Data acquisition and analysis implementation method for fluorescence immunodetection

Abstract

The invention provides a data acquisition and analysis implementation method for fluorescence immunodetection, and relates to the technical field of immunodetection, the method can realize comprehensive acquisition and analysis of various signals, including forward scattering signals, lateral scattering signals and fluorescence signals, in fluorescence immunodetection, provides rich data support for accurate detection, and improves the detection accuracy. According to the invention, the detection method is simple, the sample characteristics can be deeply understood, the detection accuracy and reliability are improved, the photodiode and the photomultiplier are used for converting optical signals with different characteristics, and the respective advantages of the photodiode and the photomultiplier, such as the rapid response of the photodiode and the high sensitivity of the photomultiplier to weak light, are fully exerted; the high efficiency and comprehensiveness of signal conversion are ensured, weak current signals are effectively converted into voltage signals through the trans-impedance amplifier and amplified, a better basis is provided for subsequent processing, the quality and identifiability of the signals are ensured, and the risk of signal loss or weakening is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and more specifically, particularly relates to a method for realizing data collection and analysis for fluorescent immunoassay. Background Art

[0002] As an important bioanalytical method, fluorescence immunoassay technology is widely used in medical diagnosis, biological research and other fields. However, traditional fluorescence immunoassay methods have certain limitations in data collection and analysis.

[0003] In previous technologies, the data acquisition system may not be perfect, and the conversion and processing accuracy of light signals is limited, making it difficult to accurately obtain multiple characteristic information of cells or particles, such as forward scatter signals, side scatter signals, and fluorescence signals, etc. This may lead to inaccurate and incomplete classification, screening, and characteristic analysis of targets in samples.

[0004] For the conversion of optical signals to electrical signals, there may be a lack of efficient and sensitive conversion devices, or the conditioning and processing of the converted signals may not be ideal, which affects the quality of subsequent data analysis.

[0005] In terms of signal analysis, there may be a lack of advanced computing processing units, which cannot quickly and accurately calculate and analyze data and cannot meet the increasingly stringent detection requirements.

[0006] At the same time, the data transmission and storage mechanisms may not be optimized enough to guarantee the real-time, stability and traceability of the data.

[0007] At present, the common data acquisition system on the market uses a microcontroller (such as STM32) plus an analog-to-digital converter (ADC) to realize data acquisition and analysis functions. This solution has the advantages of simple operation and low cost, but because the microcontroller needs to perform data acquisition, calculation and transmission functions during operation, it is difficult to achieve the goals of high stability and high sampling rate. Especially when the sample concentration is high or the sample flow rate is fast, the data acquisition and analysis system is difficult to meet the detection requirements.

[0008] With the continuous development of science and technology and the continuous improvement of the requirements for detection accuracy and efficiency, there is an urgent need for a more advanced and complete data acquisition and analysis implementation method that can comprehensively and accurately collect and analyze various signals and improve the reliability and practicality of fluorescent immunoassay. The present invention came into being in this context. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for realizing data collection and analysis for fluorescent immunoassay to solve the above problems.

[0010] A method for data collection and analysis for fluorescent immunoassay comprises the following steps:

[0011] S1: The optical signal is converted into a current signal by PD and PMT through the data acquisition and analysis system;

[0012] S2: Convert the current signal into a voltage signal via a transimpedance amplifier (TIA);

[0013] S3: Signal conditioning through a precision variable gain amplifier (VGA);

[0014] S4: The analog signal is sampled into a digital signal by an analog-to-digital converter (ADC);

[0015] S5: FPGA calculates and processes the sampled digital signal sequence, transfers data through FSMC and STM32 respectively, and the relevant historical data is stored in DDR at the same time.

[0016] Preferably, Ethernet is used between the host computer and STM32 to transfer the sampling data and the analysis results.

[0017] Preferably, the data acquisition and analysis system first configures the FPGA after being powered on.

[0018] Preferably, the FPGA workflow is as follows:

[0019] Step 1: Get started;

[0020] Step 2: Configure FPGA;

[0021] Step 3: Start collection;

[0022] Step 4: Data collection;

[0023] Step 5: Determine whether it is a pulse signal to be tested;

[0024] Step 6: Calculate pulse width, height, and area;

[0025] Step 7: Data is transferred to FI FO and DDR;

[0026] Step 8: STM32 reads data request;

[0027] Step 9: FPGA sends signal data;

[0028] Step 10: End.

[0029] Preferably, the data acquisition and analysis system further comprises an optical component for pre-processing the optical signal.

[0030] Preferably, the transimpedance amplifier (TIA) has a function of adjustable gain.

[0031] Preferably, in S3, the gain of the precision variable gain amplifier (VGA) is dynamically adjusted according to actual needs.

[0032] Preferably, in S4, the analog-to-digital converter (ADC) has a multi-channel sampling function.

[0033] Preferably, the FPGA is also used to perform filtering processing on the digital signal.

[0034] Preferably, DDR uses high-speed memory chips to increase data storage and reading speeds.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. This method can realize the comprehensive collection and analysis of multiple signals in fluorescent immunoassay, including forward scatter signals, side scatter signals and fluorescence signals, providing rich data support for accurate detection, helping to gain a deeper understanding of sample characteristics and improving the accuracy and reliability of detection.

[0037] 2. Photodiodes and photomultiplier tubes are used to convert light signals with different characteristics, giving full play to their respective advantages, such as the fast response of photodiodes and the high sensitivity of photomultiplier tubes to weak light, ensuring the efficiency and comprehensiveness of signal conversion.

[0038] 3. The weak current signal is effectively converted into a voltage signal and amplified through a transimpedance amplifier, which provides a better basis for subsequent processing, ensures the quality and recognizability of the signal, and reduces the risk of signal loss or weakening.

[0039] 4. The use of precision variable gain amplifiers makes signal conditioning more accurate and flexible, and can be adaptively adjusted according to different signal strengths and dynamic ranges, thereby maintaining signal integrity and accuracy and adapting to various complex sample conditions.

[0040] 5. The analog-to-digital converter accurately converts analog signals into digital signals, which is convenient for subsequent digital system processing and analysis, realizes the effective connection between analog and digital fields, and lays the foundation for further use and interpretation of data.

[0041] 6. FPGA undertakes the main data collection and analysis functions. Its powerful computing power and flexibility ensure the timeliness and accuracy of data processing, and can quickly respond to continuous data collection and complex computing tasks.

[0042] 7. FSMC is used to realize efficient data transmission and interaction between FPGA and STM32 in the system, which ensures the rapid flow of data between different components and improves the overall operation efficiency and collaborative working ability of the system.

[0043] 8. Data can be stored in DDR to facilitate subsequent query and analysis, providing data reserves for long-term research and monitoring, and also facilitating comparison and trend analysis of historical data.

[0044] 9. Ethernet is used for data transmission between the host computer and STM32, which ensures that a large amount of sampling data and analysis results can be transmitted at high speed and stably, meets the real-time requirements, and facilitates remote monitoring and operation.

[0045] 10. The overall method is reasonably designed, and the components work together to automatically perform pulse signal related calculations and data storage. It can respond accurately when there is a request to read data, which embodies a high degree of automation and intelligence, reduces manual intervention, and improves work efficiency and detection quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the system principle diagram of the present invention;

[0047] Figure 2 It is a schematic diagram of the process of the present invention;

[0048] Figure 3 It is the FPGA working flow chart of the present invention. DETAILED DESCRIPTION

[0049] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0050] See also Figure 1-Figure 3 The present invention provides a data collection and analysis method for fluorescence immunoassay. In the fluorescence immunoassay system, the forward scattering (FSC) signal, the side scattering (SSC) signal and the fluorescence signal are generated when the sample to be tested flows through the liquid path system. After the signals are processed by the optical system, they are respectively converted into photoelectric signals by the photodiode (PD) and the photomultiplier tube (PMT). Finally, the data collection system collects and analyzes the relevant signals. The system schematic diagram is shown in FIG. Figure 1 As shown;

[0051] The forward scatter (FSC) signal mainly reflects the physical properties of cells and other particles, such as the size. When the laser irradiates the cells or particles, part of the light will scatter forward in the same direction as the laser beam. The signal generated by this part of the scattered light is the forward scatter signal. By analyzing the FSC signal, cells or particles of different sizes can be distinguished, which is helpful for the classification and screening of the target objects in the sample, and is of great significance for accurate fluorescent immunoassay and data analysis. The side scatter (SSC) signal refers to the signal generated by the side scattering of the laser by cells or particles when the laser irradiates the sample. The side scatter signal is mainly related to the internal complexity of the cell or particle (such as particle size, the status of the organelles in the cytoplasm, etc.). It can provide information about the internal structure and morphology of cells or particles. Combined with the forward scatter signal, it can more comprehensively analyze and identify the characteristics of cells or particles, and plays an important role in accurately realizing the data collection and analysis of fluorescent immunoassay. Cells or particles of different types and states usually show a specific combination of forward scattering and side scattering characteristics, which helps to distinguish and identify them. Fluorescence signal refers to the signal generated by a specific wavelength of light emitted by a specific substance (such as labeled biological molecules, cells, etc.) after being irradiated with excitation light. When the target substance in the sample is combined with a fluorescent dye or marker, these markers will emit fluorescence under the action of appropriate excitation light. By detecting and analyzing these fluorescent signals, information about the existence, concentration, distribution, etc. of the target substance can be obtained, thereby realizing the detection, analysis and research of related biological samples. The intensity, wavelength and other characteristics of the fluorescence signal can reflect the specific properties and state of the sample, and are very critical data basis in fluorescent immunoassay;

[0052] A photodiode (PD) converts light signals into current signals. It responds to light and can convert the received light into a current proportional to the light intensity. It has a high response speed and can respond quickly to light signals. It is suitable for detecting rapidly changing light. A photomultiplier tube (PMT) has a very high detection sensitivity to weak light signals and can detect extremely weak light. It can amplify the current generated by photoelectric conversion, thereby enhancing the signal strength for subsequent processing and analysis. Photomultiplier tubes can play an important role, especially in situations where very dim light needs to be detected.

[0053] like Figure 2As shown in the figure, after the PD and PMT convert the optical signal into a current signal, the data acquisition and analysis system first converts the current signal into a voltage signal through a transimpedance amplifier (TIA), then conditions the signal through a precision variable gain amplifier (VGA), and then samples the analog signal into a digital signal through an analog-to-digital converter (ADC). FPGA calculates and processes the sampled digital signal sequence, and transmits data through FSMC and STM32 respectively, and the relevant historical data is stored in DDR at the same time. In order to ensure the data transmission requirements, Ethernet is used between the host computer and STM32 to transmit the sampled data and analysis results;

[0054] A transimpedance amplifier (TIA) is an amplifier that converts current signals into voltage signals. Its function is to convert weak current signals generated by photodiodes (PDs) and other devices into voltage signals for subsequent processing and achieve a certain degree of amplification. A precision variable gain amplifier (VGA) can accurately adjust the gain size. Its function is to adjust the amplitude of the voltage signal to adapt to different signal strength and dynamic range requirements and ensure the quality and accuracy of the signal. An analog-to-digital converter (ADC) converts analog signals into digital signals. Its function is to sample and quantize the conditioned analog voltage signals into digital form so that they can be subsequently processed and analyzed by the digital system. STM32 is a microcontroller that receives, processes and controls data and coordinates the work of various parts of the system. FSMC (Flexible Static MemoryController) is used to connect external memory devices and other devices. Its function is to achieve efficient data transmission and interaction between FPGA and STM32, so that data can be transferred between the two quickly and accurately.

[0055] The main data acquisition and analysis functions of the system are implemented by FPGA. After the system is powered on, the FPGA is first configured. After the configuration is completed, the FPGA waits for STM32 to issue a data acquisition command. After receiving the acquisition command, the FPGA starts continuous data acquisition and analysis. When it is determined that the sampled signal is a pulse signal to be measured, the width, height and area data of the pulse signal are automatically calculated. After the calculation is completed, the relevant information is transmitted to the FIFO and the original data is stored in the DDR. When the STM32 issues a request to read data, the FPGA sends the signal data separately according to the request. Refer to the FPGA workflow diagram Figure 3 shown.

[0056] This method can realize the comprehensive collection and analysis of multiple signals in fluorescent immunoassay, including forward scattering signals, side scattering signals and fluorescence signals, providing rich data support for accurate detection, helping to gain a deeper understanding of sample characteristics, and improving the accuracy and reliability of detection. Photodiodes and photomultiplier tubes are used to convert light signals with different characteristics, respectively, giving full play to their respective advantages, such as the rapid response of photodiodes and the high sensitivity of photomultiplier tubes to weak light, ensuring the efficiency and comprehensiveness of signal conversion. Weak current signals are effectively converted into voltage signals and amplified through transimpedance amplifiers, providing a better foundation for subsequent processing, ensuring signal quality and recognizability, and reducing the risk of signal loss or weakening. The use of precision variable gain amplifiers makes signal conditioning more precise and flexible, and can be adaptively adjusted according to different signal intensities and dynamic ranges, thereby maintaining signal integrity and accuracy and adapting to various complex sample conditions. The analog-to-digital converter accurately converts analog signals into digital signals, facilitating subsequent digital system processing and analysis, and realizing the effective connection between analog and digital fields. It lays the foundation for further utilization and interpretation of data. FPGA undertakes the main data acquisition and analysis functions. Its powerful computing power and flexibility ensure the timeliness and accuracy of data processing, and can quickly respond to continuous data acquisition and complex computing tasks. In the system, FPGA and STM32 realize efficient data transmission and interaction through FSMC, which ensures the rapid circulation of data between different components and improves the overall operation efficiency and collaborative working ability of the system. Data can be stored in DDR to facilitate subsequent query and analysis, providing data reserves for long-term research and monitoring, and is also conducive to the comparison and trend analysis of historical data. Ethernet is used for data transmission between the host computer and STM32, ensuring that a large amount of sampling data and analysis results can be transmitted at high speed and stably, meeting the real-time requirements and facilitating remote monitoring and operation. The overall method is reasonably designed, and each component works together to automatically perform pulse signal related calculations and data storage. It can respond accurately when there is a request to read data, which reflects a high degree of automation and intelligence, reduces manual intervention, and improves work efficiency and detection quality.

[0057] In practical applications, the construction and operation of the fluorescent immunoassay system must strictly follow a series of steps and technical specifications. First, the design and optimization of the liquid system is crucial. High-precision microfluidics technology is used to ensure that the sample to be tested can flow smoothly and evenly through the detection area to reduce sample fluctuations and errors. The pipes and joints in the liquid system are carefully selected and processed to reduce fluid resistance and avoid sample residue and cross contamination.

[0058] In terms of the optical system, high-resolution, low-scattering optical components, such as high-quality lenses and filters, are selected. These components can accurately focus and separate light of different wavelengths to ensure high-quality acquisition of forward scatter (FSC) signals, side scatter (SSC) signals, and fluorescence signals. In order to further improve the performance of the optical system, adaptive optics technology is also used to adjust the parameters of optical components in real time to meet the needs of different samples and detection environments;

[0059] There are also strict requirements for the selection and installation of photodiodes (PD) and photomultiplier tubes (PMT). According to the characteristics and strength of different signals, select the appropriate PD and PMT models. During the installation process, ensure that they are well aligned and coupled with the optical system to maximize the efficiency of receiving optical signals. At the same time, in order to reduce the impact of ambient light and electromagnetic interference on photoelectric conversion, light shielding and electromagnetic shielding measures are adopted to ensure the accuracy and stability of photoelectric conversion.

[0060] In the data acquisition and analysis system, the performance of the transimpedance amplifier (TIA) directly affects the quality of the conversion from current to voltage. The low-noise, high-gain TIA chip is used, combined with a carefully designed circuit layout and shielding measures to reduce noise interference and improve the linearity and accuracy of signal conversion. In addition, in order to adapt to optical signals of different intensities, the gain of the TIA can be dynamically adjusted through software to achieve the best conversion of the signal;

[0061] The design and control of precision variable gain amplifier (VGA) is also one of the key links. High-precision digital potentiometers or analog control circuits are used to achieve VGA gain adjustment. By real-time monitoring of the strength and dynamic range of the input signal, the gain of the VGA is automatically adjusted to ensure that the signal is within the appropriate amplitude range for subsequent processing. At the same time, VGA also has good linearity and bandwidth characteristics to ensure signal fidelity and response speed.

[0062] The selection and parameter setting of analog-to-digital converter (ADC) have a decisive influence on the quality of digital signals. We selected ADC chips with high resolution and high sampling rate, and set parameters such as sampling frequency and quantization bit number reasonably according to actual needs. Before the ADC, we also set up appropriate filtering circuits to remove high-frequency noise and interference and improve the purity of the signal. In order to ensure the accuracy and stability of the ADC, we calibrate and test it regularly to detect and correct possible deviations in time.

[0063] As a microcontroller, STM32 undertakes the core tasks of data reception, processing and control. By optimizing the program code and algorithm of STM32, its data processing capability and response speed are improved. In terms of communication with other components, high-speed and stable interface protocols such as SPI and I2C are adopted to ensure the reliability and real-time performance of data transmission. At the same time, STM32 is also responsible for communicating with the host computer and uploading the test results and analysis data in real time, which is convenient for users to conduct remote monitoring and data analysis.

[0064] The configuration and optimization of FSMC (Flexible Static Memory Control) is also the key to ensure efficient data transmission. According to the actual needs of the system, we reasonably allocate the address space and data bus width of FSMC to improve the efficiency and bandwidth of data transmission. At the same time, by setting appropriate cache and pipeline mechanisms, we can reduce the delay and jitter of data transmission and ensure fast and accurate data interaction between FPGA and STM32.

[0065] In the design and programming of FPGA, we give full play to its powerful parallel processing capabilities and flexibility. By writing efficient logic codes and algorithms, we can achieve rapid response to data acquisition, analysis and processing. For example, in the detection and analysis of pulse signals, high-speed counters and timing modules are used to accurately calculate the width, height and area of ​​pulse signals. At the same time, FPGA also has real-time data compression and encryption functions, reducing the demand for data storage space and transmission bandwidth, and ensuring data security and confidentiality.

[0066] In order to ensure the stability and reliability of the system, we have also taken a series of hardware and software protection measures. In terms of hardware, we have set up circuits such as overvoltage protection, overcurrent protection, and temperature monitoring to prevent damage to the system due to power fluctuations, overloads, or overheating. In terms of software, we have adopted error detection and correction mechanisms, data backup and recovery strategies, etc. to ensure data integrity and recoverability. In addition, we also regularly maintain and upgrade the system, update hardware equipment and software programs to adapt to the ever-changing detection needs and technological developments.

[0067] In the actual detection process, we first calibrate and initialize the system. By using standard samples or calibration materials, we calibrate components such as photodiodes, photomultiplier tubes, transimpedance amplifiers, VGAs, ADCs, etc. to ensure that their performance and parameters meet the detection requirements. At the same time, we initialize the programs of FPGA and STM32 and set relevant parameters and states.

[0068] When conducting actual sample testing, the sample to be tested flows into the detection area through the liquid system, and generates FSC signals, SSC signals and fluorescence signals under the illumination of the optical system. These signals are received by the photodiode and photomultiplier tube and converted into current signals, and then converted into voltage signals through the transimpedance amplifier, and then signal conditioned by the precision variable gain amplifier. The conditioned signal is converted into a digital signal by the analog-to-digital converter and enters the FPGA for data acquisition and analysis.

[0069] In FPGA, the continuously collected data is processed and analyzed in real time according to the set algorithm and logic. When the pulse signal to be tested is detected, the width, height, area and other data of the pulse signal are automatically calculated, and the relevant information is transferred to the FIFO for temporary storage. At the same time, the original data is stored in DDR for subsequent query and analysis.

[0070] When STM32 issues a request to read data, FPGA takes out the corresponding data from FIFO according to the request and transmits it to STM32 through FSMC. STM32 further processes and analyzes the received data to generate test results and reports. At the same time, STM32 uploads the test results to the host computer via Ethernet, and users can view the test results, analysis data and graphical display in real time on the host computer.

[0071] In order to improve the accuracy and reliability of detection, we also use a variety of data processing and analysis technologies. For example, digital filtering technology is used to remove noise and interference in the signal to improve the purity and quality of the signal. Statistical analysis technology is used to analyze and summarize a large amount of test data, extract characteristic parameters and laws, and provide a scientific basis for disease diagnosis and treatment. Machine learning and artificial intelligence technology are used to perform pattern recognition and classification on test data to achieve automatic analysis and diagnosis of complex samples.

[0072] In addition, the system performance was evaluated and optimized. By conducting comparative tests with standard detection methods or equipment, the system performance indicators such as accuracy, repeatability, and linearity were evaluated. Based on the evaluation results, the system design and parameters were optimized and adjusted to continuously improve the system performance and detection level.

[0073] This method for data acquisition and analysis for fluorescent immunoassay achieves comprehensive acquisition and accurate analysis of multiple signals in fluorescent immunoassay through reasonable system design, advanced technical means and strict quality control. This method has a high level of automation and intelligence, can quickly and accurately detect and analyze samples, and provides strong support for disease diagnosis, treatment and research. At the same time, this method also has good scalability and adaptability, and can be flexibly configured and upgraded according to different detection needs and application scenarios, and has broad application prospects and market potential.

[0074] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for data collection and analysis for fluorescent immunoassay, characterized in that: The following steps are involved: S1: The optical signal is converted into a current signal by PD and PMT through the data acquisition and analysis system; S2: Convert the current signal into a voltage signal via a transimpedance amplifier (TIA); S3: Signal conditioning through a precision variable gain amplifier (VGA); S4: The analog signal is sampled into a digital signal by an analog-to-digital converter (ADC); S5: FPGA calculates and processes the sampled digital signal sequence, transfers data through FSMC and STM32 respectively, and the relevant historical data is stored in DDR at the same time.

2. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: Ethernet is used between the host computer and STM32 to transmit sampling data and analysis results.

3. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: After the data acquisition and analysis system is powered on, the FPGA is configured first.

4. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 3, characterized in that: The FPGA workflow is as follows: Step 1: Get started; Step 2: Configure FPGA; Step 3: Start collection; Step 4: Data collection; Step 5: Determine whether it is a pulse signal to be tested; Step 6: Calculate pulse width, height, and area; Step 7: Data is transferred to FIFO and DDR; Step 8: STM32 reads data request; Step 9: FPGA sends signal data; Step 10: End.

5. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: The data acquisition and analysis system also includes an optical component for pre-processing the optical signal.

6. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: The transimpedance amplifier (TIA) has an adjustable gain function.

7. A method for implementing data collection and analysis for fluorescent immunoassay as claimed in claim 6, characterized in that: In S3, the gain of the precision variable gain amplifier (VGA) is dynamically adjusted according to actual needs.

8. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: In S4, the analog-to-digital converter (ADC) has a multi-channel sampling function.

9. A method for implementing data collection and analysis for fluorescent immunoassay as claimed in claim 4, characterized in that: FPGA is also used to filter digital signals.

10. A method for data collection and analysis for fluorescent immunoassay as claimed in claim 1, characterized in that: DDR uses high-speed memory chips to increase data storage and reading speeds.

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