CN105388283A - Immunofluorescence colored tape quantitative determination system and application thereof - Google Patents
Immunofluorescence colored tape quantitative determination system and application thereof Download PDFInfo
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- CN105388283A CN105388283A CN201510724215.2A CN201510724215A CN105388283A CN 105388283 A CN105388283 A CN 105388283A CN 201510724215 A CN201510724215 A CN 201510724215A CN 105388283 A CN105388283 A CN 105388283A
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Abstract
The invention discloses an immunofluorescence colored tape quantitative determination system. The system comprises an input module used for inputting data, an output module used for outputting data, a storage module used for storing data containing machine executable programs, and a processing module connected with the input module, the output module and the storage module and used for executing the machine executable programs, wherein the input module comprises an image acquisition device and a direct input device, an immunofluorescence colored tape image can be acquired through the image acquisition device, and related acquisition setting information can be input through the direct input device. The machine executable programs are executed in the steps of extracting the grey value of the immunofluorescence colored tape image, determining the optical density value by means of the grey value, and determining the concentration of an object to be detected according to the optical density value and the predetermined relation, wherein the predetermined relation is the function relation between the standard substance of the object to be detected and the optical density value of the immunofluorescence colored tape of the object. The system has the advantages of being simple, quick, accurate, high in sensitivity and the like when used for quantitative determination of immunochromatography substances.
Description
Technical field
The present invention relates to biochemistry detection field, particularly, the present invention relates to immunofluorescence colour band quantitative detection system and application thereof.
Background technology
Site Detection is the detection technique of a class great potential, because it has the advantages such as quick, reliable, easy and simple to handle, price is low, be suitable for the rapid field examination of batch samples, be now widely used in the fields such as clinical diagnosis, food safety detection, environment measuring.At present, on-the-spot quick diagnosis technology platform the most ripe is immunochromatography detection method, on very early pregnancy detects, achieves the whole world and generally uses.But the conventional biological tracing such as collaurum, the fluorescent dye thing used, due to deficiencies such as detection sensitivity are low, fluorescent quenching is obvious, seriously limits the development of immunochromatography technique.Based on the immunochromatography method for quick of fluorescent microsphere nano material mark, can realize detecting the ultramicron of target molecules.Be expected to break through current technical bottleneck, significantly promote field quick detection technical merit.
For realizing the quantitative determination of fluorescence immune chromatography test paper bar, the main method adopted has optical fiber scanning method, linear array CCD scanning method and image measurement method both at home and abroad at present.Optical fiber scanning method drives test strips to move by stepper motor, and Fibre Optical Sensor is scanned from the background area of test strips to p-wire.The incident light sent by light emitting diode is radiated on test strips test section, and fluorescence excitation is sent to photodetector via reception optical fiber and converts electric signal output to, and electric signal size is corresponding with the size of test section fluorescence excitation light intensity.The method needs mechanical scanner and drives Fibre Optical Sensor scanning, causes instrument volume heavy greatly; And for the situation of low concentration, because fluorescence excitation intensity variation is very faint, measurement result poor stability.Linear array CCD scanning method cold-cathode fluorescence lamp and linear CCD sensor instead of light emitting diode in optical fiber scanning method and Fibre Optical Sensor, but still remain the large and mechanical scanner of heaviness of volume.
The fluorescence developing signal of test strips is converted to picture signal by electro-optical imaging sensors by image measurement method, then uses image processing techniques to obtain the response feature amount of test strips fluorescence developing line.Image measurement method does not need stepper motor, and also can not be subject to the impact of the dynamic disturbance that moisture causes when test strips is reacted, usable image treatment technology promotes image processing effect.But be limited to existing light path design and the factor such as Linear Array CCD Image Sensor gray level resolution is low, it is to the detecting property difference of low concentration fluorescence colour band, can not the close low concentration fluorescent bands grade of good discrimination.Therefore be necessary to adopt good light path design, high-resolution imageing sensor and efficient image processing techniques to promote precision and the accuracy rate of image measurement method.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or at least provides a kind of business to select.
According to an aspect of of the present present invention, the invention provides a kind of immunochromatography fluorescence colour band quantitative detection system, as shown in Figure 1, this system 1000 comprises: load module 100, for inputting data, comprise image collecting device 110 and direct input media 130, gather the image of described fluorescence colour band by described image collecting device 110, and input described collection by described direct input media 130 and to be correlated with configuration information; Output module 200, for exporting data; Memory module 300, for storing data, comprising machine-executable program; Processing module 400, be connected with described memory module 300 with described load module 100, described output module 200, for performing described machine-executable program, performing described machine-executable program has comprised following: the gray-scale value extracting described fluorescence colour band image, utilize described gray-scale value determination optical density value, and utilizing the concentration of described optical density value and predetermined relationship determination measured object, described predetermined relationship is the funtcional relationship of the standard items of measured object and the optical density value of its fluorescence colour band.
This system on the one hand of the present invention has that the speed gathering image is fast, volume is little, stability is high and the simple feature of interface, can direct acquisition number digital image data, for the target measured object in accurate, highly sensitive quantitative sample.Can be used in realizing the good discrimination to low concentration fluorescence colour band and quantitative measurement.
According to embodiments of the invention, this system on the one hand of the present invention can also have following additional technical feature one of at least:
Alleged image collecting device 110 is scanister or is camera head.According to one embodiment of present invention, alleged image collecting device 110 comprises excitation source, imaging lens, optical filter and sensor.
For ensureing the degree of accuracy of the fluorescence information recorded, mostly be the fluorescent nano materials such as red lanthanide Nano microsphere, quantum dot, upper conversion based on the fluorescence labeling material adopted, its excitation source mostly is ultraviolet source.Therefore, according to one embodiment of present invention, in the selection of excitation source, adopt ultraviolet leds as reflected excitation light source, relative to other ultraviolet sources, ultraviolet leds has that energy consumption is low, heating less, high, operation wavelength and power stability etc. the advantage of efficiency.In addition, the volume ratio of ultraviolet leds is smaller and more exquisite, and activating system is relatively simple, is convenient to build.
For ensureing the illuminated field producing even intensity at fixing fluorescent test paper strip conversion zone, according to a preferred embodiment of the present invention, the arrangement of ultraviolet LED excitation source ring-type is distributed in around imageing sensor.Alleged ultraviolet LED light source is according to the fluorescence information of sample, and selecting peak wavelength to be about 365nm, such as, is the ultraviolet LED luminotron of 365 ± 10nm.
Alleged sensor, namely imageing sensor can select cmos sensor or ccd sensor.According to a preferred embodiment of the present invention, select cmos sensor.Cmos sensor has the features such as high degree of integration, low cost, low-power consumption, is applicable to the demand of Portable fluorescence immune test paper strip quantitative determination instrument.Although ccd sensor is better than CMOS in resolution, sensitivity and Noise measarement etc., but along with the continuous progress of science and technology, both difference will reduce day by day, simultaneously for the simple fluorescent test paper strip of digital image, existing cmos sensor also can meet its demand substantially, and therefore in general cmos sensor is the selection that cost performance is high.The image of fluorescence colour band is obtained by the imaging lens before it, the crucial imaging parameters of cmos image sensor is regulated by direct input media, as exposure, gain, white balance, frame frequency, output image data form, image clock signal polarity, window size and position etc., obtain best fluorescence colour band image and obtain high detection sensitivity.
According to one embodiment of present invention, described direct input media 130 is touch-screen, is preferably LCD touch screen.This direct input media 130 may be used for the setting of image acquisition correlation parameter, such as regulate the imaging parameters of imageing sensor, as exposure, gain, white balance, frame frequency, output image data form, image clock signal polarity, window size and position etc.
Described output module 200 comprises display or touch-screen.According to one embodiment of present invention, this output module 200 is input/output modules, alleged direct input media 130 also belongs to output module 200, in order to export and/or to show the relevant informations such as image acquisition, middle quantitative analysis results, final detection result.
Described memory module 300 comprises built-in and/or external memory module.According to one embodiment of present invention, alleged memory module 300 is connected with load module 100, and alleged memory module is SD card memory module.Alleged memory module 300 can be stored to the relevant position of memory after obtaining fluorescence colour band image from sensor, also supports the storage of SD card simultaneously, sample image can be stored in SD card, be convenient to data query.
According to one embodiment of present invention, alleged processing module 400 performs machine-executable program to extract the gray-scale value of image.This program also comprises the functional relation of the concentration utilizing MATLAB Curve Fitting Toolbox determination gray-scale value and target determinand in advance.When again measuring, inputting the test paper picture that a width collects and just automatically can calculate gray-scale value, and obtaining concentration of specimens to be detected accordingly according to the funtcional relationship preset.This machine-executable program that this processing module 400 is run has powerful image optimization and quantitative test function, can cut image, select and overturn, can zoom in or out image, brightness and contrast adjustment can be carried out to image, possess and reduce noise and sharpening function, automatically can identify the fluorescent bands in acquisition image and quantitative analysis result.
Concrete, according to a preferred embodiment of the present invention, the funtcional relationship that this machine-executable program comprises is converted into optical density value by the gray-scale value of the fluorescence colour band image by determinand standard items, and the relation then utilizing curve to go out the concentration of optical density value and standard items is decided.Optical density refers to that light is by the incident intensity I0 before solution or a certain material and this light logarithm OD=log (I0/Ib) by the transmitted intensity Ib ratio after solution or material, is a ratio.Here, in graphical analysis, incident light and transmitted intensity replace with the average gray value on two fluorescence colour band images respectively, define the average gray value of the average gray value/Quality Control fluorescence colour band of the optical density value=detection fluorescence colour band in this image analysis system.Optical density value is larger, and object color is darker, and determinand relative content is larger.
According to embodiments of the invention, as shown in Figure 2, this system 1000 on the one hand of the present invention also comprises supplementary function module 500, described supplementary function module 500 comprise can connect peripheral hardware parallel port, serial ports and/or USB interface.Peripheral hardware comprises mouse, microphone, scanner, camera etc.
According to a further aspect in the invention, the invention provides a kind of method detecting Procalcitonin (procalcitonin, PCT) concentration, the method comprises: utilize PCT to try bar and carry out fluorescence immunoassay detection to sample to be tested, obtain testing result, described testing result comprises fluorescence colour band; Utilize fluorescence colour band described in the systems axiol-ogy in one side face or any embodiment, to determine the concentration of the PCT in described sample to be tested; Described PCT tries bar and comprises the first coated film and the second coated film, one end of described first coated film is connected with one end of described second coated film, at least one piece of region in described first coated film is coated with PCT fluorescent-labeled antibody, described second coated film comprises first area and the second area of separation, described first area than described second area near described first coated film, described first area is coated with PCT coated antibody, described second area is coated with anti-PCT antibody, and described anti-PCT body can PCT fluorescent-labeled antibody described in specific binding.After utilizing PCT to try bar detection, first area alleged on it and second area are alleged fluorescence colour band, and namely the ratio of the average gray value of this two fluorescence colour band obtain alleged optical density value.
Above-mentioned to the technical characteristic of the system in one aspect of the present invention or any embodiment and the description of advantage, be suitable for the method for this one side of the present invention equally, do not repeat them here.This method of the present invention utilizes the immunofluorescence quantitative detection system of the above-mentioned digital-picture picture collecting apparatus with reflection LED ultraviolet ring-type light source, high resolution CMOS image sensor etc. etc., can Quick Acquisition image, can be direct and stable acquisition number digital image data.Inventor is based on the research of the gordian technique to the process of fluorescent test paper strip colour band Digital image analysis, comprise the research that test strips Image semantic classification, Iamge Segmentation and image feature value are extracted, and have developed the quantitative detection system of the Digital image analysis program software comprising powerful image optimization and quantitative test function.Apply this system to process Procalcitonin (PCT) fluorescent test paper strip result, analyze, result shows: the sensitivity detecting PCT reaches 0.05ng/mL, range of linearity 0.05-100ng/mL; In batch and batch between CV (coefficient of variation) be all less than 10%, achieve the demand of the quantitative measurements such as, good linear highly sensitive to PCT and repeatability.
According to embodiments of the invention, the size being coated with the region of PCT fluorescent-labeled antibody tried PCT in the first coated film in bar is not restricted, it can be whole first coated film, also can be a part for the first coated film, the first coated film be called as sample pad in one embodiment of the invention.Same, the first area of the separation in the second coated film and second area size separately are not also particularly limited, in one embodiment of the invention, as shown in Figure 3, first coated film and second coated film of this PCT examination bar are all rectangular, a broadside of the first coated film glues mutually with a broadside of the second coated film, the region being coated with PCT fluorescent-labeled antibody in first coated film is for being about 2cm, the rectangle of wide about 3mm, first area in second coated film and second area are all wire, first area is called as detection line (T line, corresponding detection fluorescence colour band), second area is called as nature controlling line (C line, corresponding Quality Control fluorescence colour band), the wire plane at place, two regions is all parallel to the bonding limit of two coated films, the width of said wire first or second area is approximately 0.5-1mm, long is the wide of film.
Alleged PCT tries bar and utilizes the first film and the second film to be coated with PCT fluorescent-labeled antibody and the PCT coated antibody of energy specific binding PCT respectively, rete is utilized to analyse after adding PCT testing sample, second film is formed double-antibody sandwich compound, namely obtain fluorescence colour band, come whether there is PCT in judgement sample based on the fluorescently-labeled fluorescence intensity detected in compound.This PCT tries the specificity that bar significantly can improve detection, shortens and detects required time.By fluorescent-labeled antibody, significantly detection sensitivity can be improved.
According to one embodiment of present invention, the other end of the second coated film of alleged PCT examination bar is connected with adsorptive pads.Adsorptive pads can be strong absorbent material, can give directed forces like this make liquid sample from directed chromatography to the second coated film of the first coated film when detecting liquid sample.
According to one embodiment of present invention, described first coated film, described second coated film and described adsorptive pads are fixed on same solid-phase matrix.Solid-phase matrix is mainly has a carrying when using this kit of the present invention, handled easily, the type of solid-phase matrix is not particularly limited, can for reacting or not affect the inert material that antigen-antibody is combined with testing sample, such as cardboard, plastic plate etc.
According to one embodiment of present invention, described first coated film is glass fibre element film, and described second coated film is nitrocellulose filter (NC film).Glass fibre membrane is chemical inertness, not containing bonding agent, adopts 100% pyrex fiber manufacture to form, it is coated with fluorescently-labeled first antibody, be beneficial to the target antigen generation specific binding in first antibody and testing sample.And NC film itself is with the addition of surfactant to improve hydrophilic ability, and had certain buffer system, there is capillary fiber structure, moisture more more than equal cellulosic filter paper can be adsorbed, flow velocity is fast, high temperature resistant, profit is wrapped the PCT coated antibody of quilt and aforesaid PCT fluorescent-labeled antibody-PCT thereon and specific binding occurs is reacted, fluorescence excitation.
According to one embodiment of present invention, described fluorescence labeling is fluorescent microsphere mark, and described PCT fluorescent-labeled antibody is marked to be combined by covalent peptide bonds by PCT antibody and described fluorescent microsphere and obtains.Like this, improve the stability of label, avoid the sterically hindered impact of antibody, be conducive to improving sensitivity and specificity.In one embodiment of the invention, described fluorescent microsphere diameter is in nano-scale range, and its diameter range is 10-500nm, and be preferably 20-300nm, on it, load has fluorescent material, and being stimulates the solia particle that can inspire fluorescence by outside energy.The fluorescent material of described fluorescent microsphere load is the fluorescent nano material converted of doped with fluorescent dyes, rare-earth complex, quantum dot etc.The fluorescent material of described fluorescent microsphere load is modified with active functional group group by high molecular polymer, and described active functional group group is carboxyl, amino, hydroxyl, sulfydryl.In one embodiment of the invention, described active functional group group is specially carboxyl, and described PCT fluorescent-labeled antibody is that the fluorescent microsphere of PCT antibody to be marked and carboxyl modified is combined the condensate formed with covalent peptide bonds.
According to one embodiment of present invention, described PCT fluorescent-labeled antibody and described PCT coated antibody are the PCT antibody be different from.These two kinds of antibody can specific binding antigen PCT, and preferably, the different surfaces determinant specific binding of two kinds of antibody capables and antigen, and what be beneficial to that antigen detects like this accurately carries out.In one embodiment of the invention, described PCT fluorescent-labeled antibody and/or PCT coated antibody are any one in PCT monoclonal antibody and PCT polyclonal antibody.It is based on the research to fluorescence labeling, antigen and antibody characteristic that this PCT tries bar, directed covalent chemical coupling is carried out by selecting the fluorescence labeling that is applicable to and specific antibody, acquisition fluorescent-labeled antibody is analyzed, and prepared by the immunoreactive various condition of optimization double-antibody sandwich.
According to one embodiment of present invention, described antiantibody is sheep anti-mouse igg antibody, its can with PCT fluorescent-labeled antibody specific binding, namely be combined with unnecessary band fluorescence labeling PCT antibody and form immune complex, fluorescence excitation, be able to obtain fluorescence colour band, with qualitative and/or quantitative this immune complex of detection.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 is the structural representation of the immunofluorescence colour band detection system in one embodiment of the present of invention.
Fig. 2 is the structural representation of the immunofluorescence colour band detection system in one embodiment of the present of invention.
Fig. 3 is the structural representation of the immunochromatography fluorescent test paper strip of detection PCT in one embodiment of the present of invention.
Fig. 4 is detected value and the concentration standard curve schematic diagram of detection PCT in one embodiment of the present of invention.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein, same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.Needs illustrate, term used herein " first ", " second " etc., only for convenience of describing, can not be interpreted as instruction or implying relative importance, having sequencing relation between can not being interpreted as.In describing the invention, except as otherwise noted, the implication of " multiple " is two or more.In this article, unless otherwise clearly defined and limited, term " is connected ", the term such as " connection " should be interpreted broadly, and such as, can be fixedly connected with, also can be removably connect, or connect integratedly; Can be mechanical connection, also can be electrical connection; Can be directly be connected, also indirectly can be connected by intermediary, can be the connection of two element internals.
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1
(1) preparation of fluorescent microsphere labelled antibody
With mean diameter be 110nm, carboxyl modified fluorescent microsphere (purchased from BangsLaboratories, Inc. company, catalog number is FC02F/10930), anti-PCT monoclonal antibody (purchased from Yunnan University's monoclonal antibody Industry Technology Center be numbered P-03), prepare fluorescent microsphere mark PCT labelled antibody by the following method:
Get the above-mentioned carboxyl modified of 15mg fluorescent microsphere MES damping fluid (0.1M, pH4.7) washing and centrifugal after, resuspended with 1mlMES damping fluid (0.1M, pH4.7), add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to final concentration be 5mM, to add NHS (N-hydroxysuccinimide) to final concentration be 10mM, room temperature lucifuge, reaction obtains the fluorescent microsphere activating rear carboxyl modified half an hour.
Wash the fluorescent microsphere of carboxyl modified after this activation with the borate buffer solution of 50mMpH8.5, after getting the above-mentioned anti-PCT antibody to be marked of 0.37mg and the above-mentioned activation of 5mg, the fluorescent microsphere of carboxyl modified is mixed in the borate buffer solution of 50mMpH8.5 and fully mixes.React 2 hours under room temperature lucifuge, allow antibody and fluorescent microsphere form stable covalent peptide bonds and combine the conjugate obtaining fluorescent microsphere and anti-PCT antibody.After reaction terminates, adding final concentration is that the BSA solution of 1% (mass percentage) is closed residual activity carboxyl site on the conjugate of fluorescent microsphere and anti-PCT antibody, and room temperature lucifuge reacts 0.5 hour.After completing, mark PCT antibody liquid with the 0.02MPBS buffer solution of pH7.4, the resuspended 5mg/ml fluorescent microsphere that obtains, 4 DEG C of preservations are stand-by.
(2) preparation of PCT immunochromatography fluorescent test paper strip
With PCT coated antibody, prepare coated film with sheep anti-mouse igg antibody, concrete grammar is as follows:
Adopt the 0.02MPBS damping fluid of pH7.4, by sheep anti-mouse igg antibody (Bo You bio tech ltd, Changsha, ABGAM-0500) concentration 1mg/ml solution is formulated as, the concentration of PCT coated antibody (purchased from Yunnan University's monoclonal antibody Industry Technology Center be numbered P-01) is formulated as concentration 2mg/ml solution, the XYZ3050 of BioDot spray membranous system is selected sheep anti-mouse igg antibody to be sprayed onto nature controlling line (C line) position of coated film (nitrocellulose filter), PCT coated antibody is sprayed onto detection line (T line) position, dehumidifier dried for standby after 4 hours is carried out in the drying plant that relative humidity is less than 10%, obtain the coated film with detection line and nature controlling line.
All-glass paper half an hour is soaked with above-mentioned film process damping fluid, the temperature of soaking is 37 DEG C, dehumidifier is carried out after 4 hours in same dehumidifier condition, mark after anti-PCT antibody content is 0.05mg/ml mixed liquor with above-mentioned film process damping fluid dilution step (one) gained fluorescent microsphere mark PCT antibody liquid to fluorescent microsphere, adopt BioDot XYZ3050 spray membranous system be sprayed into above-mentioned process glass fibre element film on preparation formed sample pad, carry out drying in same dehumidifier condition.In 100,000 grades of cleanings and dry workshop the above-mentioned dried coated film with detection line and nature controlling line, above-mentioned sample pad, adsorptive pads, backboard by carrying out after collocation assemble shown in Fig. 2, the Paperboard cutting posted is the width of 4mm/ bar by the CM4000 cutting system of employing BioDot, loads detection intermediate plate stand-by.Its structural representation as shown in Figure 2.
Embodiment 2
(1) structure of immunochromatography fluorescence colour band quantitative determination system
The invention provides a kind of immunochromatography fluorescence colour band quantitative detection system, as shown in Figure 1, this system 1000 comprises: load module 100, for inputting data, comprise image collecting device 110 and direct input media 130, gather the image of described fluorescence colour band by image collecting device 110, and input described collection by direct input media 130 and to be correlated with configuration information; Output module 200, for exporting data; Memory module 300, for storing data, comprising machine-executable program; Processing module 400, be connected with memory module 300 with load module 100, output module 200, for performing machine-executable program, performing described machine-executable program has comprised following: the gray-scale value extracting fluorescence colour band image, utilize gray-scale value determination optical density value, and utilizing the concentration of optical density value and predetermined relationship determination measured object, predetermined relationship is the funtcional relationship of the standard items of measured object and the optical density value of its fluorescence colour band.
Image collecting device 110 comprise excitation source, imaging lens, optical filter and sensor.
For ensureing the degree of accuracy of the fluorescence information recorded, mostly be the fluorescent nano materials such as red lanthanide Nano microsphere, quantum dot, upper conversion based on the fluorescence labeling material adopted, its excitation source mostly is ultraviolet source.Therefore, in the selection of excitation source, adopt ultraviolet leds as reflected excitation light source, relative to other ultraviolet sources, ultraviolet leds have that energy consumption is low, heating less, high, operation wavelength and power stability etc. the advantage of efficiency.In addition, the volume ratio of ultraviolet leds is smaller and more exquisite, and activating system is relatively simple, is convenient to build.
For ensureing the illuminated field producing even intensity at fixing fluorescent test paper strip conversion zone, the arrangement of ultraviolet LED excitation source ring-type is distributed in around imageing sensor.Alleged ultraviolet LED light source is according to the fluorescence information of sample, and selecting peak wavelength to be about 365nm, such as, is the ultraviolet LED luminotron of 365 ± 10nm.
Alleged sensor, namely cmos sensor selected by imageing sensor.Cmos sensor has the features such as high degree of integration, low cost, low-power consumption, is applicable to the demand of Portable fluorescence immune test paper strip quantitative determination instrument.Although ccd sensor is better than CMOS in resolution, sensitivity and Noise measarement etc., but along with the continuous progress of science and technology, both difference will reduce day by day, simultaneously for the simple fluorescent test paper strip of digital image, existing cmos sensor also can meet its demand substantially, and therefore in general cmos sensor is the selection that cost performance is high.The image of fluorescence colour band is obtained by the imaging lens before it, the crucial imaging parameters of cmos image sensor is regulated by direct input media, as exposure, gain, white balance, frame frequency, output image data form, image clock signal polarity, window size and position etc., obtain best fluorescence colour band image and obtain high detection sensitivity.
Direct input media 130 is touch-screen, is preferably LCD touch screen.Direct input media 130 may be used for the setting of image acquisition correlation parameter, such as, regulate the imaging parameters of imageing sensor, as exposure, gain, white balance, frame frequency, output image data form, image clock signal polarity, window size and position etc.
Output module 200 comprises display or touch-screen.Output module 200 is input/output modules, and above-mentioned direct input media 130 also belongs to output module 200, in order to export and/or to show the relevant informations such as image acquisition, middle quantitative analysis results, final detection result.
Memory module 300 comprises built-in and/or external memory module.Memory module 300 is connected with load module 100, and memory module is SD card memory module.Memory module 300 obtain fluorescence colour band image from sensor after, the relevant position of memory can be stored to, also support that SD card stores, and can be stored into sample image in SD card, be convenient to data query simultaneously.
Processing module 400 performs machine-executable program to extract the gray-scale value of image.This program also comprises the functional relation of the concentration utilizing MATLAB Curve Fitting Toolbox determination gray-scale value and target determinand in advance.When again measuring, inputting the test paper picture that a width collects and just automatically can calculate gray-scale value, and obtaining concentration of specimens to be detected accordingly according to the funtcional relationship preset.This machine-executable program that this processing module 400 is run has powerful image optimization and quantitative test function, can cut image, select and overturn, can zoom in or out image, brightness and contrast adjustment can be carried out to image, possess and reduce noise and sharpening function, automatically can identify the fluorescent bands in acquisition image and quantitative analysis result.
Concrete, the funtcional relationship that this machine-executable program comprises is converted into optical density value by the gray-scale value of the fluorescence colour band image by determinand standard items, and the relation then utilizing curve to go out the concentration of optical density value and standard items is decided.Optical density refers to that light is by the incident intensity I0 before solution or a certain material and this light logarithm OD=log (I0/Ib) by the transmitted intensity Ib ratio after solution or material, is a ratio.Here, in graphical analysis, the average gray value of the average gray value/Quality Control fluorescence colour band of the optical density value=detection fluorescence colour band in this image analysis system is defined.Optical density value is larger, and object color is darker, and determinand relative content is larger.
(2) workflow of quantitative determination system
The general work flow process of this quantitative determination system is: push in detection system in the test strips groove reacted for application of sample fluorescent test paper strip to be measured being positioned over test strips frame, direct load module 130 sends instruction to image capture module 110, open LED burst of ultraviolel light source and irradiate test strips, the fluorescent microsphere at test strips T line and C line place inspires characteristic wavelength reflected fluorescent light through UV-irradiation, its reflected light path is received by cmos image sensor after collimating mirror and narrow band pass filter process, after light signal is carried out photoelectricity and analog/digital conversion by cmos image sensor, digital data transmission after conversion is stored to memory module 300, be transferred to data processing module 400 and carry out data processing, data processing module 400 identifies the optical density value of the characteristic frequency fluorescence that test strips T line and the transmission of C line come automatically, obtain the optical density value of each checking matter, and then carry out corresponding conversion according to the ratio of optical density value of the optical density and Quality Control thing (fluorescent composition on C line) that detect thing (fluorescent composition on T line) and obtain checking matter concentration.
Embodiment 3
(1) PCT examination criteria Drawing of Curve and sensitivity and linear measurement range detect
The sensitivity of the PCT test strips of embodiment 1 is measured using PCT standard items as testing sample.
PCT standard items are formulated as series concentration (0,0.01,0.05,0.1,0.5,1,5,10,50,100ng/mL), add in the well of the PCT test strips obtained by embodiment 1, and adopt quantitative measurement device fluorescence intensity of the present invention.Detecting step: first detected sample is recovered room temperature (25 DEG C) before detection, get with accurate pipettor the well that detected sample 60 μ l vertically slowly instills the PCT test strips that embodiment 1 obtains, test with fluorescent quantitative measurement device after 10 minutes.
Its testing result is as shown in table 1 below.Can show that from testing result the sensitivity of the detection PCT of embodiment 1 is 0.05ng/mL, range of linearity 0.05-100ng/mL.
PCT ELISA test strip value and concentration curve are as shown in Figure 4.
The ELISA test strip value of the different sample concentration of table 1, PCT
(2) PCT serum sample detects
The PCT ELISA test strip normal human serum sample obtained by embodiment 1 and totally 320 parts, bacteriological infection human serum sample, measure kit (bioMerieuxSA) testing result with VIDASBRAHMSPCT to compare, linear regression: y=0.8387x+0.1321, R
2=0.9638.When threshold values is 0.5ng/mL, the coincidence rate of two kinds of methods is 97.2% (311/320), as shown in table 2.
Table 2, PCT serum sample two kinds of methods detect contrast
(3) system accuracy detects
By PCT normal concentration test card (0.1,1,100ng/mL), use quantitative determination system fluorescence intensity.Each concentration test card tests 20 times, and according to test result calculations mean deviation CV value, result is as shown in table 3.
Table 3, accuracy testing result
In the description of this instructions, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. an immunofluorescence colour band quantitative detection system, is characterized in that, comprising:
Load module, for inputting data, comprises image collecting device and direct input media, by fluorescence colour band image described in described image acquisition device, and inputs described collection by described direct input media and to be correlated with configuration information;
Output module, for exporting data;
Memory module, for storing data, comprising machine-executable program;
Processing module, be connected with described memory module with described load module, described output module, for performing described machine-executable program, performing described machine-executable program has comprised following: the gray-scale value extracting described fluorescence colour band image, utilize described gray-scale value determination optical density value, and utilizing the concentration of described optical density value and predetermined relationship determination measured object, described predetermined relationship is the funtcional relationship of the standard items of measured object and the optical density value of its fluorescence colour band.
2. the system of claim 1, is characterized in that, described image collector is set to scanister.
3. the system of claim 1, is characterized in that, described image collecting device comprises excitation source, imaging lens, optical filter and sensor.
4. the system of claim 3, is characterized in that, described excitation source is ultraviolet source, and be preferably the ultraviolet LED luminotron that peak wavelength is 365 ± 10nm, preferably, described ultraviolet LED luminotron circular row is distributed in around described sensor.
5. the system of claim 3, is characterized in that, described sensor is CMOS or ccd sensor.
6. the system of claim 1, is characterized in that, described direct input media is touch-screen, is preferably LCD touch screen.
7. the system of claim 1, is characterized in that, described output module comprises display or touch-screen.
8. the system of claim 1, is characterized in that, described memory module comprises built-in and/or external memory module, is preferably SD card memory module.
9. the system of claim 1, is characterized in that, described system also comprises supplementary function module, described supplementary function module comprise can connect peripheral hardware parallel port, serial ports and/or USB interface.
10. detect a method for PCT concentration, it is characterized in that, comprising:
Utilize PCT to try bar and carry out fluorescence immunoassay detection to sample to be tested, obtain testing result, described testing result comprises fluorescence colour band;
Utilize fluorescence colour band described in the arbitrary systems axiol-ogy of claim 1-9, to determine the concentration of the PCT in described sample to be tested;
Described PCT tries bar and comprises the first coated film and the second coated film, and one end of described first coated film is connected with one end of described second coated film, and at least one piece of region in described first coated film is coated with PCT fluorescent-labeled antibody,
Described second coated film comprises first area and the second area of separation, described first area than described second area near described first coated film,
Described first area is coated with PCT coated antibody, and described second area is coated with anti-PCT antibody, and described anti-PCT body can PCT fluorescent-labeled antibody described in specific binding.
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