CN106645708A - Quantitative detection calculation method based on fluorescent immuno-chromatographic technology - Google Patents
Quantitative detection calculation method based on fluorescent immuno-chromatographic technology Download PDFInfo
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Abstract
The invention relates to the field of immuno-chromatographic detection, in particular to a quantitative detection calculation method based on fluorescent immuno-chromatographic technology. The method includes the steps: inserting a fluorescent immuno-chromatographic test strip dripping sample solution to be measured into a fluorescent immunity analyzer; irradiating by the aid of LED (light-emitting diode) light sources; performing light filtering treatment by the aid of a light filter; detecting by the aid of a photoelectric detector; treating electric signals; performing AD conversion treatment; performing filtering algorithm treatment; performing baseline fitting; performing peak searching treatment; calculating T/C area ratio; calculating concentration; judging by the aid of calculated concentration results. The invention provides a perfect, rapid and quantitative detection calculation method based on the fluorescent immuno-chromatographic technologies. Various noise and signal interference can be filtered when concentration of objects to be measured is detected and calculated, and accurate detection results are improved. The method has the advantages of high accuracy and stability and low power consumption, and rapid and quantitative detection requirements of users are effectively met.
Description
Technical field
The present invention relates to immunochromatography detection field, and in particular to a kind of quantitative determination based on fluorescence immune chromatography technology
Computational methods.
Background technology
With the rhythm of Socialized Reading it is more and more faster, either venomous injurant in the Site Detection of disease epidemic situation, food
The field quick detection of matter, or clinical detection, the emergency of disaster medical science of the emergency rescue patient of emergency treatment and ICU
In detection rescue, and in alcohol, the detection of drug concentration of the scene of the accident;All urgent need is a kind of detects that quick, carrying is convenient, operates
Convenient field quick detection technology.But traditional Fast Detection Technique is narrow, unstable etc. due to generally existing measurable range
Defect, it is impossible to meet the requirement of quantitative quick detection well.
Field quick detection(Point Of Care Testing, POCT)It is in-vitro diagnosis(IVD)One it is emerging thin
Branch trade, is analyzed at once in sampling location, saves complex process program of the sample in laboratory inspection teacher, is quickly obtained
A kind of new method of assay.POCT, by " centered on patient ", is to realize the optimum carrier to patient's personalized service.Its with
By means of advantages such as simple, quick and low costs, extensively application is obtained clinical, be the important developing direction of IVD industries and increase most
Fast field.POCT compares traditional laboratory medicine, can save many Pretreateds, sample censorship, loaded down with trivial details equipment inspection
The steps such as survey, data processing and transfer process, are directly quickly obtained reliable result, are that further suing and labouring for doctor wins treasured
The expensive time.
Wherein, fluorescence immune chromatography detection technique is a kind of emerging detection technique of POCT.Fluorescence immune chromatography detection system
System hardware components are mainly made up of a photodetector and detection plate.Detection plate uses chromatography, will be certain density
Determinand sample is added drop-wise in sample pad, and sample solution is moved forward by chromatography effect, dissolves the mark solidified in detection line
There is specific reaction after thing therewith and form immune complex, compound is enriched with detection line and control line, the reaction of attachment
Thing content is directly proportional to testing concentration;Wherein, label certain wavelength excite light irradiation under, certain wavelength can be produced
Fluorescence.Photodetector detects the optical signal of the fluorescence, is converted into corresponding data message, and by corresponding software algorithm
Process, obtain the corresponding light intensity characteristic value of determinand(The value of detection line/control line);Finally, by light intensity characteristic value with it is to be measured
Thing concentration standard curve, can detect the concentration of determinand.
But, fluorescence analysis belongs to the detection category of small-signal, in general Instrument Design, obtains the method for signal to carry
Based on the gain of high instrument or multiplication factor, and various noises, interference are not targetedly filtered in algorithm process afterwards.
But for fluorescent quantitative detector, the signal for detecting may not be to come from fluorescent material to be detected, but from examination
Agent bar background fluorescence, the interference of external stray light;In addition, ELECTRONIC NOISE interference, mechanical oscillation interference, reagent strip production work
Interference of skill etc. can all have influence on the measurement of fluorescence intensity.Fluorescence immunoassay reagent strip detection line is very narrow, only one fine rule;When treating
Survey thing concentration it is not high when, the fluorescence signal of reagent strip detection line is very weak, due to background fluorescence, circuit noise and extraneous various
Interference can all reduce the signal to noise ratio of input signal.These signals can mix with useful signal, or even the fluorescence letter needed for flooding
Number, the sensitivity of lowering apparatus.It is typically no targetedly right in algorithm process after existing fluorescence immune chromatography technology
Noise jamming does filter operation, and the error for causing fluorescence immune chromatography to detect is larger, and the confidence level of detected value is reduced, and then is reduced
The application surface of fluorescence immune chromatography technology.
Therefore, this programme is under this background, it is proposed that a whole set of more perfect technical scheme is realizing in detection meter
During calculating testing concentration, additionally it is possible to filter out various noises, signal interference, there is provided more accurate testing result.
The content of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, there is provided a kind of to carry out at once point in sampling location
Analysis, saves complex process program of the sample in laboratory inspection teacher, quickly obtains new departure of testing result.
The present invention is achieved by the following technical solutions:
A kind of quantitative determination computational methods based on fluorescence immune chromatography technology, comprise the following steps:Dropwise addition there is into sample to be tested
Solution fluorescence immunoassay test strips insertion fluorescence immunity analyzer in, sequentially pass through LED/light source irradiation, optical filter optical filtering process,
Photodetector detection, Electric signal processing, AD conversion process, filtering algorithm process, baseline fitting, peak-seeking process, calculating T/C faces
Product ratio, the process for calculating concentration, are judged so as to pass through the concentration results for calculating.
Preferably, the filtering algorithm is processed and takes sliding window average filter algorithm.
Preferably, least square fitting is selected in the baseline fitting.
Preferably, the calculating T/C area ratios are to ask face in each M point range in T, C peak after baseline fitting or so
Product, then calculate T/C area ratios.
Preferably, comprise the following steps:
(1)Sample to be tested solution is added dropwise on fluorescence immunoassay reagent strip, after standing a period of time, in insertion fluorescence immunity analyzer
Detected;
(2)For the spectrum of fluorescent marker in reagent strip, suitable LED/light source is selected, start LED/light source irradiation reagent strip;
(3)For the excitation wavelength and wavelength of fluorescence of fluorescent marker, corresponding exciting light optical filter and fluorescence is selected to filter
Piece, wiping out background light, the interference of veiling glare;
(4)The fluorescence signal for detecting is converted into electric signal using photodetector;
(5)The low current signal that photodetector is obtained further is processed, I/V change-over circuits is first passed through and is turned current signal
Voltage signal is turned to, then is amplified by bandpass filtering amplifying circuit, increase signal to noise ratio;Then detecting circuit is passed through to obtain
Electric signal after high-precision amplification;
(6)By high-precision a/d converter, the electric signal after acquisition process is amplified, i.e. analog signal change into digital letter
Number, and the data signal of each test point is combined into order a sampling curve, i.e. raw sensor data;
(7)Sliding window average filter algorithm is taken to filter various noises and interference raw sensor data, high-frequency signal of decaying,
Remove most burr signal;
(8)For filtered data, from least square fitting baseline drift is filtered;
(9)Peak-seeking process is carried out on curve after baseline fitting, that is, determines T peaks(Correspondence detection line)With C peaks(Correspondence control
Line)Position;
(10)Quadrature in each M point range in T, C peak after baseline fitting or so, calculate T/C area ratios, obtain light intensity special
Value indicative;
(11)According to T/C light intensity characteristics value and concentration standard curve, the corresponding concentration of specimens value of T/C ratios is calculated;
(12)Concentration results to calculating judge.
Preferably, described photodetector is photodiode.
The beneficial effects of the present invention is:
In the present invention, the internal characteristicses of LED/light source are conducive to the portability of fluorescence immunity analyzer, stable, long-time to measure;And
And the power consumption of LED/light source is very low, heat is low, life-span length.For the spectrum of fluorescent marker in reagent strip, select suitable
LED/light source, during irradiation reagent strip higher fluorescence intensity can be obtained, and reduce temperature change, and then reduces temperature to detection knot
The interference of fruit.
The present invention selects corresponding exciting light optical filter and glimmering for the excitation wavelength and wavelength of fluorescence of fluorescent marker
Light optical filter is obtaining the exciting light and fluorescence of desired wavelength;The interference of bias light, veiling glare can be effective filtered out.
The present invention selects photodiode as photodetector, and its low cost, response are fast, service life is longer;And
There is high stability, low-dark current, high sensitivity.LED/light source, optical filter, photodiode and light channel structure group
Into optical system be sealed in optics camera bellows, it is possible to decrease the interference of veiling glare.
The present invention in the processing procedure that analog signal is changed into data signal, using high-precision a/d converter, and
And have the advantages that conversion rate is fast, low in energy consumption.
The characteristics of present invention is mostly random signal for the noise in raw sensor data, takes sliding window averagely to filter
Ripple algorithm, can be very good high-frequency signal of decaying, and data are played with smoothing effect, remove most burr signal, Ke Yi
Suppress to greatest extent to preserve useful signal while noise, so as to improve signal to noise ratio.
The present invention filters baseline drift from least square fitting, can effectively reduce noise jamming and baseline drift
The impact that shifting is caused to analysis result.
The light intensity value at non-selected T, C peak of the present invention as testing concentration reference point, but in order to further eliminate with
Machine noise, the method for selecting to be quadratured in T, C peak after baseline fitting or so each M point range, obtains light intensity characteristic value;Its
In, the size of M values can be arranged according to the actual conditions of reagent strip.Using both ratio ----T/C faces under same state
Product ratio further reduces noise jamming, it is ensured that certainty of measurement as final result judgement.
In the present invention, concentration standard curve is many experiments number measured in development & production link according to reagent strip
According to calculating a calibration curve using curve fitting algorithm, based on these experimental datas.
This method is based on fluorescence immune chromatography technology, and proposition devises a whole set of perfect Quantitative detection scheme;It is real
Show during detection calculates testing concentration, additionally it is possible to filter out various noises, signal interference, there is provided more accurate
Testing result.The program has the advantages that high accuracy, high stability, low-power consumption, and user's Quantitative detection is met well
Demand.
Description of the drawings
Fig. 1 is the schematic flow sheet of detection method;
Fig. 2 is original curve map in the specific embodiment of the invention;
Fig. 3 is the curve map in the specific embodiment of the invention after sliding window average filter;
Fig. 4 is the curve map in the specific embodiment of the invention after baseline fitting;
Fig. 5 is the curve map in the specific embodiment of the invention after peak-seeking;
Fig. 6 is that peak area schematic diagram is calculated in the specific embodiment of the invention;
Fig. 7 is the corresponding canonical plotting of reagent strip in the specific embodiment of the invention.
Specific embodiment
To be best understood from the present invention, with reference to embodiment and accompanying drawing, the invention will be further described, following examples
Only it is that the present invention will be described rather than it is limited.
A kind of quantitative determination computational methods based on fluorescence immune chromatography technology of the present invention, as shown in figure 1, including
Following steps:
1st, reagent strip sample-adding
Few 40 microlitres sample to be tested solution is dropped on fluorescence immunoassay reagent strip, after standing 15 minutes, is started in inserting instrument
Detection.
2nd, the hardware handles flow process in instrument is as follows:
(1)Test paper window on LED/light source irradiation reagent strip, the excitation wavelength of the fluorescent material on reagent strip is concentrated mainly on
365nm or so, the wavelength of fluorescence concentrates on 613nm or so after being stimulated.Therefore, LED light of this programme from 365nm wavelength
Source.When starting detection reagent bar, LED/light source irradiates exciting light to the test paper window of reagent strip first.The inherence of LED/light source
Feature is conducive to the portability of fluorescence immunity analyzer, stable, long-time to measure;And the power consumption of LED/light source is very low, heat
Low, life-span length.For the spectrum of fluorescent marker in reagent strip, suitable LED/light source is selected, can be obtained during irradiation reagent strip
Higher fluorescence intensity, reduces temperature change, and then reduces interference of the temperature to testing result.
(2)Optical filtering is processed:For the excitation wavelength and wavelength of fluorescence of fluorescent marker, corresponding exciting light is selected to filter
Piece and fluorescent optical filter are obtaining the exciting light and fluorescence of desired wavelength.LED/light source is sent after light, first by LED/light source
The exciting light optical filter of front end(It is 365 ± 20nm by optical wavelength), filter out the exciting light of other wavelength that may be present;Swash
Luminous to be irradiated to reagent strip, the fluorescent material on test paper is stimulated generation fluorescence;Fluorescence is by the front end of photodetector
Fluorescent optical filter(It is 610 ± 20nm by optical wavelength), effective filter out the interference of bias light, veiling glare;It is follow-up to ensure
Photodetector can obtain useful fluorescence signal.
(3)Photodetector is detected:This programme selects photodiode to believe the fluorescence for getting as photodetector
Number electric signal is converted into, its low cost, response is fast, and service life is longer;And with high stability, low-dark current, highly sensitive
The advantages of spending.The optical system of LED/light source, optical filter, photodiode and light channel structure composition is sealed in optics camera bellows
In, to reduce the interference of veiling glare.In detection process, the motor of instrument internal drives reagent strip linear uniform motion, due to examination
The distribution of fluorescent material on agent bar is change, and the fluorescence signal that photodetector is obtained on each test point of test paper is also
Change.
(4)Electric signal processing:The low current signal that photodetector is obtained further is processed;First, by I/V
Current signal is converted into voltage signal by change-over circuit, then is amplified by bandpass filtering amplifying circuit, increases signal to noise ratio;So
Electric signal after detecting circuit is to obtain high-precision amplification afterwards(Remain analog signal).
(5)AD conversion process:By high-precision a/d converter, it has the advantages that conversion rate is fast, low in energy consumption, it
The analog signal that pre-treatment is obtained changes into data signal;And the data signal of each test point is passed in order instrument
Client software, be combined into a sampling curve(I.e.:Raw sensor data, such as Fig. 2), so as to further algorithm process.
3rd, filtering process:The raw sensor data for mainly passing over to hardware module, is filtered calculating process;With
In further filter out it is actually detected during, the dark current that exists, veiling glare, the background fluorescence noise of reagent strip, current noise,
Photon noise, the electromagnetic noise of motor, etc..The characteristics of this programme is mostly random signal for above-mentioned noise, takes slip
Window average filter algorithm, can be very good high-frequency signal of decaying, and data are played with smoothing effect, remove most burr letter
Number, useful signal can be preserved while noise is suppressed to greatest extent, so as to improve signal to noise ratio.Filtered curve such as Fig. 3.
4th, baseline fitting is processed:Filtered data are needed with the further process operation for removing baseline.In actual inspection
Due to various noise reasons such as temperature change, reagent strip fluorescence uneven concentration during survey, curve occurs serious drift,
Have a strong impact on the calculating of concentration value.Need to be solved by baseline fitting algorithm;A most young waiter in a wineshop or an inn is selected through considering the system
Multiplication is fitted to filter baseline drift, can effectively reduce the impact that noise jamming and baseline drift are caused to analysis result.
Curve after baseline fitting such as Fig. 4.
5th, peak-seeking is processed:To the curve after baseline fitting, in addition it is also necessary to further peak-seeking process, i.e. searching T peaks and C peaks
Position.This programme is adopted in the range of specific crest, and the method for maximizing obtains T peaks(Correspondence detection line)With C peaks(It is right
Answer control line).Curve after peak-seeking such as Fig. 5.
6th, reference area:Calculate T/C area ratios;The light intensity value at T, C peak can be selected dense as determinand in theory
The reference point of degree, but for further Removing Random No, this programme selects T, C peak after baseline fitting or so each M
The method quadratured in point range, obtains light intensity characteristic value;For the peak feature of this reagent strip, M values are set to 35(Peak area takes
Value scope such as Fig. 6).Calculated T peak areas are:3217878, C peak areas are:14013014.In actual measurement process
Always there are various destabilizing factors, including vibrations, fixed, the reagent strip fluorescent signal decay of flashing clicked on etc., cause T with
The overall fluctuation of C area values, thus using under same state both ratio ----T/C area ratios are sentenced as final result
It is disconnected, further reduce noise jamming, it is ensured that certainty of measurement.Calculating T/C values is:0.229635.
7th, concentration is calculated:I.e. according to T/C light intensity characteristics value and concentration standard curve;Reagent strip per batch all can dispatch from the factory
Front demarcation calibration curve, the corresponding calibration curve point of reagent strip of this experiment is:(0,0.041317)(1.5,0.056911)
(6.25,0.214792)(12.5,0.399957)(25,0.72784)(50,1.225726)(100,2.188615)(200,
3.749999)(Concentration unit is:μg/L)Calibration curve such as Fig. 7.T/C values 0.229635 will be calculated in 6th step and bring this into
Calibration curve, calculated sample to be tested concentration is:6.2617μg/L.And the reality of the sample to be tested solution used in testing
Concentration is 6.25 μ g/L, and the error of this reagent strip is only 0.19%, and error range very little, accuracy of detection is very high.
The above embodiment is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, technical side of the those of ordinary skill in the art to the present invention
Various modifications and improvement that case is made, all should fall into the protection domain of claims of the present invention determination.
Claims (6)
1. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology, it is characterised in that comprise the following steps:Will drop
In fluorescence immunoassay test strips insertion fluorescence immunity analyzer added with sample to be tested solution, sequentially pass through LED/light source irradiation, filter
Piece optical filtering process, photodetector detection, Electric signal processing, AD conversion process, filtering algorithm process, baseline fitting, at peak-seeking
Reason, the process for calculating T/C area ratios, calculating concentration, are judged so as to pass through the concentration results for calculating.
2. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In:The filtering algorithm is processed and takes sliding window average filter algorithm.
3. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In:Least square fitting is selected in the baseline fitting.
4. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In:The calculating T/C area ratios are quadratured in each M point range in T, C peak after baseline fitting or so, then calculate T/C
Area ratio.
5. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In comprising the following steps:
(1)Sample to be tested solution is added dropwise on fluorescence immunoassay reagent strip, after standing a period of time, in insertion fluorescence immunity analyzer
Detected;
(2)For the spectrum of fluorescent marker in reagent strip, suitable LED/light source is selected, start LED/light source irradiation reagent strip;
(3)For the excitation wavelength and wavelength of fluorescence of fluorescent marker, corresponding exciting light optical filter and fluorescence is selected to filter
Piece, wiping out background light, the interference of veiling glare;
(4)The fluorescence signal for detecting is converted into electric signal using photodetector;
(5)The low current signal that photodetector is obtained further is processed, I/V change-over circuits is first passed through and is turned current signal
Voltage signal is turned to, then is amplified by bandpass filtering amplifying circuit, increase signal to noise ratio;Then detecting circuit is passed through to obtain
Electric signal after high-precision amplification;
(6)By high-precision a/d converter, the electric signal after acquisition process is amplified, i.e. analog signal change into digital letter
Number, and the data signal of each test point is combined into order a sampling curve, i.e. raw sensor data;
(7)Sliding window average filter algorithm is taken to filter various noises and interference raw sensor data, high-frequency signal of decaying,
Remove most burr signal;
(8)For filtered data, from least square fitting baseline drift is filtered;
(9)Peak-seeking process is carried out on curve after baseline fitting, that is, determines the position at T peaks and C peaks;
(10)Quadrature in each M point range in T, C peak after baseline fitting or so, calculate T/C area ratios, obtain light intensity special
Value indicative;
(11)According to T/C light intensity characteristics value and concentration standard curve, the corresponding concentration of specimens value of T/C ratios is calculated;
(12)Concentration results to calculating judge.
6. according to a kind of arbitrary described quantitative determination computational methods based on fluorescence immune chromatography technology of claim 1-5, its
It is characterised by:Described photodetector is photodiode.
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