7 It is the sectional view of the excitation light and photoelectric detection module of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0030] Figure 8 It is the software control flow chart of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0031] Figure 9 It is the pin diagram of the main control chip STMF103C8T6 of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0032] Figure 10 It is the transimpedance amplifier circuit diagram in signal acquisition of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0033] Figure 11 It is the voltage follower circuit diagram in signal acquisition of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention.

[0034] Figure 12 It is the data output result diagram of a miniaturized portable fluorescence microplate reader for instant fluorescence detection proposed by the present invention. Specific embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Such as Figure 1As shown in the figure, the upper computer software of the present invention serves as the user interaction interface, which not only provides experiment settings and data collection, but also has the functions of result display and storage. The microcontroller, as the core of the system, is responsible for parsing the instructions from the upper computer and precisely controlling each connected hardware module to ensure the accurate execution of the experimental process. The automatic temperature control module uses a DS18B20 temperature sensor to monitor and adjust the temperature of the experimental environment in real time, providing stable temperature conditions for the microbial reaction. The light source driver controls the brightness of the light source to simulate natural light or perform fluorescence excitation to meet different detection requirements. The motor driver controls the movement of mechanical components, ensuring the entry and exit of the sample tank and the fixed-point measurement of the sample. The infrared photoelectric sensor and the photodiode are respectively used for non-invasive measurement and optical signal conversion, providing quantitative data of microorganisms for the system. In addition, the system also includes a signal processing and data processing module, which performs necessary processing and analysis on the original signals and finally generates easy-to-understand reports and charts.

[0037] As Figure 2 shown, in the signal processing process of the present invention, first, the feedback optical signal is converted into an electrical signal by the photodiode; then, the weak current signal generated by the photodiode is converted into a voltage signal by a transimpedance amplifier circuit (TIA circuit) and preliminarily amplified to ensure that the signal intensity is sufficient for subsequent circuit processing; next, the integrated operational amplifier (OP amplifier circuit) further amplifies and conditions the signal. Through operations such as gain adjustment, noise reduction, filtering, and voltage stabilization, the signal quality is optimized and noise interference is reduced, making the signal clearer and more stable; finally, the microcontroller (MCU) collects the amplified and conditioned analog signal and converts it into a digital signal. The MCU uses built-in algorithms to perform more in-depth analysis and processing on the digital signal to extract key information on the microbial quantity and growth status.

[0038] As Figure 3 shown, the present invention uses a high-performance excitation light LED as the light source of the system to ensure that the system can provide stable and intense enough excitation light, that is, Figure 3 the light sources 1 and 2 in the figure. The excitation light emitted by the light source first passes through the filter 1 to select light of a specific wavelength. The filtered light is then reflected by the dichroic mirror 1 to the lens 2. The lens 2 converges the excitation light to the optical fiber. The optical fiber is used to transmit the excitation light to the sample. The optical fiber can not only effectively transmit light but also flexibly adjust the light path and direction. After the sample is irradiated by the excitation light, emitted light is generated. The emitted light returns to the lens 2 through the optical fiber. The lens 2 transmits the converged light through the dichroic mirror 1. The dichroic mirror has a high reflectivity and high transmittance at a specific wavelength, ensuring the effective separation of the excitation light and the emitted light. The emitted light passes through the output filter 2 and the lens 1 and finally reaches the photodiode.

[0039] As Figure 4As shown in the figure, 1 is a centrifuge tube for loading a sample solution, 2 is an eight-channel tube seat, 3 is a radiator, 4 is an optical fiber, 5 is a temperature sensor, 6 is a main control board, 7 is a semiconductor refrigeration chip, 8 is a main control board fixing bracket, 9 is a shielding sheet, 10 is an optical fiber fixing seat, 11 is an excitation light and photoelectric detection module, 12 is a metal base plate, 13 is a synchronous belt, 14 is a motor, and 15 is a cooling fan. The centrifuge tube 1 is placed in the eight-channel tube seat 2, and the excitation light and emission light of the sample are transmitted out and back through the optical fiber 4 to the excitation light and photoelectric detection module 11. The motor 14 drives the synchronous belt 13, and the synchronous belt drives the excitation light and photoelectric detection module to realize the switching detection of different optical fibers, that is, the switching detection between 8 channels. The semiconductor refrigeration chip heats or cools the eight-channel tube seat to maintain the sample temperature stable. The radiator 3 and the cooling fan 15 dissipate heat from the semiconductor refrigeration chip. The temperature sensor 5 monitors the real-time temperature of the eight-channel tube seat.

[0040] As Figure 5 shown in the figure, 1 is an optical fiber fixing seat, 2 is a driven wheel, 3 is a synchronous belt, 4 is a metal base plate, 5 is an optical fiber, 6 is a motor, 7 is a light shielding sheet, 8 is a home photoelectric switch, 9 is a travel photoelectric switch, 10 is an excitation light and photoelectric detection module, 11 is a wire pressing piece for the cable, and 12 is a cable for the optoelectronic board. After the motor 6 is driven, it drives the excitation light and photoelectric detection module 10 to move horizontally through the synchronous belt 3. When moving, the travel photoelectric switch 9 is triggered at the slit of the light shielding sheet 7, and the optical path inside the excitation light and photoelectric detection module and the optical fiber 5 are aligned and stop moving. At most, two optical paths and two optical fibers can be aligned simultaneously. The excitation light is emitted, and the emission light of the sample is detected. After the detection is completed, it continues to move to the slit of the next light shielding sheet and stops for detection. After a round of detection, the motor drives the excitation light and photoelectric detection module to return to the origin, starts the next round of movement, and detects the fluorescence signal.

[0041] As Figure 6 shown in the figure, 1 is a shielding cover for the optoelectronic detection board, 2 is an optoelectronic detection board, 3 is an optoelectronic fixing seat, 4 is an optoelectronic processing board, 5 is a socket for the optoelectronic processing board, and 6 is a light source board. The optoelectronic processing board controls the light source board to emit a fixed monochromatic light, receives the voltage transmitted back after the optoelectronic detection board converts the emission light into an electrical signal, and processes it. The shielding cover of the optoelectronic detection board covers and is fixed on the optoelectronic detection board.

[0042] As Figure 7As shown in the figure, 1 is the guide rail connection seat, 2 and 19 are the first plano-convex lenses, 3 is the first filter, 4 is the first light source board, 5 is the first light source LED, 6 is the first dichroic mirror, 7 is the lens fixing sleeve, 8 and 15 are the second plano-convex lenses, 9 is the second filter, 10 is the photoelectric detection board, 11 is the shield of the photoelectric detection board, 12 and 13 are photodiodes, 14 is the third filter, 16 is the fourth filter, 17 is the second light source, and 18 is the fourth filter. The guide rail connection seat 1 is connected to the synchronous belt of the motor, so the motor drives the excitation light and the photoelectric detection module. The excitation light of the first LED 5 is filtered into monochromatic light by the filter 3, and then reflected by the dichroic mirror 6 to the lens 2 for focusing. The focused monochromatic light is transmitted to the sample and excites the sample. The light emitted by the sample is transmitted back to the lens 2. The lens 2 converts the point light source of the emitted light into parallel light, penetrates the dichroic mirror 6, and then is focused by the lens 8. After passing through the filter 9, a purer monochromatic emitted light is focused on the photodiode 12. The photoelectric detection board 10 converts the optical signal into an electrical signal, and the shield 11 eliminates signal interference. The left and right halves of the excitation light and the photoelectric detection module are symmetric structures, with the same optical path principle and independent of each other.

[0043] As Figure 8 shown, the software control flow chart of the present invention starts from the user setting the temperature, detection mode, and setting the standard curve. After these preparations are completed, the detection starts. The lower computer sends the detection data to the upper computer and judges whether the sending is successful; if the data sending fails, the data is resent. If the data sending is successful, it judges whether the detection is over; if the detection is over, the whole process will terminate; if there are still remaining detection tasks, the instrument will loop through the detection steps.

[0044] As Figure 9 shown, the main control module of the present invention uses a specific existing chip. We use the STM32F103C8T6 chip with a Cortex-M3 core as the main control chip, and also design a crystal oscillator circuit, a reset circuit, an ST-LINK download circuit, etc. around the chip. The crystal oscillator circuit uses an 8MHZ active crystal oscillator to provide an oscillation signal for the chip. The chip has rich peripheral resources. Using the ADC module of the chip, it can simultaneously collect signals from 2 channels, use DMA to process data, without occupying the CPU, and improve the instruction execution speed; use interrupts and timers to generate PWM waves to control the rotation of the motor to complete the relevant instructions of the upper computer; use the GPIO port to read the data of the sensor, and then realize temperature control.

[0045] As Figure 10As shown, the transimpedance amplifier circuit (TIA circuit) in the signal acquisition circuit of the present invention can convert the weak current signal generated by the photodiode into a voltage signal, and perform processing such as signal amplification and noise reduction. The signal gain, signal bandwidth, and noise gain of the TIA circuit restrict each other. The key parameters affecting the TIA circuit include the junction capacitance of the photodiode, the common-mode input capacitance and differential-mode input capacitance inside the operational amplifier, etc. The gain of the TIA circuit for the signal has the dimension of transresistance, and the resistors R51, R64, and R65 are the transresistance of the TIA circuit, representing the DC gain of this circuit.

[0046] As Figure 11 shown, the voltage follower circuit in the signal acquisition circuit of the present invention does not consume energy and does not change the signal amplitude, and can perfectly transfer the signal to the next stage, greatly reducing the mutual influence between the front and rear stage circuits and playing a role in buffering the signal; the capacitors C45 and C46 below the circuit diagram are filter capacitors, which can filter out the influence of high-frequency and low-frequency components on the power supply on the operational amplifier and protect the chip.

[0047] As Figure 12 shown, it is the experimental result of using this instrument to verify whether the CRISPR-Cas12j protein has cleavage activity. The reaction system contains Target, sgRNA, CRISPR-cas12j, FQ, Buffer, and the total sample volume is 50 uL, and the negative control group has no Target. 12-a is the kinetic curve of the fluorescence intensity change during the positive and negative nucleic acid amplification processes of this reaction system detected by the instrument. The fluorescence signal of the positive sample starts to increase from close to 0 and increases up to 12000 at most, while the fluorescence curve of the negative sample remains at a very low value, not greater than 5. It can be clearly seen that the curve increases in this reaction, and the contrast between negative and positive is obvious. The fluorescence probe of this detection system is fluorescein with an excitation light of 475 nm and an emission light of 525 nm. 12-b is the repeatability test.

[0048] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A miniaturized portable fluorescence microplate reader for instant fluorescence detection, comprising a detection instrument body, characterized in that: The detection instrument comprises an MCU microcontroller, a voltage conversion module, a light source driving module, a motor driving module, a photoelectric detection module, an AD sampling module, an automatic temperature control module based on a PID algorithm, a signal processing system and a data processing system.

2. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The MCU microcontroller is based on STM32F103C8T6 as the main control chip. The chip has a core width of 32 bits and a maximum main frequency of 72MHZ. It has a rich built-in peripheral interface, including but not limited to communication interfaces such as SPI, I2C, USART, and rich GPIO pins, which provide flexible control and communication capabilities for various functional modules of the instrument, can handle complex algorithms and high-speed data transmission, and ensure the efficiency and stability of the instrument operation. In addition, the MCU microcontroller has low power consumption characteristics, which enables the instrument to effectively reduce energy consumption during long-term operation and extend the service life of the equipment. In summary, STM32F103C8T6, as the MCU microcontroller of the detection instrument, not only provides powerful computing and control capabilities, but also has the advantages of low power consumption and high compatibility, providing a solid foundation for realizing an efficient, stable and portable instant fluorescence detection device.

3. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The voltage conversion module has high adaptability and stability. Through precise voltage stabilization circuit design, it can convert the input 24V voltage into a series of stable voltages of different amplitudes, including but not limited to +12V, -12V, +7V, -7V, 5V, 4.2V and 3.3V. This multi-level voltage output design not only ensures that each module inside the microplate reader can obtain the precise voltage they need, but also enhances the compatibility and reliability of the entire system. In order to adapt to different usage environments and power supply conditions, the voltage conversion module also has the characteristics of a wide input voltage range. Even in the case of large power fluctuations, it can maintain a stable output to ensure the normal operation of the microplate reader. This feature makes the microplate reader not only suitable for laboratory environments, but also meets the needs of on-site testing, greatly expanding its application range. In addition, the design of the voltage conversion module also takes electromagnetic compatibility into consideration. Through optimized circuit layout and shielding measures, electromagnetic interference to other modules is reduced, and the microplate reader's resistance to external electromagnetic interference is also improved, providing a strong guarantee for the efficient operation and long-term stability of the microplate reader.

4. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The light source driving module can be controlled by software to flexibly select 470nm blue light and 630nm red light as excitation light sources to meet the detection requirements of different fluorescent markers. The corresponding emission lights are 525nm and 680nm, respectively, ensuring the effective excitation and collection of fluorescent signals. The excitation light is efficiently and stably transmitted to the sample detection area through the optical fiber, and the emission light of the sample is transmitted to the photoelectric detection module for detection. The module also has temperature compensation and current control functions to ensure the stability and reliability of the light source.

5. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The motor drive module is used to receive the control signal of the GPIO port of the microcontroller and output the motor drive current to meet the driving requirements of the stepper motor. This module not only provides a stable motor drive function, but also has precise motor control capabilities to ensure the precise positioning and operation of the sample during the detection process. It also takes into account the compatibility of the motor and has built-in over-current, over-voltage and over-heating protection mechanisms to improve the stability and safety of the system.

6. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The photoelectric detection module, which is composed of two high-performance photodiodes and their corresponding signal processing circuits, can detect samples from two channels at a time and quickly complete a comprehensive analysis of samples from eight channels. The module ensures a high signal-to-noise ratio and a low noise level, has high sensitivity, and can accurately detect changes in tiny light signals in the sample slot, thereby achieving accurate measurement of microbial concentrations.

7. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The AD sampling module is composed of an AD7682 chip and its corresponding circuits. The AD7682 is a 16-bit, 4-channel, 250kSPS ADC chip. The chip is powered by a single power supply and has the characteristics of multi-channel, low power consumption and high resolution, which greatly improves the efficiency and flexibility of data acquisition.

8. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The automatic temperature control module based on the PID algorithm is composed of a semiconductor refrigeration sheet, a heat sink, a fan, a centrifugal tube seat, a temperature sensor, a corresponding drive circuit and a PID software algorithm. It can stably maintain the cavity temperature at a temperature value set by the user. The temperature range is 20°C to 65°C, the step is 1°C, and the temperature accuracy is ±0.2°C. The PID algorithm monitors the cavity temperature in real time and compares it with the target temperature set by the user. The PID algorithm dynamically adjusts the working state of the semiconductor refrigeration sheet to achieve precise control of the temperature. The module can accurately control the temperature of the sample slot and provide stable environmental conditions for microbial reactions.

9. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The signal processing system, which consists of a TIA circuit, an OP amplifier circuit and a signal conditioning circuit, can convert the weak current signal generated by the photodiode into a voltage signal, and amplify, reduce noise, filter and stabilize the signal for ADC sampling. The module ensures the quality and stability of the signal.

10. A miniaturized portable fluorescence microplate reader for instant fluorescence detection according to claim 1, characterized in that: The data processing system analyzes and interprets the data collected by the ADC, presents the change process of sample concentration through kinetic curves, can also detect the concentration of the sample in real time, and provide relevant statistical and analytical results.

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