CN106841133A - A kind of quantitative determination computational methods based on fluorescence immune chromatography technology - Google Patents
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Abstract
The invention belongs to immunochromatography detection field, specifically a kind of quantitative determination computational methods based on fluorescence immune chromatography technology.Comprise the following steps：To be added dropwise in the fluorescence immunoassay test strips insertion fluorescence immunity analyzer for having sample to be tested solution, sequentially pass through LED light source irradiation, optical filter optical filtering treatment, photodetector detection, Electric signal processing, AD conversion treatment, baseline correction, L M algorithm process, areal calculation, the process of concentration calculating.The inventive method focuses on rebuilding signal by using algorithm, has both eliminated the influence of the various interference such as noise, can also make signal processing undistorted again.The result of high accuracy and low error can be obtained.I.e. the present invention can be quick, convenient and accurately measures concentration results.
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 development of modern science and technology, increasing portable field detection device is developed, either in doctor
In quick detection, or can be used to portable inspectiont device in the detection of food security.And portable inspectiont
Also referred to as field quick detection, is a kind of industry of subdivision that in-vitro diagnosis are evolving middle generation, by the scene
Sampling is analyzed at once, eliminates the numerous and diverse detection process using laboratory inspection teacher and prolonged wait, can be fast
A kind of new method for obtaining assay of speed.Wherein in portable inspectiont fluorescence immune chromatography detection technique be apply it is glimmering
Light technology and a kind of emerging detection technique that produces.
Fluorescence immune chromatography technology is main to be made up of including photodetector and detection plate two parts.Chromatography is applied
Place be exactly that, in detection plate, detection is dripped in detection plate sample when starting, sample is moved under chromatography effect, while will be solid
Label i.e. the fluorescent material dissolving of change, react and are formed immune complex, and compound be then enriched to detection line and
On control line, the content and testing concentration of reactant are directly proportional；Because the characteristic of fluorescent material, swash in the irradiation of certain wavelength
Give, the fluorescent emission of certain wavelength can be produced.And photodetector is translated into correspondence when the fluorescence of transmitting is received
Data message, by the respective handling of software algorithm, it is possible to obtain the light intensity characteristic value i.e. detection line and control line of sample
Ratio；Finally, the concentration of test sample can be calculated by light intensity characteristic value and gone out with the standard curve of sample concentration.
Because the luminous light intensity of fluorescent material is relatively low, so being all by the gain of instrument during general test in fluorescence analysis
Improved with multiplication factor, to obtain light-intensity test signal high.But, without for various influences in the algorithm process after
Factor is processed, and causes the fluorescence signal for obtaining to be more than the launching light containing fluorescent material, also includes agent plate
The veiling glare such as background fluorescence and electronic circuit interference influence.And because the detection line of reagent strip is very narrow, when detection sample
When concentration is not high, the light intensity signal of fluorescent material transmitting is very weak, along with the interference of various influence factors, can be blended in useful
In signal, or even useful signal is flooded, reduce signal to noise ratio and the sensitivity of signal.And L-M methods are employed herein
Treatment hardware data, L-M algorithms are collectively referred to as Levenberg-Marquardt, and Chinese is the literary Burger-Ma Kuaertefa of row, is one
The algorithm for optimizing is planted, is the most widely used Nonlinear Least-Square Algorithm.It can make number in processing data process
According to reconstruction, can be good at remaining the characteristic of data.It is widely used in economics, management optimization, network analysis, optimal
Design, mechanically or electrically design etc..
The content of the invention
This programme under above-mentioned background condition, in order to avoid the deficiency of above-mentioned technology, it is proposed that at a different algorithm
Reason method, while applying the area computation method in this algorithm, eliminates various influence factors during algorithm process, and
Keep the undistorted property of data.And it is very simple and convenient to be applied to the areal calculation in this algorithm, can convenient and quick detection obtains
To high accuracy, the result of low error.
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 is had to be measured
In the fluorescence immunoassay test strips insertion fluorescence immunity analyzer of sample solution, sequentially pass through LED light source irradiation, at optical filter optical filtering
Reason, photodetector detection, Electric signal processing, AD conversion treatment, baseline correction, L-M algorithm process, areal calculation, densimeter
The process of calculation.
Preferably, described areal calculation is that primitive curve is fitted into two Gaussian curves of standard, is intended by calculating
The coefficient ratio of two curves after conjunction is so as to calculate original area under the curve ratio.
Preferably, least square method is selected in the baseline correction.
Preferably, the described method comprises the following steps：
(1) sample to be tested solution is added dropwise on fluorescence immunoassay reagent strip, after standing a period of time, fluoroimmunoassay is inserted
Detected in instrument；
(2) for the spectrum of fluorescent marker in reagent strip, suitable LED light source is selected, starts LED light source irradiation reagent
Bar；
(3) for the excitation wavelength and launch wavelength of fluorescent marker, corresponding exciting light optical filter and launching light are selected
Optical filter, wiping out background light, the interference of veiling glare；
(4) fluorescence signal that will be detected using photodetector is converted into electric signal；
(5) low current signal that photodetector is obtained further is processed, first passes through I/V change-over circuits and believe electric current
Number voltage signal is converted into, then is amplified by bandpass filtering amplifying circuit, increase signal to noise ratio；Then by detecting circuit with
Obtain the analog electrical signal of high-precision amplification；
(6) converter is used, the analog electrical signal after acquisition process is amplified is converted to data signal, and will conversion
The data signal come is combined into a collection sample curves for raw sensor data according to the detection ordering of each test point；
(7) data after AD conversion are carried out into baseline correction, uses least square fitting to carry out baseline correction to disappear
Except the influence that curves shift is caused；
(8) raw sensor data after baseline correction is carried out into L-M treatment；
(9) after above-mentioned algorithm and correcting process are treated, the Gaussian curve at T peaks and C peaks is formd, two for being formed
Individual Gaussian curve is written as the product of peak factor and the Gaussian curve that area is 1, and the area of Gaussian curve is than being exactly bent Gauss
The ratio of the peak factor in line formula, while the result of ratio also is designated as into light intensity characteristic value；
(10) according to T/C light intensity characteristics value and concentration standard curve, the corresponding testing sample concentration of T/C ratios is calculated.
Preferably, step (2) the suitable LED light source of selection refers to selecting launch wavelength exciting with fluorescent material
Wavelength identical LED light source.
Preferably, the computational methods detailed process of the light intensity characteristic value described in step (9), formula are as follows：
T peaks fitting after Gaussian curve function be：
C peaks fitting after Gaussian curve function be：
The T peaks and the area ratio at C peaks calculated by above-mentioned Gaussian curve function are：
Preferably, described photodetector is photodiode.
The beneficial effects of the present invention are：
In the present invention, the internal characteristicses of LED light source are conducive to the portability of fluorescence immunity analyzer, stabilization, survey for a long time
Amount；And the power consumption of LED light source is very low, heat is low, long lifespan.For the spectrum of fluorescent marker in reagent strip, selection is closed
Suitable LED light source, fluorescence intensity higher can be obtained during irradiation reagent strip, reduce temperature change, and then reduce temperature to inspection
Survey the interference of result.
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 obtains the exciting light and fluorescence of desired wavelength；The interference of bias light, veiling glare can be effective filtered out.
, used as photodetector, its low cost, response are fast, service life is longer for present invention selection photodiode；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 converter, and
And have the advantages that conversion rate is fast, low in energy consumption.
Used L-M algorithms during algorithm process in the present invention, L-M algorithms as the algorithm process for optimizing,
Make data reconstruction in processing procedure, and it is to be rebuild useful signal to rebuild data, can be by number in its process of reconstruction
The influence signal such as noise in is completely eliminated, and has both eliminated the influence signal big for data influence, in turn ensure that data
It is complete true.
The present invention filters baseline drift from least square fitting, can effectively reduce noise jamming and baseline drift
The influence that shifting is caused to analysis result.
Areal calculation in the present invention is based on the optimum results of L-M algorithms, and initial data is synthesized to two by L-M algorithms
The Gaussian curve of bar standard, and the calculating of this area ratio is the calculating of the area ratio of standard gaussian curve, as marks
The ratio between peak factor of quasi- Gaussian curve.The method with simple and fast, and can accurately calculate the ratio of area under the curve.
The inventive method focuses on rebuilding signal by using algorithm, has both eliminated the various interference such as noise
Influence, can also make signal processing undistorted again, can obtain high accuracy and low error result.I.e. the present invention can it is quick,
It is convenient and accurately measure concentration results.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of detection method；
Fig. 2 is the sample curves figure of raw sensor data of the present invention；
Fig. 3 is by the curve map after algorithm process and baseline correction；
Fig. 4 is the standard curve of the reagent strip that the present invention is used.
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.
The present invention is a kind of quantitative detecting method based on fluorescence immune chromatography technology, implementing procedure figure as shown in figure 1, tool
Body step is as follows：
1st, sample addition
At least 40 microlitres of sample to be tested solution is added dropwise on fluorescence immunoassay reagent strip, after standing 15 minutes, inserting instrument
In start detection.
2nd, hardware handles：
(1) lighting process：Using LED light source as the light source for being irradiated to test paper on reagent strip, LED light source is conducive to fluorescence
The portable and stabilization of immunity analysis instrument.The excitation wavelength of fluorescent material is mainly in 365nm or so on reagent strip, glimmering after exciting
Light launch wavelength is in 613nm or so.During from LED light source, launch wavelength is selected with the excitation wavelength identical of fluorescent material
The LED light source of 365nm.During detection reagent bar, LED light source is pointed into the test paper window of reagent strip, sends exciting light.Meanwhile, it is right
In selection and the excitation wavelength identical LED light source of fluorescent material, be conducive to obtaining the transmitting fluorescence higher of fluorescent material
Intensity, and cause that range of temperature interval diminishes, so as to reduce influence of the temperature to testing result.
(2) filtering process：Selection corresponding to fluorescent marker excitation wavelength and launch wavelength exciting light filter plate and
Launching light filter plate, to obtain preferable excitation wavelength and launch wavelength.Specific in example, LED light source send exciting light with
Afterwards, first pass through before LED be by optical wavelength 365 ± 20nm exciting light filter plate, filter other of non-excitation wavelength
LED light；Exciting light is irradiated on reagent strip, and the fluorescence labeling material on test paper launches fluorescence after being stimulated；That launches is glimmering
Light by photodetector front end be by optical wavelength 610 ± 20nm launching light optical filter, it is wiping out background light, spuious
Light, only passes through the launching light produced by fluorescent material to reduce interference；Ensure that the fluorescence letter that follow-up photodetector is obtained
Number purity.
(3) detection process：The photodetector as detection of this programme selection is photodiode, will can be detected
Fluorescence signal output be converted into electric signal, for the output of signal the advantages of because of its high stability, low-dark current, high sensitivity
There are the influence of minimum, and with low cost, fast response time, long service life；Will be by LED light source, optical filter, the pole of photoelectricity two
The optical system that pipe and light channel structure are constituted is sealed in optics camera bellows, for reducing the influence of veiling glare.Detection process
In, the motor of instrument internal drives test agent bar linear uniform motion, due to the difference of the fluorescent material distribution on reagent strip,
The fluorescence signal that photodetector is obtained on each test point on test paper is also different.
(4) signal transacting：Signal by being obtained in a upper detection process is further processed；First, will obtain
Current signal be input to I/V change-over circuits and be converted to voltage signal, reuse band-pass filter amplifier and amplify signal, while
Also increase signal to noise ratio；Then the analog electrical signal of high-precision amplification is obtained by detecting circuit.
(5) AD conversion treatment：Using fast, the low in energy consumption high-precision converter of conversion speed, preceding signal is processed
Later analog signal is converted to data signal；And will change come data signal according to each test point detection ordering according to
The secondary client software for passing to instrument, forms a collection sample curves (Fig. 2) for raw sensor data, is easy to calculation below
Method treatment.
3rd, baseline correction：Data after conversion carry out baseline correction.It is small due to being had in actual detection process
The reason for various influence factors such as temperature change, fluorescence intensity be uneven, the drift of curve is caused, have influence on final result
Calculate.So the system design employs least square fitting to carry out baseline correction to eliminate the shadow that curves shift is caused
Ring.
4th, L-M algorithm process：L-M algorithms are a kind of optimization algorithms.Data separate L-M algorithms after baseline correction are entered
Row treatment；Background fluorescence noise, secretly veiling glare, electricity that can be by data in the reagent strip existing for detection and hardware handles process
The error component of the influence signal identification of stream, the electromagnetic noise of motor etc. is filtered.And due to the randomness of its noise, the system
The L-M algorithms of use, that is, Nonlinear Least-Square Algorithm, can be very good to rebuild data, remove error component
Influence；And the undistorted of data can also be well kept in processing procedure, it is ensured that the accuracy of result.Made an uproar in elimination
Original property of signal can also be kept while sound, so as to improve signal to noise ratio.By figure after algorithm process and baseline correction such as
Shown in Fig. 3.
5th, areal calculation：Calculate the ratio of T peaks and C peak areas, the method that the system is used for：By above-mentioned correction stream
After journey and L-M algorithm process, the signal graph at T peaks and C peaks is fitted to the Gaussian curve of canonical form.The Gauss of canonical form
Curvilinear function is multiplied by a peak factor and constitutes by the Gaussian curve that area is 1, so its area ratio is exactly amplitude system
Several ratio.
T peaks fitting after Gaussian curve function be：
C peaks fitting after Gaussian curve function be：
The area ratio for going out T peaks and C peaks calculated by above-mentioned Gaussian curve function is：
The area ratio for obtaining is used as light intensity characteristic value；Simultaneously as the influence of the various factors in measurement process.Meeting
The fluctuation for causing the peak value at T peaks and C peaks overall, so selecting the knot that the C peaks at T peaks are under same state when processing and calculating
Fruit carries out areal calculation and ratio, and the method may also operate as certain noise when final result judges and eliminate and essence
Degree keeps.T, C peak area ratio are in the present embodiment：4.0145.
6th, concentration is calculated：Each reagent strip can demarcate a T/C light intensity characteristics value before dispatching from the factory and the standard of concentration is bent
Line, light intensity characteristic value i.e. T, C peak area ratio as obtained by T/C is calculated are substituted into curve, it is possible to obtained to be measured
Sample concentration.The standard curve of the reagent strip that this experiment is used is (1,0.04) (3.125,0.1293) (6.25,0.247)
(12.5,0.4932) (25,1.0119) (50,2.1012) (100,4.0145) (concentration unit is：μ g/mL) standard curve such as figure
4.Area ratio obtained by above calculating substitutes into standard curve, and being calculated concentration results is：104.3627 μ g/mL, experiment
In the concentration of actually used sample be：100μg/mL.Error range is only：4.36%.It can be seen that the high precision of algorithm
Degree and small error range.
The above implementation method is only that the preferred embodiment of the present invention is described, not to model of the invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, those of ordinary skill in the art are to technical side of the invention
Various modifications and improvement that case is made, all should fall into the protection domain of claims of the present invention determination.
Claims (7)
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 treatment, photodetector detection, Electric signal processing, AD conversion treatment, baseline correction, L-M algorithm process, area meter
The process that calculation, concentration are calculated.
2. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In：Described areal calculation is that primitive curve is fitted into two Gaussian curves of standard, by two songs after digital simulation
The peak factor ratio of line is so as to calculate original area under the curve ratio.
3. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 1, its feature exists
In：Least square method is selected in the baseline correction.
4. 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, starts LED light source irradiation reagent strip；
(3) for the excitation wavelength and launch wavelength of fluorescent marker, corresponding exciting light optical filter and launching light is selected to filter
Piece, wiping out background light, the interference of veiling glare；
(4) fluorescence signal that will be detected using photodetector is converted into electric signal；
(5) low current signal that photodetector is obtained further is processed, first passes through I/V change-over circuits and turn 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
The analog electrical signal of high-precision amplification；
(6) converter is used, the analog electrical signal after acquisition process is amplified is converted to data signal, and will change what is come
Data signal is combined into a collection sample curves for raw sensor data according to the detection ordering of each test point；
(7) data after AD conversion are carried out into baseline correction, uses least square fitting to carry out baseline correction to eliminate song
The influence that line drift is caused；
(8) raw sensor data after baseline correction is carried out into L-M treatment；
(9) after above-mentioned algorithm and correcting process are treated, the Gaussian curve at T peaks and C peaks is formd, two for being formed are high
This curve is written as the product of peak factor and the Gaussian curve that area is 1, and the area of Gaussian curve is than being exactly public Gaussian curve
The ratio of the peak factor in formula, while the result of ratio also is designated as into light intensity characteristic value；
(10) according to T/C light intensity characteristics value and concentration standard curve, the corresponding testing sample concentration of T/C ratios is calculated.
5. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 4, its feature exists
In：Step (2) the suitable LED light source of selection refers to selecting launch wavelength with the excitation wavelength identical of fluorescent material
LED light source.
6. a kind of quantitative determination computational methods based on fluorescence immune chromatography technology according to claim 4, its feature exists
In the computational methods detailed process of the light intensity characteristic value described in step (9), formula are as follows：
T peaks fitting after Gaussian curve function be：
C peaks fitting after Gaussian curve function be：
The T peaks and the area ratio at C peaks calculated by above-mentioned Gaussian curve function are：
7. according to a kind of any described quantitative determination computational methods based on fluorescence immune chromatography technology of claim 1-6, its
It is characterised by：Described photodetector is photodiode.
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Cited By (13)
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| CN109030321A (en) * | 2018-05-23 | 2018-12-18 | 中国科学院苏州生物医学工程技术研究所 | Data processing method for flow cytometer |
| CN109557300A (en) * | 2019-01-17 | 2019-04-02 | 湖北中医药高等专科学校 | A kind of full-automatic fluoroimmunoassay system and method |
| CN110927136A (en) * | 2019-12-25 | 2020-03-27 | 杭州微策生物技术有限公司 | Quality detection device of dry-type immunofluorescence POCT detection instrument and use method thereof |
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| CN111190004A (en) * | 2020-01-10 | 2020-05-22 | 上海泰辉生物科技有限公司 | Instant detection system for immunochromatography test strip |
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| CN112345502A (en) * | 2020-10-27 | 2021-02-09 | 天津市英贝特航天科技有限公司 | Detector for eliminating fluorescence noise in fluorescence immunoassay analyzer |
| 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 |
| CN113376384A (en) * | 2021-05-19 | 2021-09-10 | 棒糖科技(杭州)股份有限公司 | Method for calculating hormone concentration of fluorescent test paper |
| CN114544568A (en) * | 2022-01-14 | 2022-05-27 | 苏州和迈精密仪器有限公司 | Direct chemiluminescence fluorescence spectrum theoretical model |
| CN116973563A (en) * | 2023-09-22 | 2023-10-31 | 宁波奥丞生物科技有限公司 | Immunofluorescence chromatography determination method and device based on quadrature phase-locked amplification |
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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 |
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