A ccd-type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system

A technology of immunochromatography and quantitative analysis, which is applied in the direction of material analysis, material analysis through optical means, and measuring devices, etc. It can solve problems such as inability to quantitatively analyze, facilitate function upgrades and expansions, solve subjective deviations, and ensure stability Effect

Inactive Publication Date: 2011-12-14
杭州顿力医疗器械有限公司
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AI-Extracted Technical Summary

Problems solved by technology

Every time the machine is turned on, it is necessary to manually operate the standard color plate to calibrate the system; in addition, the photodiode is used as...
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Abstract

The invention relates to a CCD-type quantitative analysis system of colloidal gold immunity chromatography diagnosis test papers and an analysis method. According to the invention, a CCD digital image technology is used for carrying out instant shooting, processing and reading upon tested targets. The system comprises an image collecting device, a data processing device, and a result outputting device. Bilateral handshaking is respectively carried out between a motherboard handshaking terminal and image processing and reading software, and between a motherboard handshaking terminal and an industrial camera in the image collecting device. The system and the method are advantaged in that: an illumination light source with a patent technology, a self calibrate algorithm, and an image noise reduction algorithm are adopted, such that the stability, consistency and accuracy of reading results can be effectively ensured, and subjective deviation caused by human eye interpretation can be solved; an integral industrial control computer application architecture, with which the invention applied for an independent intellectual property right, is integrated in the instrument system; the parts such as modularized LED illumination, CCD camera, data processing system, can be selected according to user requirements of different performances and prices; according to the invention, parts of the software are developed on a mainstream Windows platform, such that subsequent function upgrading and expanding can be promoted.

Application Domain

Technology Topic

Ccd cameraCalibration algorithm +20

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  • A ccd-type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system
  • A ccd-type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system
  • A ccd-type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system

Examples

  • Experimental program(2)

Example Embodiment

[0035] Example 1: Refer to the attached Figure 1-11. A CCD type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system, which is composed of an image collector, a data processor, and a result output; the signal exchange end of the main board in the data processor is respectively connected with the image processing and reading software and the image collector The industrial camera in the two-way data exchange. The image collector is composed of a target tray, an LED lighting room, an optical plane mirror, and an industrial camera. The receiving surface and the reflecting surface of the optical plane mirror are distributed at 45 degrees and the LED lighting room located below the optical plane mirror illuminates the measured target The above light is reflected vertically to the 45-degree distributed optical plane reflecting mirror, and the measured target image is reflected to the industrial camera lens through the 45-degree distributed optical plane reflecting mirror. The low-cost and high-reliability 12-channel white LED ring illumination light source, using ring-shaped 12-channel independently adjustable white LEDs, and a white diffuse reflection coating with a reflection coefficient of more than 95% in the visible light band, realizes the detection of the target More uniform and stable lighting. Thereby improving the stability and consistency of the system. The software part of the image processing and reading, running under the Windows system, the execution process is as follows Figure 4 Shown. By placing the standard color card in the viewing area of ​​the CCD camera, the software system can analyze the standard color card area in the obtained digital image to obtain image brightness data; by comparing the preset brightness reference value with the actual brightness data, You can know the current brightness deviation of the instrument; the software system controls the electronic shutter speed, adjusts the exposure time, and can change the brightness level of the digital image. According to the current brightness deviation, increase or decrease the exposure time appropriately to limit the brightness deviation of the instrument to a small interval, so as to achieve the stability of the instrument reading. The red, green, and blue (R, G, B) channels of the colloidal gold immunochromatographic diagnostic test paper test line area have different brightness attenuation amplitudes. Since the G channel changes the most, we can set R, G, B three The weight of the channel brightness data is: 0, 1, 0. Only use G channel brightness data for analysis and processing, thereby improving the detection sensitivity of the instrument system for this type of test strip. The structure of the colloidal gold immunochromatographic diagnostic test paper mainly includes a sample pad, a binding pad, an analysis membrane, a test wire, a material control wire, and a water-absorbing pad. Among them, the gold nano particles are combined with biologically active molecules and fixed on the binding pad. Twelve white LEDs are placed in a circular transparent base. The inner and outer surfaces of the base are evenly covered with diffuse reflection coating. The inner side is thinner and the outer layer is thicker; the tested target-the test line and quality control of the colloidal gold immunochromatographic diagnostic test paper The line is placed under the center of the illumination source; after the light emitted by the white LED is reflected by the diffuse reflection coating on the inner and outer sides of the transparent base for multiple times, it will form a relatively uniform circular illumination area in the base to achieve the detection of the target Even lighting.
[0036] The "CCD type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system" has the advantages of fast, simple, quantitative and accurate, and can be widely used in major hospitals and human health inspection institutions for early pregnancy, influenza, drugs and other different categories or different manufacturers. Test strips to determine the results. Utilizing the advantages of high measurement accuracy, stability and consistency of this system, it can also be used in disease monitoring institutions, medical laboratories, etc. figure 2 Schematic diagram of the composition of the instrument.
[0037] The light is emitted from the LED lighting chamber and irradiated on the test strip after the reaction. After diffuse reflection on the surface of the test strip, the light is reflected by the optical flat mirror to the industrial camera. The industrial camera shoots and generates digital images, which are then sent to computer software for analysis and processing. Finally, the reading values ​​of the test line and the quality control line on the colloidal gold immunochromatographic diagnostic test paper are obtained.
[0038] On the test strip test line and quality control line, due to the light absorption characteristics of the gold nanoparticles, the scattered light signal at the test line (T line) and quality control line (C line) will be smaller than that of other areas of the test strip signal. After analyzing the brightness of the real-time image, there should be two low value areas on the brightness distribution curve corresponding to the test line and the quality control line. In principle, the presence and concentration of the target object can be judged based on the brightness values ​​of these two areas.
[0039] The image processing and reading software of this instrument first calculates the brightness value T_1 of the test line and the brightness value C_1 of the quality control line according to the collected test strip image data; at the same time, according to the area outside the test line without colloidal gold particles (background ) The background brightness value R_1 of the scattered light on the test line obtained by the brightness value; considering the visual characteristics of human eyes, we use the difference between the background brightness value and the brightness value of the test line and the quality control line: R_1-T_1, R_1- C_1, to indicate the strength of the test line and quality control line on the test strip. When there are more nano-gold particles fixed on the test line, the human eye will judge that the test line is stronger, and the reading obtained by the instrument system will be larger; on the contrary, when there are fewer nano-gold particles fixed on the test line, the human The eye interpretation is that the test line is weak, and the readings obtained by the instrument system will be smaller.
[0040] By testing a series of standard samples with known concentrations, the corresponding relationship between the readings of the instrument system and the concentration of the standard samples can be obtained. The process of establishing correspondence is called calibration. Different types of colloidal gold immunochromatographic diagnostic test papers produced by different manufacturers have different consistency, stability and distinguishing ability for samples of different concentrations. Therefore, the calibration result is related to the type of test strip. The image processing and reading software design of this instrument allows users to set the calibration results as working parameters for actual reading. And allows users to set multiple sets of working parameters at the same time, corresponding to multiple types of test strips, to facilitate the selection during actual work.
[0041] The "CCD type colloidal gold immunochromatographic diagnostic test paper quantitative analysis system" is composed of hardware and software. The software part of the image processing and reading, running under the Windows system, the execution process is as follows Figure 4 Shown. Taking into account the requirements of the application environment of the instrument, a light source with stable illuminance, low heat generation and long-term continuous operation is required. Light-emitting diodes have become the first choice because of their small size, low power consumption, high luminous efficiency, and easy control of brightness. In the project instrument, this application uses Nichia Chemical (NICHIA) high-quality white LED. In order to make the light emitted by the LEDs be more evenly distributed on the detection target, the application adopts a combination of multiple LEDs, a circular distribution and diffuse reflection paint; to ensure stable LED light emission, a dedicated LED power supply circuit is designed. Taking into account the performance requirements and versatility requirements of this system, this application adopts a targeted design for image processing.
[0042] Noise reduction: Due to the working characteristics of electronic equipment, the digital image of the detected target has a certain amount of noise. Through a large number of experiments, this application analyzes the regular characteristics of noise distribution and designs a software noise reduction algorithm. Effectively improve the image quality taken by the CCD camera and ensure the accuracy of the reading results;
[0043] Such as Image 6 As shown, the brightness attenuation amplitudes of the red, green, and blue (R, G, B) channels in the test line area of ​​the colloidal gold immunochromatographic diagnostic test paper are different. Since the G channel changes the most, we can set the weights of the R, G, and B three-channel brightness data as: 0, 1, 0. Only use G channel brightness data for analysis and processing, thereby improving the detection sensitivity of the instrument system for this type of test strip.
[0044] Figure 7 The following describes the process plan measures from the production process, key processes and assurance measures, and process improvement plans.
[0045] Among them, the assembly of optical components is the focus. The assembly process of this component is ( Figure 8 ):
[0046] Innovation point 1: High-precision real-time brightness, contrast, and white balance self-calibration. Make full use of the powerful data processing capabilities of the computer system. By using standard color cards, real-time image detection, automatic feedback control of camera brightness, contrast, and white balance parameters, it eliminates the need for electronic components for image acquisition related parts of the instrument system of this project when working for a long time. The signal drift caused by the working characteristics improves the accuracy and stability of the system.
[0047] The reading result of this instrument system is based on the digital image obtained by the image acquisition component. Therefore, the instability of the image acquisition related components during operation will cause a certain difference in the digital image obtained, thereby affecting the final reading. After a lot of experiments, it is found that the following two aspects affect the measurement accuracy and stability of the instrument system:
[0048] (1) The stability of the lighting source. As electronic devices such as LEDs and LED power supply circuits work for a long time, working parameters drift. For example, the luminous efficiency of the LED, the current and voltage of the power supply circuit, etc.; in addition, the white paint used in the diffuse reflection coating of the lighting room will also experience aging and discoloration as time increases. These factors will lead to unstable lighting conditions during measurement, manifested as abnormal brightness, color cast, etc.;
[0049] (2) Stability of CCD camera. Since the working principle of a CCD camera is to convert light signals into electrical signals through optoelectronic devices, when the instrument system works for a long time, the working indicators of the electronic devices will also change to a certain extent. Eventually, it will show the instability of the output digital image brightness, contrast, R, G, B three-channel color. The price of CCD cameras with better working stability is also higher. The price of a 3-CCD camera with better color reproduction is much higher than that of a single CCD camera for common use.
[0050] By placing the standard color card in the viewing area of ​​the CCD camera, the software system can analyze the standard color card area in the obtained digital image to obtain image brightness data; by comparing the preset brightness reference value with the actual brightness data, You can know the current brightness deviation of the instrument; the software system controls the electronic shutter speed, adjusts the exposure time, and can change the brightness level of the digital image. According to the current brightness deviation, increase or decrease the exposure time appropriately to limit the brightness deviation of the instrument to a small interval, so as to achieve the stability of the instrument reading.
[0051] Feature 1: Break through the traditional test strip quantitative analysis system that requires regular manual maintenance. Realizes the self-calibration and maintenance-free of the instrument system; Feature 2: The standard color card is included in the digital image taken by the system. At the same time, it can be used as a basis for judging whether the system's working status is normal, and it provides a powerful reference for handling abnormalities. Picture 9 Shown.
[0052] Such as Picture 10 As shown, the system has undergone 3500 consecutive readings (5 seconds between each reading) on ​​the standard grayscale test card (Edmund Optics a53-712 level 1, level 10), which proves that the self-calibration function can convert the image taken by the CCD camera The changes in brightness, contrast, and white balance are stable within a small range.
[0053] Innovation point 2: low-cost and highly reliable 12-channel white LED ring lighting source. Adopting 12 independently adjustable white light LEDs distributed in a ring shape and a white diffuse reflection coating with a reflection coefficient of more than 95% for the visible light band, to achieve a more uniform and stable illumination of the detected target. Thereby improving the stability and consistency of the system. The digital image taken by the CCD camera on the detected target (ie, colloidal gold immunochromatographic diagnostic test paper) is the basis of the analysis and calculation of this system. The CCD camera imaging, in principle, is that after the detected target is illuminated by the light source, the reflected light enters the camera lens and is collected by the camera's photoelectric device. Therefore, the lighting conditions of the detected target will directly affect the quality of the final digital image output by the CCD camera. LED (Lighting Emitting Diode) lighting is light emitting diode lighting, which is a semiconductor solid light emitting device. It uses a solid semiconductor chip as a luminescent material, in the semiconductor through the recombination of carriers to release excess energy to cause photon emission, directly emitting red, yellow, blue, green, cyan, orange, purple and white light. LED has the characteristics of energy saving, environmental protection, long life, small size, etc., making it the preferred lighting source solution for this system. However, to use LED lighting in the actual colloidal gold immunochromatographic diagnostic test paper quantitative analysis system, it is also necessary to solve the problems of uniformity and stability of lighting.
[0054] Such as Picture 11 As shown, 12 white LEDs are placed in a circular transparent base, and the inner and outer surfaces of the base are evenly covered with a diffuse reflection coating (the inner side is thinner and the outer layer is thicker). The test line and quality control line of the detected target (colloidal gold immunochromatographic diagnostic test paper) are placed under the center of the illumination light source. After the light emitted by the white LED is repeatedly reflected by the diffuse reflection coating on the inner and outer sides of the transparent base, a relatively uniform circular illumination area will be formed in the base, thereby achieving uniform illumination of the detected target.
[0055] LEDs are typical current-type components. When the operating voltage is turned on, small changes in voltage may bring about drastic changes in current, resulting in drastic changes in input power and output light intensity. For such current-type components, from a purely technical point of view, it is best to use a constant current source for power supply. However, the constant current source power supply circuit has a complicated structure and high cost, and there are not many applications. By choosing the resistance value of the current-limiting resistor reasonably, a constant voltage source can also be used to power the LED. This driving method has a simple structure and flexible use.
[0056] This project adopts multi-channel independent voltage regulation design, and can realize millivolt output fine adjustment. After the instrument system has been used for a period of time, the output voltage of the power supply circuit can be adjusted to correct the changes in the LED operating indicators.
[0057] Innovation point 3: Abnormal numerical filtering algorithm based on dispersion analysis
[0058] This system is based on digital image processing technology. When the software system works, the pixel is the basic unit. The test line and quality control line of the colloidal gold immunochromatographic diagnostic test paper appear as a specific area on the acquired image. Since the area usually contains multiple pixels, the brightness value of the area has a problem with the sampling method of the brightness values ​​of multiple pixels. The normal practice is to take the average. That is: the area brightness value = the sum of the brightness values ​​of the pixels in the area / the number of pixels. However, in actual work, due to the manufacturing process of the colloidal gold immunochromatographic diagnostic test paper, there is a problem of uneven distribution of gold nanoparticles on the test line and quality control line; at the same time, because the test sample (urine, blood, saliva, etc.) is usually Containing impurities will form stains on the test strip; in addition, due to the operator and operating environment, occasionally impurities such as floating dust and hair will be brought on the test strip. The above-mentioned influence is reflected in the brightness value of the test line and the quality control line area, and it will appear as an abnormal (high or low) brightness value of multiple pixels.
[0059] In order to reduce the reading deviation caused by such factors, the software of this project has designed an abnormal value filtering algorithm based on dispersion analysis. When the brightness value of some pixels in the test line and quality control line area is lower or higher than the limited range (determined by the dispersion control parameter preset by the user), the data of the pixel position will be excluded from the calculation. Not as a sampling point. For example, the brightness value of the common broken wire area will be significantly higher; the brightness value of the colloidal gold accumulation area will be significantly lower.
[0060] For example, such as Figure 14 Shown. Test strips A, B, and C after the reaction with the same concentration standard. The reading result of the algorithm is turned off and on.
[0061] Test strip A Test strip B Test strip C average value Turn off the reading of the algorithm 65.94 66.02 66.59 66.18 Turn on the reading of the algorithm 73.61 71.12 73.77 72.83
[0062] It can be seen from the reading value that the opening algorithm can effectively reduce the influence of the disconnection area pulling down the reading.
[0063] After a lot of experimental verification, this algorithm has the following advantages compared to the conventional area sampling algorithm
[0064] Advantage 1: It can improve the reliability of the system during operation, reduce the frequency of abnormal reading results, and greatly enhance the practical value of the system;
[0065] Advantage 2: Effectively enhance the tolerance of the system to test strips of different production quality provided by different manufacturers. When reading test strips with uneven distribution of gold nanoparticles, the system results can also be highly consistent with the human eye interpretation results.

Example Embodiment

[0066] Example 2: On the basis of Example 1, a CCD type colloidal gold immunochromatographic diagnostic test paper quantitative analysis method, the light is emitted from the LED lighting chamber, irradiated on the test strip after the reaction, and the surface of the test strip is diffused. After reflection, the light is reflected by the optical plane mirror to the industrial camera, and the digital image is taken by the industrial camera, and then sent to the computer software for analysis and processing, and finally the reading values ​​of the test line and the quality control line on the colloidal gold immunochromatographic diagnostic test paper are obtained. On the test strip test line and quality control line, due to the light absorption characteristics of gold nanoparticles, the scattered light signal at the test line-T line and quality control line-C line will be less than that of other areas of the test strip Signal, after the brightness analysis of the real-time image, there should be two low value areas on the brightness distribution curve corresponding to the test line and the quality control line. In principle, the brightness value of these two areas can be used to determine the target object. Presence and concentration, that is: when testing, the sample containing the target test substance is dropped onto the sample pad, the sample enters the binding pad through penetration and siphoning, flows through the analysis membrane, and flows to the absorbent pad. In this process In the analysis membrane test line and quality control line, a complex immune response will occur; after the immunochromatographic reaction is over, gold nanoparticles are fixed on the test line and the quality control line, and the content of the gold nanoparticles on the test line is The concentration of the target analyte in the sample has a corresponding relationship; if the sample does not contain the target analyte, there is no gold nanoparticles bound to the test line.
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