Detection system and detection method based on fluorescence immunity POCT (Point of Care Testing)

Abstract

The invention relates to the technical field of fluorescence immunity, and provides a detection system and a detection method based on fluorescence immunity POCT, the detection system comprises a fluorescence detection light path module, an embedded control system and a program design and detection method, and the detection method comprises the following steps: S1, inputting information; s2, detecting: after information input is completed, selecting an instruction of starting detection, and automatically executing a detection process by a system; s3, receiving the fluorescence signal voltage, and converting the fluorescence signal into an electric signal to the greatest extent by adopting a double-side oblique light path design, thereby reducing the interference of ambient light noise on the system, and ensuring the stability of excitation efficiency and transmission efficiency at the same time; the optical detection table remarkably improves the collection efficiency of fluorescence signals of the sample test paper card, so that the equipment can more accurately capture and analyze the fluorescence signals of the sample, the detection accuracy and reliability are improved, the optical detection table has the advantages of portability, usability and the like, the biological sample reagent adding method is simple, the equipment sealing is good, and the detection cost is low. And safe and reliable operation is ensured, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence immunoassay technology, and in particular to a fluorescence immunoassay POCT detection system and a detection method. Background Art

[0002] In China, the incidence and mortality of malignant tumors are increasing year by year, and the medical expenses caused by them exceed 220 billion yuan each year. In order to improve the efficiency of cancer screening and reduce the mortality rate as much as possible, it is crucial to develop advanced detection technologies in clinical practice.

[0003] Traditional laboratory testing methods require expensive and complex equipment, cumbersome procedures and long testing cycles, which cannot meet the urgent care needs of critically ill patients, and their monitoring efficiency and accuracy are low.

[0004] Therefore, in response to the above problems, a fluorescence immunoassay POCT detection system and detection method are proposed to solve the above problems. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention develops a fluorescence immunoassay POCT detection system and a detection method, which has high sensitivity and accuracy and is simple, convenient, rapid and cost-effective.

[0006] The technical solution for solving the technical problem of the present invention is as follows: the present invention provides a fluorescence immunoassay POCT detection system, including a fluorescence detection optical path module, an embedded control system and a host computer. The fluorescence detection optical path module is used to collect fluorescence signals, and the fluorescence signals are converted into voltage signals by the embedded control system. The voltage signals are processed by an algorithm by the host computer to complete the qualitative and quantitative analysis of the sample solution.

[0007] As an optimization, the fluorescence detection optical path module includes a double-sided oblique entry optical path, which includes two excitation light sources, two bandpass filters, an InGaAs photodetector and a high-pass filter; the light-receiving surface of the InGaAs photodetector is parallel to the front of the test paper card, and the excitation light source is irradiated at an angle of 45° to the plane where the front of the test paper card is located and is perpendicular to the detection line; the bandpass filter is perpendicular to the excitation light source, and the high-pass filter is parallel to the light-receiving surface of the InGaAs photodetector.

[0008] As an optimization, the distance H between the InGaAs photodetector and the test paper card is set to 2.45 mm.

[0009] As an optimization, the embedded control system includes a main controller and a serial communication module, a power module, a display module, a program download and A / D conversion connected to the main controller. The main controller is connected to the host computer through serial communication; the main controller transmits signals to the light source driver, and the light source driver transmits signals to the fluorescence detection optical path module. The collected fluorescence signal is received by the photoelectric detector and converted into an electrical signal. After signal processing, it is converted into a digital signal by A / D, and uploaded to the display screen and the host computer after data processing in the program design.

[0010] A detection method based on the fluorescent immunoassay POCT detection system applied to any of the above, comprising the following steps: S1: Start; S2: Power-on system self-test; S3: Read instrument parameters; S4: Determine whether the system is normal; S5: insert the sample; S6: Sample testing: After completing the information input, select the "Start Testing" command, and the system will automatically execute the testing process; S7: signal acquisition; S8: A / D sampling; S9: data processing; S10: Upload to the display screen and host computer.

[0011] As an optimization, in S5, information is input through the host computer, and the input information includes the patient's name, ID, sample batch, number and sample type.

[0012] As an optimization, in S7, when outdoors or when it is not suitable to use the host computer, the detection program is started by directly inserting the test paper card. After the test paper card is inserted to start the detection program, the light source LD1 is activated.

[0013] As an optimization, the laser beam emitted by LD1 irradiates the T line position on the test paper card, and then the detector captures the fluorescent signal ST emitted on the T line. The system continues for a fixed time t1, and the LD1 light source is turned off to complete a fluorescent signal acquisition process; after LD2 is activated and waits for t0 time, the detector begins to capture the fluorescent signal SC emitted by the C line on the test paper card, and then the system enters a fixed time t1 again.

[0014] As an optimization, in S9, when the number of sampled data reaches the set number, it stops working, receives the collected fluorescence signal voltage through the signal acquisition circuit, obtains a set of data points by collecting the fluorescence signal voltage S (x), and uses the curve fitting method to fit the baseline of the fluorescence data curve to obtain the fitting polynomial P (x), which is expressed as follows: , in, are the fitting parameters, is the order of the polynomial, is a point on the baseline; subtract the fitted baseline data from the fluorescence signal voltage S(x) to obtain the net fluorescence signal F(x) = S(x) - P(x); all points greater than zero in the net fluorescence signal F(x) constitute the peak area of ​​the T line and the C line, and all points greater than zero in the peak area are accumulated to obtain the area value A of the T line and the C line T and A C , the formula is as follows: , , in, represents the peak area of ​​the T line, Represents the peak area of ​​the C line; use MATLAB peak finding function to identify the peak and determine the maximum value of the T line and C line, that is, the peak value; perform qualitative and quantitative analysis of the sample solution based on the peak value and area value.

[0015] The effects provided in the content of the invention are only the effects of the embodiments, rather than all the effects of the invention. The above technical solution has the following advantages or beneficial effects: 1. The present invention adopts a double-sided oblique optical path design, aiming to convert the fluorescence signal into an electrical signal to the greatest extent, thereby reducing the interference of ambient light noise on the system. At the same time, the device ensures the stability of excitation efficiency and transmission efficiency; 2. In terms of optical path packaging, the present invention designs an efficient fluorescence detection optical path module, which significantly improves the collection efficiency of the fluorescence signal of the sample test paper card, so that the equipment can more accurately capture and analyze the fluorescence signal of the sample, thereby improving the accuracy and reliability of the detection.

[0016] 3. The present invention has the advantages of portability and ease of use, the method of adding biological sample reagents is simple, the equipment is well sealed, and safe and reliable operation is ensured, and it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0018] Figure 1 It is a design block diagram of the overall system of the present invention; Figure 2 It is a schematic diagram of the double-side oblique entry optical path in the system of the present invention; Figure 3 Modeling and design of the optical path support platform in the system of the present invention; Figure 4 This is the main program flow chart of the system of the present invention; Figure 5 Design interface for host computer program of the system of the present invention; Figure 6 This is the linear fitting of the detection concentration of carcinoembryonic antigen (CEA) by the system of the present invention. DETAILED DESCRIPTION

[0019] In order to clearly illustrate the technical features of the present solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings. The disclosure below provides many different embodiments or examples for realizing different structures of the present invention. In order to simplify the disclosure of the present invention, the components and settings of specific examples are described below. In addition, the present invention may repeat reference numbers and / or letters in different examples. This repetition is for the purpose of simplification and clarity, and does not itself indicate the relationship between the various embodiments and / or settings discussed. It should be noted that the components illustrated in the accompanying drawings are not necessarily drawn to scale. The present invention omits the description of known components and processing techniques and processes to avoid unnecessary limitations on the present invention.

[0020] Example 1 In this embodiment, Figure 1 As shown, the fluorescence immunoassay POCT detection system includes a fluorescence detection optical path module, an embedded control system and a host computer. The fluorescence detection optical path module is used to collect fluorescence signals. The fluorescence signals are processed by the embedded control system and converted into voltage signals. The voltage signals are processed by the host computer through algorithms to complete qualitative and quantitative analysis of the sample solution.

[0021] Complete the PCB design according to the schematic diagram and realize the production of the embedded control system mainboard.

[0022] As one of the important components of the immunofluorescence quantitative detection system, the core of the fluorescence detection optical path module is to convert the fluorescence signal into an electrical signal to the greatest extent. It is necessary not only to minimize the interference of ambient light noise on the system as much as possible, but also to ensure the excitation efficiency and transmission efficiency. Therefore, the fluorescence detection optical path module includes a double-sided oblique entry optical path, the structure of which is as follows: Figure 2As shown in the figure, the double-sided oblique optical path consists of two excitation light sources (Laser diode1 / 2), two bandpass filters (Bandpass optical filter), an InGaAs photoelectric sensor and a high pass filter (High pass Optical filter). The excitation light source is 808nm, the light-receiving surface of the InGaAs photoelectric sensor is parallel to the front of the test paper card, the excitation light source is irradiated at a 45° angle to the plane where the front of the test paper card is located and is perpendicular to the detection line; the bandpass filter is perpendicular to the excitation light source, and the high pass filter is parallel to the light-receiving surface of the InGaAs photoelectric sensor. The distance H between the InGaAs photoelectric sensor and the test paper card is 2.45 mm.

[0023] The working process is as follows: In the first step, the 808 nm excitation light emitted by LD1 is vertically irradiated to the bandpass filter to first filter the excitation light, and then the excitation light irradiates the T line. After the fluorescent probe captured on the T line is excited by the excitation light, it will emit fluorescence greater than 1000 nm. These fluorescences will diverge upward. After the fluorescence is filtered by the high-pass filter, only the fluorescence with a wavelength greater than 1000 nm can pass, and other stray light is filtered out. Finally, the fluorescence signal captured by the photodetector is detected and recorded. In the second step, LD1 is turned off and LD2 is started, which is exactly the same as the one-step process, exciting the C line, capturing fluorescence, and then detecting and recording. Through the above steps, the double-sided oblique optical path can effectively capture the fluorescence signal and provide high-precision detection results after analysis by the detection system.

[0024] The dark current of the fluorescence detection optical module is only 1 nA at the rated voltage, which greatly reduces the impact of background noise on the detection results. Its response range is wide, from 800 nm to 1700 nm, which can cover the fluorescence signal range of various objects to be tested, and the responsivity in the range of 1000-1600 nm is more than 0.8. Its excellent performance fully meets the design requirements and can reliably capture and convert fluorescence signals.

[0025] The embedded control system is responsible for integrating and scheduling various embedded control systems in the system. After the InGaAs detector collects the fluorescence signal and converts it into a voltage signal, it first passes through the LM358D voltage follower and then sends it to the A / D. The voltage follower has a high input impedance and a very low output impedance. The embedded control system includes a main controller and a serial communication module, a power module, a display module, a program download and an A / D conversion connected to the main controller. The main controller is connected to the host computer through serial communication; the main controller transmits the signal to the light source driver, and the light source driver transmits the signal to the fluorescence detection optical path module. The collected fluorescence signal is received by the photoelectric detector and converted into an electrical signal. After signal processing, it is converted into a digital signal by the A / D, and uploaded to the display screen and the host computer after data processing in the program design.

[0026] A detection method based on the fluorescent immunoassay POCT detection system applied to any of the above, comprising the following steps: S1: Start; S2: Power-on system self-test; S3: Read instrument parameters; S4: Determine whether the system is normal S5: insert the sample; S6: Sample testing: After completing the information input, select the "Start Testing" command, and the system will automatically execute the testing process; S7: signal acquisition; S8: A / D sampling; S9: data processing; S10: Upload to the display screen and host computer.

[0027] In S5, information is input through the host computer, and the input information includes the patient's name, ID, sample batch, number and sample type.

[0028] In S7, when it is outdoors or not suitable to use the host computer, the detection program is started by directly inserting the test paper card. After the test paper card is inserted to start the detection program, the light source LD1 is activated.

[0029] The laser beam emitted by LD1 irradiates the T line position on the test paper card, and then the detector captures the fluorescent signal ST emitted on the T line. The system continues for a fixed time t1, and the LD1 light source is turned off to complete a fluorescent signal acquisition process; after LD2 is activated and waits for t0 time, the detector begins to capture the fluorescent signal SC emitted by the C line on the test paper card, and then the system enters a fixed time t1 again.

[0030] In S9, when the number of sampled data reaches the set number, the operation stops, and the collected fluorescence signal voltage is received through the signal acquisition circuit, and the baseline of the fluorescence data curve is fitted by curve fitting to obtain a fitting polynomial that meets the requirements. The maximum value of the obtained data is the peak value of the T line and the C line by subtracting the fitted limit data from the original data. All points greater than zero in the peak area are accumulated to obtain the area value of the T line and the C line. The MATLAB peak finding function is used to identify the peak, calculate the area of ​​the T line and the C line, and complete the qualitative and quantitative analysis of the sample solution.

[0031] Example 2 In this embodiment, first, key information such as the patient's name, ID, sample batch, number, and sample type are input through the host computer to ensure accurate matching with the patient information to be tested. After completing the information input, select the "Start Testing" command, and the system will automatically execute the testing process. Outdoors or in other environments where it is not suitable to use a host computer, the test program can be started by directly inserting the test paper card. The overall software structure design of the system is shown in detail in Figure 4 In the test, after the test card is inserted to start the detection process, the light source LD1 is immediately activated. At this time, considering that the light source needs to go through a preheating period when it is initially started, the system waits for a duration of t0 to ensure that the light source is preheated. The laser beam emitted by LD1 directly irradiates the T line position on the test card, and then the detector immediately captures the fluorescence signal ST emitted on the T line. In order to ensure that the intrinsic light of the fluorescent nanoprobe used in the detection can be completely attenuated during data acquisition, the system will continue for a fixed time t1, after which the LD1 light source is turned off to complete a fluorescence signal acquisition process. Then LD2 is activated and waits for t0 time, and the detector begins to capture the fluorescence signal SC emitted by the C line on the test card. After this step, the system enters a fixed waiting cycle again, that is, t1 duration. When the number of sampled data reaches the specified number, it stops working, receives the collected fluorescence signal voltage through the signal acquisition circuit, and then performs subsequent algorithm processing.

[0032] Based on the detection of the fluorescence lifetime of the NIR-II fluorescent nanoprobe, it is known that it is about 20 μs, so the t1 duration is set to 40 μs to ensure that the fluorescence signal is completely quenched. After the fluorescence signal collected by the detector is calculated and fitted, the test results will be intuitively displayed on the touch screen and the host computer interface. The host computer software interface is displayed on Figure 5 middle.

[0033] NIR-II fluorescent nanoprobes doped with rare earth nanoparticles coupled with carcinoembryonic antigen (CEA) were used as experimental reagents. The performance of the instrument was tested in four aspects: accuracy test, linear fitting, repeatability test and error analysis. The experiment configured 10 groups of 10 standard solutions with different concentrations (0.1, 1, 10, 20, 50, 90, 150, 220 and 300 ng / ml) as samples. According to the experimental data and the detected fluorescence signal intensity, the fitting curve obtained by linear fitting using the least squares method is shown in the figure below. Figure 5 As shown. The fitting curve expresses the linear relationship between the sample concentration and the ratio of the signal intensity of the T line to the C line. The vertical axis represents the sample concentration, and the horizontal axis represents the ratio of the signal intensity of the T line to the C line. After fitting, the linear relationship between the two is expressed as follows: Through the analysis of experimental data, it was found that there was a good linear relationship between sample concentration and the ratio of T-line to C-line fluorescence signal intensity in the range of 1-300 ng / ml. The goodness of fit R2 was 0.995, which means that the fitting curve can explain about 99.5% of the variance in sample concentration changes and has a high fitting accuracy. In order to conduct systematic repeatability analysis, the following measurement conditions need to be met: (1) Use samples of the same concentration for repeated measurements. (2) Ensure that the steps, sequence and conditions of each measurement are the same. (3) Use measuring equipment of the same model and specifications. (4) All measurement operations are performed by the same person. (5) Measurements are performed under the same laboratory environment. To ensure the credibility of the repeatability test, samples with different concentration levels can be configured for repeated testing.

[0034] In this section, three groups of samples with concentrations of 1, 10, and 50 ng / mL were used as test samples, and each group of samples was measured 10 times within 12 hours. The experimental results are presented in Figure 6 In the figure, the three colors represent samples of different concentrations. In general, the standard deviation S and the coefficient of variation CV are used to measure the degree of dispersion of the data. The calculation formulas for the two are as follows: According to the experimental data, the standard deviation and coefficient of variation of the sample concentrations of the three sets of repeatability tests are shown in Table 1. As the test concentration increases, the coefficient of variation becomes smaller, indicating that the test results of the system are more stable. And the overall coefficient of variation is less than 3%, which meets the design requirements of the system.

[0035] Table 1 Detection values, standard deviations and coefficients of variation of sample solutions with different concentrations.

[0036] In Table 1, CEA is the actual value; Detected is the detected value; SD is the standard deviation; CV is the coefficient of variation.

[0037] Although the above describes the specific implementation mode of the invention in conjunction with the drawings, it is not intended to limit the scope of protection of the invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. Based on the fluorescence immunoassay POCT detection system, its characteristics are: It includes a fluorescence detection optical path module, an embedded control system and a host computer. The fluorescence detection optical path module is used to collect fluorescence signals. The fluorescence signals are processed by the embedded control system and converted into voltage signals. The voltage signals are processed by algorithms in the host computer to complete qualitative and quantitative analysis of the sample solution.

2. The fluorescence immunoassay POCT detection system according to claim 1 is characterized in that: The detection optical path module includes a double-sided oblique optical path, which includes two excitation light sources, two bandpass filters, an InGaAs photodetector and a high-pass filter; the light-receiving surface of the InGaAs photodetector is parallel to the front of the test paper card, and the excitation light source is irradiated at an angle of 45° to the plane where the front of the test paper card is located and is perpendicular to the detection line; the bandpass filter is perpendicular to the excitation light source, and the high-pass filter is parallel to the light-receiving surface of the InGaAs photodetector.

3. The fluorescence immunoassay POCT detection system according to claim 1 is characterized in that: The distance H between the InGaAs photodetector and the test paper card is 2.45 mm.

4. The fluorescence immunoassay POCT detection system according to claim 1 is characterized in that: The embedded control system includes a main controller and a serial communication module, a power module, a display module, a program download and A / D conversion connected to the main controller. The main controller is connected to a host computer through serial communication; the main controller transmits signals to a light source driver, and the light source driver transmits signals to a fluorescence detection optical path module. The collected fluorescence signals are received by a photoelectric detector and converted into electrical signals. After signal processing, they are converted into digital signals by A / D, and are uploaded to the display module and the host computer after data processing by the program in the program download.

5. A detection method based on the fluorescent immunoassay POCT detection system according to any one of claims 1 to 4, characterized in that: Includes the following step, S1: Start; S2: Power-on system self-test; S3: Read instrument parameters; S4: Determine whether the system is normal; S5: insert the sample; S6: Sample testing: After completing the information input, select the "Start Testing" command, and the system will automatically execute the testing process; S7: signal acquisition; S8: A / D sampling; S9: data processing; S10: Upload to the display screen and host computer.

6. The detection method according to claim 5, applied to the fluorescent immunoassay POCT detection system according to any one of claims 1 to 4, characterized in that: In S5, information is input through the host computer, and the input information includes the patient's name, ID, sample batch, number and sample type.

7. The detection method according to claim 6, applied to the fluorescent immunoassay POCT detection system according to any one of claims 1 to 4, characterized in that: In S7, when it is outdoors or not suitable to use the host computer, the detection program is started by directly inserting the test paper card. After the test paper card is inserted to start the detection program, the light source LD1 is activated.

8. The detection method according to claim 7, applied to the fluorescent immunoassay POCT detection system according to any one of claims 1 to 4, characterized in that: The laser beam emitted by LD1 irradiates the T line position on the test paper card, and then the detector captures the fluorescent signal ST emitted on the T line. The system continues for a fixed time t1, and the LD1 light source is turned off to complete a fluorescent signal acquisition process; after LD2 is activated and waits for t0 time, the detector begins to capture the fluorescent signal SC emitted by the C line on the test paper card, and then the system enters a fixed time t1 again.

9. The detection method according to claim 8, applied to the fluorescent immunoassay POCT detection system according to any one of claims 1 to 4, characterized in that: In S9, when the number of sampled data reaches the set number, the operation stops, and the collected fluorescence signal voltage is received through the signal acquisition circuit. By collecting the fluorescence signal voltage S (x), a set of data points is obtained, and the baseline of the fluorescence data curve is fitted using the curve fitting method to obtain the fitting polynomial P (x), which is expressed as follows: , in, are the fitting parameters, is the order of the polynomial, is a point on the baseline; subtract the fitted baseline data from the fluorescence signal voltage S(x) to obtain the net fluorescence signal F(x) = S(x) - P(x); all points greater than zero in the net fluorescence signal F(x) constitute the peak area of ​​the T line and the C line, and all points greater than zero in the peak area are accumulated to obtain the area value A of the T line and the C line T and A C , the formula is as follows: ， ， in, represents the peak area of ​​the T line, Represents the peak area of ​​the C line; use MATLAB peak finding function to identify the peak and determine the maximum value of the T line and C line, that is, the peak value; perform qualitative and quantitative analysis of the sample solution based on the peak value and area value.

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