CN108802376A - A kind of quantitative detecting method and device of up-conversion fluorescence test paper - Google Patents
A kind of quantitative detecting method and device of up-conversion fluorescence test paper Download PDFInfo
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Abstract
The invention discloses a kind of quantitative detecting method and device of up-conversion fluorescence test paper, this method includes：Under the excitation of 980nm near-infrared lasers, the fluorescence signal sent out using imaging sensor capture test paper standard product, and the brightness value of T bands and C with region corresponding pixel points is extracted from imaging sensor, the ratio for calculating T region average brightness values and the regions C average brightness value, obtains the linear relationship of the ratio and project corresponding concentration to be measured；Take a plurality of test paper to be measured for detecting disparity items, extract the brightness value of T bands and C with region corresponding pixel points on each test paper to be measured, calculate the ratio of the regions T average brightness value and the regions C average brightness value on each test paper to be measured, according to the linear relationship that step (1) obtains, the concentration of each project to be measured is obtained.The method of the present invention and device detect while can not only realizing a plurality of up-conversion fluorescence test paper, additionally it is possible to realize that quantification detects, detection accuracy is high.
Description
Technical field
The present invention relates to the quantitative measurement technology field of up-conversion fluorescence test paper more particularly to a kind of up-conversion fluorescence test paper
Quantitative detecting method and device.
Background technology
Up-conversion luminescence is a kind of fluorescence radiation mechanism with anti-Stokes effect, usually long in 980nm near-infrareds
Go out the fluorescence of short wavelength under wavelength light irradiation by Up-conversion emission, and emits under different element dopings and different doping ratios
The fluorescence of different wave length.Since the fluorescent emission of substance in nature all meets Stokes effect, different material is swashing
It can emit certain fluorescence under shining, also result in the appearance of background of matter fluorescence.And up-conversion fluorescence is used because avoiding
To the fluorescence excitation of material itself, only to combine the object of transition probe can send out corresponding fluorescence signal,
Therefore the sensitivity of fluorescence signal detection is greatly improved.
Up-conversion luminescent material is a kind of novel fluorescence label material that can launch visible light under near infrared light excitation
Material, its special characteristics of luminescence make it play exclusive characteristic in detection field：First, it is glimmering that up-conversion fluorescence belongs to excitation
Light, it is small in this Band Interference, good signal-to-noise ratio and sensitivity can be obtained；Second, upconverting fluorescent material is avoided that sample
Corrode luminescent material, ensures the stability of signal；The material of third, up-conversion fluorescence detection belongs to nothing to human body and environment
Evil, is the direction of future development.
Research about up-conversion fluorescence test paper has a large amount of reports now, such as：
(1) application for a patent for invention that application publication number is CN105548121A discloses a kind of multifunctional food and quickly detects
Instrument, which includes the motor for driving up-conversion fluorescence test strips to move, for irradiating up-conversion fluorescence test strips
Adjustable wavelength laser module, and each up-conversion fluorescence test strips sets and have unique ID and identify, and also set
It is equipped with the bar code scanner module for obtaining up-conversion fluorescence test strips ID identification informations, and is provided with and turns for acquiring
Change the optical detection unit for the visible light that fluorescent test paper strip is sent out, wherein the signal input part of the motor and adjustable wave
The signal input part of long laser module is separately connected the control signal output of control unit, the bar code scanner module and
The signal output end of optical detection unit is separately connected the signal input part of control unit, and described control unit also passes through internet
Module connects the cloud database for being stored with data corresponding with the up-conversion fluorescence test strips ID identification informations, the control
The information output of unit connects the SD storage cards for storing output information, and the information output of described control unit is also connected with
LCD touch screen for showing output information.
(2) application for a patent for invention that application publication number is CN105717080A discloses a kind of for up-conversion fluorescence test paper
The detection method of item, the detection method set unique mark ID in each up-conversion fluorescence test strips, including：It obtains respectively
Drop has the corresponding value of electrical signals of id information, fluorescent intensity of the up-conversion fluorescence test strips of sample to be tested and is used when determining detection
The time of up-conversion fluorescence test strips；From server obtain up-conversion fluorescence test strips the corresponding batch of id information upper turn
Change the decaying transfer rate of fluorescent photon, the time of making the product；Decaying transfer rate is substituted into function of time inversion formula, is obtained described
The light intensity attenuation parameter of up-conversion fluorescence test strips；Value of electrical signals and light intensity attenuation parameter are substituted into retrieving concentration formula, obtained
The concentration of respective substance in sample.This method can accurate quantitative analysis detect a variety of and relevant substance of food security content,
Detection accuracy, which is quantified, for raising up-conversion fluorescence provides new breakthrough mouth.
Currently, the detection of up-conversion fluorescence test paper is usually scanned by mechanical translation platform, to obtain T, C signal intensity
Value.Such method has the advantages such as high sensitivity, technology maturation, but cannot achieve the synchronous inspection of a plurality of up-conversion fluorescence test strips
It surveys, the occasion for synchronizing multiple Indexs measures, efficiency far is needed to cannot be satisfied multiple determination for tumor marker analyte detection etc.
Needs.
Therefore, it is necessary to be improved to up-conversion fluorescence detecting system, to realize that the synchronous of multiple indexs is detected.
Invention content
The present invention provides a kind of quantitative detecting methods and dress for detecting the up-conversion fluorescence test paper of tumor marker
It sets, this method and device detect while can not only realizing a plurality of up-conversion fluorescence test paper, additionally it is possible to realize that quantification detects,
Detection accuracy is high.
Specific technical solution is as follows：
A kind of quantitative detecting method of up-conversion fluorescence test paper, including：
(1) sample known to project concentration to be measured is taken, the up-conversion fluorescence test paper standard product of series concentration are prepared；?
Under the excitation of 980nm near-infrared lasers, the fluorescence signal sent out using imaging sensor capture test paper standard product, and passed from image
The brightness value of T bands and C with region corresponding pixel points is extracted in sensor, and it is flat with the regions C to calculate T region average brightness values
The ratio of equal brightness value, obtains the linear relationship of the ratio and project corresponding concentration to be measured；
(2) it uses up-conversion fluorescence test paper to acquire sample to be tested, carries out test paper reaction, obtain test paper to be measured；
(3) several test paper to be measured for detecting disparity items are taken, are captured simultaneously using imaging sensor each to be tested
The fluorescence signal that paper is sent out, and correspond to picture from T bands and C bands region on each test paper to be measured is extracted in imaging sensor respectively
The brightness value of vegetarian refreshments calculates the ratio of the regions T average brightness value and the regions C average brightness value on each test paper to be measured, according to
The linear relationship that step (1) obtains, obtains the concentration of each project to be measured
Cmos sensor, ccd sensor and sCMOS sensors etc. may be used in imaging sensor of the present invention, this
The advantages of class high sensitivity image sensor can acquire fluorescent emission and be formed by image, this mode is the side by image
Formula can acquire the fluorescence of multiple test strips bands simultaneously, therefore be obtained than the flux of the photoelectric sensor scan mode of usual form
It greatly improves, greatly facilitates multi objective and synchronize detection.
Since up-conversion fluorescence test paper with sample to be tested progress fluorescent test paper after reacting, the C bands on test paper to be measured and T bands
It will appear the fluorescent bands of different brightness.Wherein, C bands is, containing fixed concentration sheep anti-mouse antibody, T bands are fixed excessive anti-
Former monoclonal antibody, therefore T takes shown band fluorescence intensity and measured target antigen concentration is proportional, C bands are made
For relative quantification reference, the monoclonal antibody amount combined every time is certain, therefore T/C is with ratio and the proportional relationship of antigen concentration.
Fluorescence intensity is higher, and the fluorescent image brightness responded on CCD is higher.
Preferably, described image sensor is ccd sensor.Such as SONY ICX285, SONY ICX674 are giving
Higher fluorescence detection efficiency is capable of providing under CCD refrigerating conditions.
For tumor marker, it is typically necessary a variety of marker types of detection, existing detection method and device
A project can only all be detected and can not be detected simultaneously, the present invention can detect multiple projects simultaneously, institute in the process of the present invention and
The detection of the particularly suitable tumor sample of device.Preferably, in step (2), the sample to be tested is tumor sample.
Further, the tumor sample is venous blood sample.
The present invention also provides a kind of quantitative testing devices of up-conversion fluorescence test paper, including：
Detection platform, for placing a plurality of test paper to be measured；
Excitation light source sends out 980nm near-infrared lasers for the test paper to be measured in detection platform；
Imaging sensor, the fluorescence letter sent out for capturing a plurality of test paper to be measured simultaneously because being excited by near-infrared laser
Number, and the fluorescence signal is stored in the form of black white image；
Data processor corresponds to picture for extracting T bands and C bands region on the test paper to be measured stored in imaging sensor
The brightness value of vegetarian refreshments, and calculate the ratio of the regions T average brightness value and the regions C average brightness value.
The quantitative detecting method that the present invention uses relies on above-mentioned quantitative testing device and realizes.
Preferably, described image sensor is cmos sensor, ccd sensor or sCMOS sensors.
It is further preferred that described image sensor is ccd sensor.
Compared with prior art, the invention has the advantages that：
The present invention can capture the fluorescence information of a plurality of up-conversion fluorescence test paper by imaging sensor simultaneously, and be passed from image
The brightness value of T bands and C with region corresponding pixel points is extracted in sensor, and it is flat with the regions C to calculate T region average brightness values
The ratio of equal brightness value, obtain the ratio and project corresponding concentration to be measured linear relationship and sample to be tested in project to be measured
Concentration, detected while can not only realizing a plurality of up-conversion fluorescence test paper, additionally it is possible to realize that quantification detects, detection is accurate
Property it is high.
Description of the drawings
Fig. 1 is the structural schematic diagram of the quantitative testing device of up-conversion fluorescence test paper.
Fig. 2 is the structural schematic diagram of up-conversion fluorescence test strips.
Fig. 3 is the standard curve of the sample of detection marker 1 (AFP albumen) in application examples 1.
Specific implementation mode
Embodiment 1
A kind of quantitative testing device of up-conversion fluorescence test paper, it is specific as follows：
Detection platform 1, for placing a plurality of up-conversion fluorescence test paper 6 to be measured；
Excitation light source 2 sends out the near-infrared laser of 980nm for the test paper to be measured in detection platform；
Imaging sensor 3 is located at the surface of detection platform 1, for capturing a plurality of test paper to be measured simultaneously because by close red
Outer laser excitation and the fluorescence signal sent out, and the fluorescence signal is stored with image format；The imaging sensor is adopted
With highly sensitive CCD camera, optical filter 5 is equipped between CCD camera lower section and detection platform；
Data processor 4 is corresponded to for extracting T bands and C bands region on the test paper to be measured stored in imaging sensor
The brightness value of pixel, and calculate the ratio of the regions T average brightness value and the regions C average brightness value.
A plurality of up-conversion fluorescence test paper, the tumour that each up-conversion fluorescence test paper is detected quantitatively are detected using above-mentioned apparatus
Marker project is different, and the specific method is as follows：
(1) standard items of the tumor marker of various concentration gradient are prepared, fluorescent test paper reaction is carried out, are obtained containing different dense
Spend the up-conversion fluorescence test paper standard product of gradient tumor marker；A plurality of test paper standard product are positioned in detection platform simultaneously,
Under the excitation of 980nm near-infrared lasers, the fluorescence signal sent out using the ccd sensor capture test paper standard product of sensitivity,
And the brightness value of T bands and C with region corresponding pixel points is extracted from the image information that imaging sensor stores, calculate T bands
The ratio of region average brightness value and the regions C average brightness value obtains the ratio and tumor marker corresponding concentration to be measured
Linear relationship；
(2) it uses up-conversion fluorescence test paper to acquire sample to be tested, carries out test paper reaction, obtain test paper to be measured；
(3) a plurality of test paper to be measured for detecting disparity items is taken, each test paper to be measured is captured using imaging sensor simultaneously
The fluorescence signal sent out, and from extracting T bands and C band region respective pixel on each test paper to be measured in imaging sensor respectively
The brightness value of point calculates the ratio of the regions T average brightness value and the regions C average brightness value on each test paper to be measured, according to step
Suddenly the linear relationship that (1) obtains, obtains the concentration of each project to be measured.
Application examples 1
The application example carries out the quantitative inspection of the up-conversion fluorescence test paper of tumor marker using method described in embodiment 1
It surveys.
Include two band of T bands and C bands on the nitrocellulose filter of the test strips (such as Fig. 2) of use, wherein T takes packet
There is a certain amount of tumor marker antigen to correspond to antibody, C, which takes, is coated with anti-mouse antibody mab, on test strips glass fibre membrane
Drop has the antitumor marker antigen pairing antibody of enough label UCP.When the work that the serum containing antigen is chromatographed in lateral flow
Under, when being moved on nitrocellulose filter by glass fibre element film, UCP probes are reacted with antigen in serum, and
It is reacted respectively with T lines and C lines on nitrocellulose filter, passes through the ratio and reference standard curve of T/C, you can obtained corresponding
Antigen concentration.
Fluorescent brightness value by acquiring T bands and C bands obtains T/C ratio datas, by ratio data and calibration over-richness
Standard curve compare, substitute into calibration formula, to obtain the concentration of specimens value of corresponding detection.
Wherein, by taking marker 1 (AFP albumen) as an example, pattern detection standard curve such as Fig. 3 institutes of marker 1 (AFP albumen)
Show, the results are shown in Table 1 for concentration conversion.
The T/C values and concentration value of 1 marker 1 (AFP albumen) of table
The application example also use ccd sensor simultaneously detect marker 1~5 (be respectively AFP, CEA, PS A, CA125,
CA19-9) totally five projects；The results are shown in Table 2：
2 imaging sensor of table detects the T/C values and concentration value of multiple markers simultaneously
Claims (8)
1. a kind of quantitative detecting method of up-conversion fluorescence test paper, including：
(1) sample known to project concentration to be measured is taken, the up-conversion fluorescence test paper standard product of series concentration are prepared；It is close in 980nm
Under infrared laser excitation, the fluorescence signal sent out using imaging sensor capture test paper standard product, and from imaging sensor
The brightness value of T bands and C with region corresponding pixel points is extracted, T region average brightness values and the regions C average brightness are calculated
The ratio of value obtains the linear relationship of the ratio and project corresponding concentration to be measured；
(2) it uses up-conversion fluorescence test paper to acquire sample to be tested, carries out test paper reaction, obtain test paper to be measured；
(3) a plurality of test paper to be measured for detecting disparity items is taken, capturing each test paper to be measured simultaneously using imaging sensor is sent out
The fluorescence signal gone out, and from T bands and C is extracted on each test paper to be measured in imaging sensor respectively with region corresponding pixel points
Brightness value calculates the ratio of the regions T average brightness value and the regions C average brightness value on each test paper to be measured, according to step (1)
The linear relationship of acquisition obtains the concentration of each project to be measured.
2. quantitative detecting method as described in claim 1, which is characterized in that described image sensor is cmos sensor, CCD
Sensor or sCMOS sensors.
3. quantitative detecting method as described in claim 1, which is characterized in that described image sensor is ccd sensor.
4. quantitative detecting method as described in claim 1, which is characterized in that in step (2), the sample to be tested is tumour sample
This.
5. quantitative detecting method as claimed in claim 4, which is characterized in that the tumor sample is venous blood sample.
6. a kind of quantitative testing device of up-conversion fluorescence test paper, which is characterized in that including：
Detection platform, for placing a plurality of up-conversion fluorescence test paper to be measured；
Excitation light source sends out the near-infrared laser of 980nm for the test paper to be measured in detection platform；
Imaging sensor, the fluorescence signal sent out for capturing a plurality of test paper to be measured simultaneously because being excited by near-infrared laser,
And the fluorescence signal is stored with image format；
Data processor, for extracting T bands and C bands region corresponding pixel points on the test paper to be measured stored in imaging sensor
Brightness value, and calculate the ratio of the regions T average brightness value and the regions C average brightness value.
7. quantitative testing device as claimed in claim 6, which is characterized in that in step (3), described image sensor CMOS
Sensor, ccd sensor or sCMOS sensors.
8. quantitative testing device as claimed in claim 6, which is characterized in that described image sensor is ccd sensor.
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| CN113281319A (en) * | 2021-06-04 | 2021-08-20 | 成都云芯医联科技有限公司 | Detection method of up-conversion fluorescent test strip based on Gaussian function |
| CN115327119A (en) * | 2022-10-18 | 2022-11-11 | 深圳市强大创新科技实业有限公司 | Antigen detector and detection method thereof |
| CN115980333A (en) * | 2023-03-22 | 2023-04-18 | 湖南伊鸿健康科技有限公司 | Fluorescence immunoassay method and optical system |
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| CN113281319A (en) * | 2021-06-04 | 2021-08-20 | 成都云芯医联科技有限公司 | Detection method of up-conversion fluorescent test strip based on Gaussian function |
| CN113281319B (en) * | 2021-06-04 | 2023-09-15 | 成都云芯医联科技有限公司 | Detection method of up-conversion fluorescent test strip based on Gaussian function |
| CN115327119A (en) * | 2022-10-18 | 2022-11-11 | 深圳市强大创新科技实业有限公司 | Antigen detector and detection method thereof |
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