CN107367610A - Fluorescence immune chromatography tests the processing method of base-line data - Google Patents

Abstract

The invention provides a kind of method for handling fluorescence immune chromatography test base-line data, including providing an immuno-chromatographic test paper strip, an excitation source and one is excited optical pickup device, the excitation source is set to produce exciting light, it is radiated on the near-end of the chromatograph test strip, so as to form hot spot, and the stimulated light sent by stimulated light reading device reading spot area；Hot spot is controlled, along the near-end of chromatograph test strip to distal direction, to be moved to the next position from current location, and read the stimulated light that spot area at the next position is sent in test strips by the control device；Light signal strength is excited based on reading, curve where determining baseline using fitting base-line method.

Description

Fluorescence immune chromatography tests the processing method of base-line data

Technical field

The invention belongs to detection field, specifically, at a kind of fluorescence immune chromatography test data optimization A kind of reason method, more particularly it relates to base-line data processing method of fluorometric investigation instrument.

Background technology

In the spectrogram of fluorescence detection equipment output, it will usually using the difference of signal value and baseline value as this group of sample Valid signal strengths, it can so eliminate due to caused by the systematic errors such as test piece lot number difference, immune response differences between batches Disturbing factor.Fluorescent microsphere in test strips using capillarity movement initial stage, because amounts of particles is more, often now base Line number is worth higher (the red dotted line baselines of such as Fig. 1 (b))；When passing through p-wire with fluorescent microsphere, with antibody binding on p-wire Afterwards, fluorescent microsphere quantity is reduced, and now baseline value is relatively low (the blue dotted line baselines of Fig. 1 (b)).When signal peak is higher, choosing One of uneven baseline or both average values at left and right sides of signal peak are taken, valid signal strengths are influenceed little；But work as signal peak When being worth relatively low, the different reading manner of baseline value, valid signal strengths result is disturbed extremely obvious.

In the spectrogram of output, the signal peak of varying strength is generally divided into high-value signal peak and lower value signals peak, such as Fig. 2 (a) and shown in Fig. 2 (b).

The mode migrated based on fluorescent microsphere in test strips, after the baseline value before signal peak would generally be than signal peak Baseline value is high, and this phenomenon is particularly evident on lower value signals peak, usually causes lower value signals peak that non-gaussian distribution state is presented.

Therefore, this area especially improves low there is an urgent need to work out Baseline Survey mode new in a kind of fluoroscopic examination The signal results in signal peak area, the degree of accuracy and sensitivity of numerical value are improved, so as to the degree of accuracy of optimizing detection result and sensitive Degree.

The content of the invention

It is an object of the invention to provide a kind of method of new determination fluoroscopic examination result baseline.

The first aspect of the present invention, there is provided a kind of method for handling fluorescence immune chromatography test base-line data, including step：

(1) one immuno-chromatographic test paper strip of offer, an excitation source and one are excited optical pickup device, wherein,

The excitation source is used to produce exciting light, and the exciting light is irradiated onto on the chromatograph test strip, so as to shape Into hot spot；

And described excitation source is furnished with control device, for controlling described immuno-chromatographic test paper strip and described exciting The relative position of light source, so that length direction of the hot spot along the chromatograph test strip moves；

Wherein, described immuno-chromatographic test paper strip is provided with p-wire, and the test strips length is L₀, width W₀, the survey Examination line length is L_t；

(2) excitation source is produced exciting light, be radiated on the near-end of the chromatograph test strip, so as to form light Spot, and the stimulated light sent by stimulated light reading device reading spot area；

(3) control hot spot in test strips along the near-end of chromatograph test strip to distal direction by the control device, from Current location is moved to the next position, and reads the stimulated light that spot area at the next position is sent；

(4) repeat step (3) Z-1 times, Z is >=10 positive integer, until the inswept p-wire of the hot spot；

(5) light signal strength is excited based on reading, if the arithmetic mean of instantaneous value of the signal strength values of n point is before signal peakIf the arithmetic mean of instantaneous value of the signal strength values of m point is after signal peakWhen When, adopt Curve where determining baseline with fitting base-line method, wherein, n >=2, and m >=2.

In another preference, whenWhen, using fitting base-line method Curve where determining baseline, wherein, I_peakFor the signal value of signal peak vertex correspondence.

In another preference, 3≤n≤12.

In another preference, 3≤m≤12.

In another preference, the difference of maxima and minima is Δ in n signal strength values before the signal peak_n, And Δ_n>=15% (I_peak–n_min)。

In another preference, after the signal peak in m signal strength values maxima and minima difference DELTA_m, and And Δ_m>=15% (I_peak–m_min)。

In another preference, in the step (5), the fitting base-line method calculates according to equation below (i)：

In formula, y₀, A₁, A₂, t₁, t₂For function parameter, y₀Represent offset, A₁And A₂It is pre-exponential factor, t₁And t₂Represent to relax The Henan time.In another preference, software used in fitting base-line method is the letter in the Origin8.0 that OriginLab companies release Number fitting function.

In another preference, described each time mobile moving step length is not equal or not etc., preferably equal.

In another preference, the preceding n point of described signal peak or rear m point are continuous or discrete, are preferably Continuously.

In another preference, the stimulated light is fluorescence.

In another preference, the Z is >=36 positive integer.

In another preference, in the step (1), the hot spot presses equal moving step length S_t0Inswept immunity-chromatography test The whole length of paper slip, the fluorescent value of N number of data point is read, and moving step length is determined by formula (ii)：

In another preference, the test line length L_tScope be 0.45-1.55mm, preferably 0.50- 1.50mm, it is more preferably 0.80-1.30mm, is most preferably 0.95-1.05mm.

In another preference, the test line width W₀Scope is 1.5-5.0mm, preferably 2.0-4.5mm, more preferably Ground is 2.5-4.0mm, is most preferably 3.0-3.5mm.

In another preference, the length L of the test strips₀Scope be 12.5-14.5mm, preferably 13.0- 14.0mm, it is more preferably 13.2-13.7mm.

In another preference, the scope of the data point N is 100-500, preferably 120-450, more preferably for 150-400, it is most preferably 170-300.

In another preference, the step-length S_tScope be 0.025-0.145mm, preferably 0.030-0.113, more It is 0.034-0.09mm goodly, is most preferably 0.045-0.079mm.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and specific in below (eg embodiment) It can be combined with each other between each technical characteristic of description, so as to form new or preferable technical scheme.As space is limited, herein No longer tire out one by one and state.

Brief description of the drawings

Fig. 1 shows fluorometric investigation instrument dependence test principle schematic, and (a) is that fluorescence lateral flow chromatographs immune response principle Schematic diagram, (b) are fluorometric investigation instrument Cleaning Principle schematic diagram.

Fig. 2 shows signal peak spectrogram, and (a) is high-value signal peak, and (b) is lower value signals peak.

Fig. 3 shows that mean value method handles baseline schematic diagram, and (a), (b) are whole story mean value method, after (c), (d) are preceding ten Ten mean value methods.

Fig. 4 shows the calculated by peak area mode of signal peak.

Fig. 5 shows fitting base-line method process signal peak base, and (a) is sample 1, and (b) is sample 2.

Fig. 6 shows that fitting base-line method calculates effective peak area of signal peak, and (a) is sample 1, and (b) is sample 2.

Fig. 7 shows calibration object experimental result, and (a) is mean value method, and (b) is fitting base-line method.

Embodiment

The present inventor and in-depth study, is surprised to find that a kind of new fluorescence immune chromatography test base first by extensive The processing method of line number evidence so that all data can correctly reflect useful signal value, i.e. signal value and baseline all on baseline The difference of value.The present invention is completed on this basis.

Term explanation

Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as art of the present invention with scientific terminology The identical meanings that are generally understood that of those of ordinary skill.

As used herein, in use, term " about " means that the value can be from enumerating in the numerical value specifically enumerated is mentioned Value, which changes, is not more than 1%.For example, as used herein, statement " about 100 " include 99 and 101 and between whole values (for example, 99.1st, 99.2,99.3,99.4 etc.).

As used herein, term " containing " or " including (including) " can be open, semi-enclosed and enclosed.Change Yan Zhi, the term also include " substantially by ... form " or " by ... form ".

Fluoroscopic examination

Fig. 1 shows that in double antibodies sandwich fluorescence detection method fluorescence immune chromatography tester produces fluorescence signal peak Principle schematic.

Fig. 1 (a) describes the double antibodies sandwich principle of the upper fluorescent-labeled microspheres of fluorescence lateral flow chromatography and antibody binding, mark Antibody on microballoon forms double antibodies sandwich compound by identifying another antibody capture being tested after object on film；Through Cross LASER Light Source to excite, feed back certain signal intensity, signal intensity is directly proportional to fluorescent microsphere quantity, reflects mesh in proportion Mark the content of thing.

Fig. 1 (b) is described after laser facula inswept p-wire, and detector feeds back the glimmering of dash area in the form of a voltage Optical signal, abscissa represent the data point (i.e. the distance of hot spot movement) that instrument is read, and ordinate represents the fluorescence of Voltage Feedback Signal.Meanwhile highest signal peak peak height value is shown in Fig. 1 (b).

Baseline Survey method

As used in the present invention, " fitting base-line method ", " matched curve method " have identical implication, are used interchangeably.

As shown in Fig. 2 (a) and (b), in spectrogram is exported, the signal peak of varying strength can be divided into high-value signal peak and low Value signal peak.Due to the mode that fluorescent microsphere migrates in test strips, baseline value before signal peak often than signal peak after Baseline value it is high, this phenomenon is particularly evident on lower value signals peak, usually causes lower value signals peak that non-gaussian distribution is presented State.

At present, there are two kinds of Baseline Survey methods in the prior art：

Method one：Whole story mean value method, from the average value of signal peak starting point and terminating point as baseline value (Fig. 3 (a)、(b))；

Method two：Ten mean value methods after preceding ten, take the average value and signal of the anterior continuous 3-10 data point of signal peak The average value of the continuous 3-10 data point in peak rear portion, then calculate the average value of the rwo as baseline value (Fig. 3 (c), (d))。

Above two method is that the signal value for reading instrument deducts baseline value (i.e. dotted line numerical value in Fig. 3), to count Calculate signal peak valid signal strengths, the obtained effective peak area of signal peak is shadow region as shown in Figure 4, i.e., baseline with On blue shading part be just effective peak area value, the red dash area below baseline is bears effect peak area value, most Effectively peak area value is the numerical difference of baseline above and below shaded area eventually.As shown in table 1, both processing methods are for height The influence at value signal peak is little, deviation about 1%, but the influence for lower value signals peak is clearly, deviation about 1900%, even There is irrational negative value.

Ten mean value method process signal peak area result after the whole story mean value method of table 1 and preceding ten

As can be seen that handling baseline using mean value method, partial data especially can calculate lower value signals under baseline Deviation during peak is larger, fitting base-line method of the invention so that all data can correctly reflect effective letter all on baseline Number value (difference of signal value and baseline value).

Main advantages of the present invention are：

1. the fitting base-line method of the present invention so that all data can correctly reflect useful signal value all on baseline (difference of signal value and baseline value).

2. using fitting base-line method processing data signal, it is particularly possible to the sensitivity and the degree of accuracy in low value area are improved, for Fluorometric reagent vitro detection is significant.

3. fitting base-line method is not limited to immune detection platform, it is equally applicable to handle other signal peaks as caused by particle Quantitative computational problem.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are mass percent and quality Number.

Experiment material and reagent used can obtain from commercially available channel unless otherwise instructed in following examples.

1. it is fitted baseline：

3~10 data points after 3~10 data points and signal peak are chosen before signal peak, do curve matching；

According to shown in table 2,2 groups of data (sample 1 and sample 2), each 180 data points of every group of data, intercept signal are chosen Peak region (the 33rd~71 data point), obtains curve shown in Fig. 2.

3~10 data points after 3~10 data points and signal peak are chosen before signal peak, are carried out curve fitting.It is high herein Each 10 data points before and after signal peak are chosen at value signal peak, and 9 data points before signal peak are chosen at lower value signals peak, 3 after signal peak Individual data point, fitting data point obtain curve shown in Fig. 5, and wherein Fig. 5 (a) is the data point curve-fitting results of sample 1, Fig. 5 (b) For the data point curve-fitting results of sample 2.

The initial data of the sample 1 of table 2 and sample 2

The matched curve model used is as follows for ExpAssoc functions, formula：

Curvilinear equation where baseline

In formula, x is data point everywhere；y₀, A₁, A₂, t₁, t₂For the relevant parameter of function, the function is that double exponential dampings are intended Close function, y₀Represent offset, A₁And A₂It is pre-exponential factor, represents amplitude, t₁And t₂Represent the relaxation time.

ExpAssoc exponential type functions, residual sum of squares (RSS) analysis is carried out according to principle of least square method in software, to ginseng After number sets initial value, the parameter information of curve where matching baseline is obtained by the iterative calculation of at least 50 times.

The sample data of Gaussian Profile (sample 1) and non-gaussian distribution (sample 2) can be handled according to pattern function, is fitted The equation parameter information that data point obtains is as shown in table 3.

The ExpAssoc function models of table 3 are fitted the parametric results of baseline

Y in table₀、A₁、t₁、A₂And t₂Parameter respectively in ExpAssoc functions, R²Represent the exponential function to baseline number The fitting effect at strong point, sees from the above, no matter sample is Gaussian Profile or non-gaussian distribution, fitting base-line method obtains Fitting effect can reach more than 0.99, i.e. residual sum of squares (RSS) very little.

2. calculate baseline value at each data point

After being fitted baseline curve by ExpAssoc function models, the baseline value at each data point is calculated, concrete numerical value is such as Shown in table 4：

The ExpAssoc models fitting base-line data values of table 4

3. calculate the useful signal value deducted at each data point after baseline

Signal intensity at each data point is deducted by the baseline value after the fitting of ExpAssoc function models, obtains the number Useful signal value T at strong point_e(formula (iii)), is as a result shown in Table 5 below.

Useful signal value T_e=signal value I_t- baseline value I_baselineFormula (iii)

In formula：Signal value I_tTo be excited light signal strength；

Baseline value I_baselineThe baseline value for often locating data point is calculated for ExpAssoc function models.

The useful signal value after baseline is deducted at 5 each data point of table

4. calculating two groups of data deducts effective peak area after baseline

Useful signal peak area

By taking sample 1 and sample 2 as an example,

Useful signal peak area

Effective peak area (Fig. 6) after deducting baseline is calculated, and by effective peak area and two kinds of average value described previously Method compares, as a result as shown in table 6.

As can be seen that curve where effective peak area can accurately find out baseline is calculated using the fitting base-line method of the present invention Equation, compared with mean value method, when handling high-value signal peak (sample 1), three kinds of method divergences are little, and deviation is less than 5%；And When handling lower value signals peak (sample 2), handled using mean value method, irrational negative result easily occur, and used and intend Closing baseline rule has fabulous improvement.

Effective peak area result at 6 three kinds of method process signal peaks of table

5. it is fitted base-line method processing calibration object data instance

5.1 calibration objects are tested

(1) that calibration object mother liquor is diluted into following concentration is stand-by：0、0.05、0.1、0.5、1ng/mL；

(2) after 0.4 μ L fluorescent microspheres (5mg/mL) are added in 0.5mL centrifuge tubes, the calibration that 90 μ L prepare concentration is added Product；

(3) 30s is blown and beaten repeatedly with liquid-transfering gun, take 60 μ L mixed liquors to be added in test strips, after reacting 900s, by test strips It is placed on fluorometric investigation instrument and reads result；

(4) each concentration parallel determination 5 times, the average value assessment performance of 5 results of final choice.

5.2 experimental data

Table 7 is calibration object experimental data, as can be seen that the data result handled using fitting base-line method from data It can count into all useful signal values, therefore its result can be slightly larger than the result of mean value method, but such calculation obtains The coefficient of variation (CV) can be smaller than mean value method, and will higher (i.e. Fig. 7 (b) using the sensitivity of fitting base-line method calculating data In slope it is relatively bigger), fitting degree (R²=0.9761) it is also relatively preferable.

The calibration object Data Processing in Experiment result of table 7

Mean value method result is calculated using ten mean value methods after preceding ten stated above herein.

6. method brief summary

It can be seen from the results above that a large amount of signal values can be effectively prevented from using fitting base-line method process signal intensity Below baseline, particularly with lower value signals peak, the size of effective peak area can be accurately obtained, improves the spirit in low value area Sensitivity and the degree of accuracy, it is significant for fluorometric reagent vitro detection.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims (10)

1. a kind of method for handling fluorescence immune chromatography test base-line data, including step：

(1) one immuno-chromatographic test paper strip of offer, an excitation source and one are excited optical pickup device, wherein,

The excitation source is used to produce exciting light, and the exciting light is irradiated onto on the chromatograph test strip, so as to form light Spot；

And described excitation source is furnished with control device, for controlling described immuno-chromatographic test paper strip and the excitation source Relative position so that length direction of the hot spot along the chromatograph test strip moves；

Wherein, described immuno-chromatographic test paper strip is provided with p-wire, and the test strips length is L₀, width W₀, the p-wire Length is L_t；

(2) excitation source is produced exciting light, be radiated on the near-end of the chromatograph test strip, so as to form hot spot, and The stimulated light sent by stimulated light reading device reading spot area；

(3) control hot spot in test strips along the near-end of chromatograph test strip to distal direction by the control device, from current Position is moved to the next position, and reads the stimulated light that spot area at the next position is sent；

(4) repeat step (3) Z-1 times, Z is >=10 positive integer, until the inswept p-wire of the hot spot；

(5) light signal strength is excited based on reading, if the arithmetic mean of instantaneous value of the signal strength values of n point is before signal peak If the arithmetic mean of instantaneous value of the signal strength values of m point is after signal peakWhen When, use Fitting base-line method determines curve where baseline, wherein, n >=2, and m >=2.

2. the method as described in claim 1, it is characterised in that when When, curve where determining baseline using fitting base-line method, wherein, I_peakFor the signal value of signal peak vertex correspondence.

3. the method as described in claim 1, it is characterised in that 3≤n≤12 and/or 3≤m≤12.

4. the method as described in claim 1, it is characterised in that before the signal peak in n signal strength values maximum with most The difference of small value is Δ_n, and Δ_n>=15% (I_peak–n_min)；And/or

After the signal peak in m signal strength values maxima and minima difference DELTA_m, and Δ_m>=15% (I_peak– m_min)。

5. the method as described in claim 1, it is characterised in that in the step (5), the fitting base-line method is according to following public affairs Formula (i) calculates：

<mrow> <msub> <mi>I</mi> <mrow> <mi>b</mi> <mi>a</mi> <mi>s</mi> <mi>e</mi> <mi>l</mi> <mi>i</mi> <mi>n</mi> <mi>e</mi> </mrow> </msub> <mo>=</mo> <msub> <mi>y</mi> <mn>0</mn> </msub> <mo>+</mo> <msub> <mi>A</mi> <mn>1</mn> </msub> <mrow> <mo>(</mo> <mn>1</mn> <mo>-</mo> <msup> <mi>e</mi> <mrow> <mo>-</mo> <mfrac> <mi>x</mi> <msub> <mi>t</mi> <mn>1</mn> </msub> </mfrac> </mrow> </msup> <mo>)</mo> </mrow> <mo>+</mo> <msub> <mi>A</mi> <mn>2</mn> </msub> <mrow> <mo>(</mo> <mn>1</mn> <mo>-</mo> <msup> <mi>e</mi> <mrow> <mo>-</mo> <mfrac> <mi>x</mi> <mrow> <mi>t</mi> <mn>2</mn> </mrow> </mfrac> </mrow> </msup> <mo>)</mo> </mrow> <mo>-</mo> <mo>-</mo> <mo>-</mo> <mrow> <mo>(</mo> <mi>i</mi> <mo>)</mo> </mrow> <mo>,</mo> </mrow>

In formula, y₀, A₁, A₂, t₁, t₂For function parameter, y₀Represent offset, A₁And A₂It is pre-exponential factor, t₁And t₂When representing relaxation Between.

6. the method as described in claim 1, it is characterised in that the moving step length of each time described movement be it is equal or 's.

7. the method as described in claim 1, it is characterised in that the preceding n point of described signal peak or rear m point are continuous or not Continuously.

8. the method as described in claim 1, it is characterised in that the Z is >=36 positive integer.

9. the method as described in claim 1, it is characterised in that in the step (1), the hot spot presses equal moving step length S_t0The whole length of inswept immuno-chromatographic test paper strip, the fluorescent value of N number of data point is read, and mobile step is determined by formula (ii) It is long：

<mrow> <msub> <mi>S</mi> <mrow> <mi>t</mi> <mn>0</mn> </mrow> </msub> <mo>=</mo> <mfrac> <msub> <mi>L</mi> <mn>0</mn> </msub> <mi>N</mi> </mfrac> <mo>-</mo> <mo>-</mo> <mo>-</mo> <mrow> <mo>(</mo> <mi>i</mi> <mi>i</mi> <mo>)</mo> </mrow> <mo>.</mo> </mrow>

10. the method as described in claim 1, it is characterised in that the test line length L_tScope be 0.45-1.55mm； And/or；

The test line width W₀Scope is 1.5-5.0mm；And/or

The length L of the test strips₀Scope be 12.5-14.5mm；And/or

The scope of the data point N is 100-500；And/or

The step-length S_tScope be 0.025-0.145mm.