CN108844951B - Mercury ion detection product and method and smartphone imaging analysis system - Google Patents
Mercury ion detection product and method and smartphone imaging analysis system Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention provides a mercury ion detection product and method and a smartphone imaging analysis system. The invention provides a lateral flow chromatographic sensor based on nucleic acid base mismatch, which comprises a sample No. SEQ ID in a sequence table: 1-3, and has good detection specificity and high sensitivity. The intelligent mobile phone imaging analysis system used in cooperation with the lateral flow chromatographic sensor can convert the detection result displayed on the lateral flow chromatographic sensor into the concentration value of mercury ions through a mobile phone, and the quantitative detection of the mercury ions in a sample is simply and efficiently realized. The mercury ion detection product, the mercury ion detection method and the intelligent mobile phone imaging analysis system are very suitable for untrained personnel to carry out field test, and provide great convenience for field tests of food safety, environmental safety and the like.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a mercury ion detection product and method and a smart phone imaging analysis system.
Background
The water-soluble divalent mercury ions are common heavy metal risk factors in food safety and drinking water safety, have strong biological enrichment and great harm to human bodies, and can damage nervous systems, digestive systems, brain tissues and kidney tissues even under the condition of extremely low concentration. Many countries and organizations regulate the maximum allowable upper limit of mercury ions in drinking water samples, for example, the World Health Organization (WHO) specifies that the maximum allowable limit of mercury ions in drinking water does not exceed 6ng mL-1(30nM), the Environmental Protection Agency (EPA), stipulates that an acceptable limit of mercury ions in drinking water is 2ng mL-1(10nM), European Union (EU) Drinking Water standards and the department of health of China all specify a maximum allowable limit of mercuric ions of not more than 1ng mL-1(5 nM). Therefore, the detection of trace mercury ions is a global concern. At present, the mainstream research trend is to construct a sensor for detecting mercury ions based on a "nucleic acid base mismatch" recognition system, which means that two thymine bases of DNA can be mismatched and combined with one mercury ion to form a stable "T-Hg (II) -T" structure.However, most of these sensors face the dilemma that the system is complicated and the quantitative detection is not easy.
As a novel rapid detection platform, the lateral flow chromatographic sensor has the characteristics of rapidness, simplicity, specificity, accuracy, sensitivity and the like. However, the current lateral flow chromatographic sensor can only realize qualitative or semi-quantitative detection, an additional special instrument is needed during quantitative detection, and a technology or a portable instrument which can directly read quantitative detection data from the lateral flow chromatographic sensor simply and conveniently does not exist at present.
Disclosure of Invention
The invention provides a mercury ion detection product, a mercury ion detection method and a smartphone imaging analysis system, which can convert a detection result displayed on a lateral flow chromatographic sensor into a concentration value of mercury ions through a mobile phone so as to at least realize simple and efficient quantitative detection of the concentration of the mercury ions in a sample. The nucleic acid base mismatch-based lateral flow chromatographic sensor and/or the intelligent mobile phone imaging analysis system used in cooperation are/is very suitable for untrained personnel to carry out field test, and provide great convenience for field detection of food safety, environmental safety and the like.
It is an object of the present invention to provide a composition comprising at least one of the following 1) to 3):
1) SEQ ID No: 1; or the SEQ ID No: 1 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID No: 1 has the same function; specifically, the function includes at least one of the following (1) to (4): (1) can specifically identify or combine with SEQ ID No: 3; (2) can specifically identify or combine the sequences of SEQ ID No: 3 through substitution and/or deletion and/or addition of one or more nucleotides; (3) when mercury exists, the mercury can react with mercury and a sample No. SEQ ID in a sequence table: 2 to form a T-Hg (II) -T structure; (4) when mercury exists, the mercury can react with mercury to form a mercury-free mercury alloy with the: 2, the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides to form a T-Hg (II) -T structure;
2) SEQ ID No: 2; or the SEQ ID No: 2 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID No: 2 has the same function; specifically, the function includes at least one of the following (1) to (2): (1) when mercury exists, the mercury can react with mercury and a sample No. SEQ ID in a sequence table: 1 forms a T-Hg (II) -T structure; (2) when mercury exists, the mercury can react with mercury to form a mercury-free mercury alloy with the: 1, the nucleotide sequence is subjected to substitution and/or deletion and/or addition of one or more nucleotides to form a T-Hg (II) -T structure;
3) SEQ ID No: 3; or the SEQ ID No: 3 is substituted and/or deleted and/or added by one or more nucleotides, and is compared with the nucleotide sequence shown in SEQ ID No: 3 has the same function. Specifically, the function includes at least one of the following (1) to (2): (1) can specifically identify or combine with SEQ ID No: 1; (2) can specifically identify or combine the sequences of SEQ ID No: 1 by substitution and/or deletion and/or addition of one or more nucleotides.
It is a further object of the invention to provide a lateral flow chromatographic sensor comprising the composition of any of the above.
It is yet another object of the present invention to provide a method for detecting mercury and/or mercury ions. The method comprises detecting using the composition or lateral flow sensor described above.
It is still another object of the present invention to provide a method for obtaining a concentration of an analyte from a lateral flow chromatographic sensor, in which a sample to be detected is dropped on a sample pad area of the lateral flow chromatographic sensor, and a detection result is displayed on a detection line of the lateral flow chromatographic sensor, and then a quantitative analysis is performed to obtain the concentration of the analyte, the method further comprising:
1) acquiring and/or displaying a detection image of a detection result of the lateral flow chromatography sensor through a mobile phone;
2) calculating and/or outputting a gray intensity value and a peak area S formed by a detection line region of the flow measuring chromatography sensor in the detection image;
3) inputting the quantitative detection standard curve S of the lateral flow chromatographic sensor into mobile phone software manually, wherein the curve S is 693.71lgC-1360.4, and R is20.9868, wherein lgC is a logarithm value of the concentration of the analyte, and S is the peak area S in the step 2);
4) substituting the peak area S value obtained in the step 2) into the quantitative detection standard curve in the step 3), calculating and outputting the concentration value of the object to be detected in the sample to be detected, and finishing the detection work;
wherein the calculation method of the gray intensity value formed by the detection line region of the flow measuring chromatographic sensor in the detection image and the peak area S value comprises the following steps:
taking the flowing direction of a sample to be detected on the flow measurement chromatographic sensor in the detection image as the direction of an abscissa, wherein the ordinate is vertical to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the detection image is recorded as a column gray intensity value Y, and a gray intensity P (x) function curve is established by using the obtained column gray intensity value Y and the abscissa value x;
the gray value Y calculation method comprises the following steps: y is 0.299R +0.587G +0.114B, where R, G, B is the R, G, B value of the pixel;
the gray scale intensity function curve of the detection image with the resolution of mxn is as follows:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the detected image;
and selecting a peak surface of the gray intensity function curve in the detection line area, and calculating the peak area through integration to obtain a peak area S.
It is a further object of the invention to provide a storage medium comprising a stored program, wherein any of the methods of the invention is performed by a processor when the program is run.
It is still another object of the present invention to provide a quantitative detection analysis system comprising:
the system comprises an image acquisition module, an image interception module, a regional image processing module and a standard curve module, wherein the image acquisition module is used for calling a camera to shoot images or reading images from a mobile phone storage device, the image interception module is used for intercepting a part needing to be detected in the images, the regional image processing module is used for calculating the pixel gray value of the part, constructing a gray intensity function, selecting a peak surface according to the gray intensity function and calculating the area S of the peak surface; the standard curve module is used for inputting a standard curve and calculating and/or outputting the concentration of a to-be-detected product;
the calculation method of the gray intensity function and the peak area S comprises the following steps:
taking the flowing direction of a sample to be detected in the image on the flow measurement paper-based chromatographic sensor as the direction of an abscissa, wherein the ordinate is vertical to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the image is recorded as a gray intensity value Y of a column, and a gray intensity function curve is established by using the obtained gray intensity value Y of the column and the abscissa value x;
the gray value Y calculation method comprises the following steps: y is 0.299R +0.587G +0.114B, where R, G, B is the R, G, B value of the pixel;
the gray scale intensity function curve of the image with the resolution of mxn is:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the image;
and selecting a peak surface of the gray intensity function curve in a detection line area on the flow measurement paper-based chromatographic sensor in the image, and integrating and calculating the peak area to obtain a peak area S.
Specifically, the method for obtaining the standard curve comprises the following steps:
1) providing a plurality of standard samples, wherein the concentration of the substance to be detected in the plurality of standard samples is diluted by the same multiple;
2) respectively detecting the plurality of standard sample samples by using a lateral flow paper-based chromatographic sensor, and respectively obtaining and/or displaying detection images of detection results of the lateral flow paper-based chromatographic sensor by using a mobile phone;
3) calculating and/or outputting a plurality of peak areas S formed by detection line regions of the flow measurement paper-based chromatographic sensor in the detection images of the plurality of standard samples;
4) and (3) taking the concentration value C of the object to be detected in a plurality of standard sample samples or the logarithm lgC of the concentration value C of the object to be detected as an abscissa, taking a plurality of peak areas S values corresponding to different concentration values of the object to be detected obtained in the step 3) as an ordinate to form a graph, obtaining a plurality of discrete points, connecting the plurality of discrete points into a straight line, wherein the slope of the straight line is the slope value a in a standard curve S (a x C + b) or S (a x lgC + b), and the intercept of the straight line and the abscissa is the intercept value b, wherein C is the concentration of the object to be detected and S is the peak area S.
It is a further object of the invention to provide a use of any of the compositions of the invention, any of the lateral flow sensor of the invention, any of the method of the invention, any of the storage medium of the invention, or any of the system of the invention.
Specifically, the application comprises qualitative detection or quantitative detection of mercury ions
Compared with other detection technologies, the detection method at least has the following advantages:
(1) nucleic acid base mismatch lateral flow chromatographic sensor: the gold nanoparticles are used as signals, a thymine base (T) -rich nucleic acid sequence capable of specifically recognizing mercury ions and the mercury ions in a water sample to be detected form a 'T-Hg (II) -T' structure, a red line which can be recognized by naked eyes is presented on a detection line, the depth of the line color is in positive correlation with the concentration of the mercury ions, and the problems of rapid recognition of the mercury ions in water and rapid conversion of the concentration of the mercury ions into reliable optical signals are at least solved.
(2) Imaging analysis system of smart phone: the system is developed based on an android system, comprises a human-computer interaction interface and an image processing algorithm design, and is used for realizing the rapid quantitative detection of the lateral flow chromatographic sensor, and a user can directly read the concentration value of a target object to be detected, which is detected by the lateral flow chromatographic sensor through the system, so that the problem that an instrument which is large in size, high in price and incapable of moving is additionally used in the traditional quantitative method is at least solved.
(3) The nucleic acid base mismatch-based lateral flow chromatography sensor and/or the intelligent mobile phone imaging analysis system used in cooperation only generate signal response to mercury ions, and the detection specificity is good; the lowest detection line which can be realized is 10nM mercury ion, the quantitative detection of the mercury ion in the liquid can be carried out in the linear range of 10nM to 1mM, and the detection sensitivity is high.
(4) The nucleic acid base mismatch-based lateral flow chromatographic sensor and/or the intelligent mobile phone imaging analysis system used in cooperation are/is very suitable for untrained personnel to carry out field test, and provide great convenience for field detection of food safety, environmental safety and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application, are incorporated in and constitute a part of this application, and are not intended to limit the application. In the drawings:
FIG. 1 is a schematic diagram of a nucleic acid base mismatch-based lateral flow chromatography sensor, in which reference numerals 1 to 5 denote a plastic lower liner, an NC membrane, a conjugate pad, a water-absorbent pad (paper), and a sample pad in this order.
FIG. 2 is a diagram showing the results of a specificity experiment of a nucleic acid base mismatch-based lateral flow chromatography sensor, in which reference numerals 1 to 13 denote the results of detection of Hg (II), Zn (II), Mg (II), Pb (II), Fe (III), Fe (II), Cu (II), K (I), Ca (II), Mn (II), Ag (I), Au (III) and Ni (II) solutions, respectively, in this order.
FIG. 3 is a photograph of a lateral flow sensor showing the results of detection.
Fig. 4 is a graph of optical density distribution.
FIG. 5 is a photograph showing the results of detection by the lateral flow chromatography sensor, in which reference numerals 0 to 9 denote the results of detection by the negative mercury ion concentration, 1nM, 10nM, 100nM, 1. mu.M, 10. mu.M, 100. mu.M, 1mM, 10mM, and 100mM, respectively, in this order.
FIG. 6 is an optical density distribution graph, in which reference numerals 0 to 9 denote negative, 1nM, 10nM, 100nM, 1. mu.M, 10. mu.M, 100. mu.M, 1mM, 10mM, and 100mM, respectively, in this order.
FIG. 7 is a graph showing the relationship between the peak area and the concentration of mercury ions in a mercury standard solution.
FIG. 8 is a standard graph of peak area versus mercury ion concentration.
FIG. 9 is a schematic diagram of a quantitative detection and analysis system.
Detailed Description
As used in the following examples, the experimental procedures used were all conventional ones unless otherwise specified.
The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples and their detailed description are presented to explain and understand the present application and are not to be construed as unduly limiting the present application.
Example 1 preparation of a lateral flow chromatography sensor based on nucleic acid base mismatches
(I) design of nucleotide sequence for detection
Sequence 1 (nucleotide sequence on gold nanoparticles): 5 '-Thiomo 6-GGTGGTGGTGGTGG-3'
Sequence 2 (nucleotide sequence on detection line): 5 '-Biotin-CCCCCCCTCCTCCTCCTCC-3'
Sequence 3 (nucleotide sequence on quality control line): 5 '-Biotin-CCCCCCCACCACCACCACC-3'
All the nucleotide sequences designed above were obtained by artificial synthesis. Wherein the sequence 1 is a sequence shown in SEQ ID No: 1 is obtained by thioMC6 sulfhydryl modification at the 5' end of the nucleotide sequence shown in the specification; the sequence 2 is the sequence of SEQ ID No: 2 is obtained after biotin labeling is carried out on the 5' end of the nucleotide sequence shown in the step (2); the sequence 3 is the sequence of SEQ ID No: 3 is labeled with biotin at the 5' end of the nucleotide sequence shown in the specification.
(II) preparation of lateral flow chromatography sensor based on nucleic acid base mismatch
1. The above designed thymine base-rich nucleic acid sequence designated as sequence 2 was immobilized on a detection Line (Test Line, T Line) of an NC membrane by a specific immobilization process in the following references Nan Cheng, Yuancong Xu, Kunlun Huang, Yuting Chen, Zhanshen Yang, Yunbo Luo, Wentao Xu, one-step compatible molecular flow biosensor running on an independent quantification system for sample phosphor base section in-situ detection of sample Hg (II) in water. food Chemistry,2017,214:169 175.
2. Coupling the designed thymine base-rich nucleic acid sequence named as sequence 1 with gold nanoparticles; the preparation and coupling process of gold nanoparticles can be referred to the reference in step 1.
3. The nucleic acid sequence coupled with the gold nanoparticles is immobilized on the bonding pad, and the specific immobilization process can refer to the reference in step 1.
4. The nucleotide sequence rich in adenine base designated as sequence 3 is immobilized on the Control Line (Line C) of NC membrane, and the reference in step 1 is also referred to for the specific immobilization process.
5. The prepared NC membrane and the prepared combining pad are prepared into the lateral flow chromatography sensor according to the existing method, and the method specifically comprises the following steps: as shown in fig. 1, the prepared NC film is fixed to the middle of the plastic lower liner 1; covering the prepared bonding pad to one end of the NC film 2 such that the bonding pad 3 is partially overlapped with the NC film 2; covering the other end of the NC film 2 with a water absorbent pad (paper) 4 so that the NC film 2 and the water absorbent pad (paper) 4 are partially overlapped; covering the sample pad 5 to the end of the conjugate pad 3 remote from the NC membrane 2, with the conjugate pad 3 partially overlapping the sample pad 5; and finally covering a protective film to prepare the lateral flow chromatography sensor for later use.
The NC membrane 2, the combination pad 3, the sample pad 5 and the absorbent pad (paper) 4 are made of materials such as a nitrocellulose membrane, a glass fiber membrane and absorbent paper in sequence.
(III) detection principle and process of lateral flow chromatographic sensor based on nucleic acid base mismatch
The detection principle of the nucleic acid base mismatch lateral flow chromatographic sensor is based on a sandwich structure (thymine base-rich nucleic acid sequence-mercury ion-thymine base-rich nucleic acid sequence), as shown in fig. 1, one thymine base-rich nucleic acid sequence is fixed on a detection line, the other thymine base-rich nucleic acid sequence is coupled with gold nanoparticles and fixed on a binding pad, and one adenine base-rich nucleic acid sequence is fixed on a quality control line. In a standard assay, a sample containing a concentration of mercury ions is first dropped onto a sample pad, after which the solution will move up to the conjugate pad due to capillary forces (i.e., suction from the absorbent pad or paper) in the direction of the chromatographic sensor; the compound which is coupled with the thymine-base-rich nucleic acid sequence and the gold nanoparticles on the combination pad continuously moves upwards along the direction of the chromatographic sensor to reach a detection line; on a detection line, mercury ions with a certain concentration in a sample are combined with two sections of thymine-base-rich nucleic acid sequences to form a T-Hg (II) -T structure, so that gold nanoparticles are grabbed and accumulated on the detection line, a red line which can be identified by naked eyes appears on the detection line, and the larger the concentration of the mercury ions in the sample is, the deeper the red color is; and the excessive compound coupled with the gold nanoparticles and the thymine-rich base nucleic acid sequence continuously moves upwards to reach the quality control line, the gold nanoparticles are grabbed and accumulated on the quality control line through the base complementary pairing combination of thymine and adenine, and a red line which can be identified by naked eyes appears on the quality control line. If the sample does not contain mercury ions with certain concentration, a T-Hg (II) -T structure cannot be formed on the detection line, and no gold nanoparticles are accumulated, so that a red line which can be identified by naked eyes cannot appear on the detection line.
Example 2 specificity test of lateral flow chromatography sensor based on nucleic acid base mismatch
The specificity of the sensor was tested by detecting different metal ion solutions using the nucleobase mismatch-based lateral flow chromatography sensor prepared in example 1, in which Hg (II) was 1. mu.M.other metal ions were 1 mM.
The sample to be tested is dropped on the sample pad, and after about 5 minutes, the lateral flow chromatography sensor can display the detection result. The result of the specificity experiment is shown in FIG. 2, the lateral flow chromatography sensor based on the nucleic acid base mismatch only generates signal response to mercury ions, and the method is proved to have good specificity.
When the lateral flow chromatography sensor according to the embodiment of the present disclosure, that is, the lateral flow chromatography sensor described in example 1, detects mercury ions, a red line appears on the detection line and/or the quality control line. Therefore, by using the image containing the red line and establishing a standard curve corresponding to the mercury ion concentration in the mobile phone, the mercury ion concentration can be quantitatively detected by using the shot image containing the red line (or the image stored in the mobile phone in advance or downloaded by the mobile phone).
Creation of a (first) standard curve
Preparing a series of mercury standard solutions with known concentrations by dilution according to multiple times, wherein the concentrations of mercury ions in different mercury standard solutions are 0, 1nM, 10nM, 100nM, 1 muM, 10 muM, 100 muM, 1mM, 10mM and 100mM in sequence.
The prepared mercury standard solutions with different concentrations are respectively dripped on 10 sample pads of the lateral flow chromatography sensor based on nucleic acid base mismatch prepared in example 1, and after about 5 minutes, the lateral flow chromatography sensor can show the detection result.
The camera of the mobile phone respectively photographs the detection results displayed by the lateral flow tomography sensor, so that an image showing a red line on the quality control line and/or the actual measurement line as shown in fig. 3 can be obtained, and the image can be an image pre-stored in the mobile phone or an image obtained by downloading through the mobile phone.
Then, taking the longitudinal extending direction of the lateral flow sensor as the abscissa and the average gray-scale value of the column corresponding to each abscissa as the ordinate, a curve image (i.e., an optical density distribution curve) as shown in fig. 4 can be obtained.
And selecting a curve of the position of the detection line, and calculating the peak area of the curve (namely performing integral operation on the curve), so as to obtain the peak area value corresponding to the specific mercury ion concentration.
In the above manner, from the results of photographing shown in fig. 5, the optical density distribution curve shown in fig. 6 was obtained, and from the obtained curve, peak area values corresponding to different mercury ion concentration values (for example, 0, 1nM, 10nM, 100nM, 1 μ M, 10 μ M, 100 μ M, 1mM, 10mM, 100mM) were respectively calculated. As shown in fig. 7, the obtained peak area was used to prepare a curve of the concentration value of mercury ions in a known mercury standard solution. Finally, a standard curve shown in fig. 8 is obtained through Excel manual fitting calculation: 693.71lgC-1360.4, R20.9868, wherein lgC is the logarithm of the concentration of mercury ions in the analyte, and S represents the peak area.
(II) establishment of imaging analysis system of smart phone
And (3) calculating the fitting to obtain a standard curve: 693.71lgC-1360.4 (R)20.9868) is built into the smartphone imaging analysis system;
dropping a sample to be detected on the sample pad, after about 5 minutes, displaying a detection result by the lateral flow chromatography sensor, and taking a picture of the detection result by a camera of the mobile phone to obtain a detection image, wherein the picture taking result can be stored in the mobile phone or can be directly used;
the calculation method for detecting the gray intensity value and the peak area S value in the image comprises the following steps:
taking the flowing direction of a sample to be detected on the flow measurement chromatographic sensor in the detection image as the direction of an abscissa, wherein the ordinate is vertical to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the detection image is recorded as a column gray intensity value Y, and a gray intensity P (x) function curve is established by using the obtained column gray intensity value Y and the abscissa value x;
the gray value Y calculation method comprises the following steps: y is 0.299R +0.587G +0.114B, where R, G, B is the R, G, B value of the pixel;
the gray scale intensity function curve of the detection image with the resolution of mxn is as follows:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the detected image;
and selecting a peak surface of the gray intensity function curve in the detection line area, and calculating the peak area through integration to obtain a peak area S.
The obtained peak area S is input to a built-in standard curve, and an output concentration value C, that is, a concentration value corresponding to the detection line in the photographed detection result photograph is output.
In a specific implementation scheme, the smartphone imaging analysis system of the embodiment can be developed and designed based on an android system.
(III) sensitivity of mercury ion quantitative detection of nucleic acid base mismatch-based lateral flow chromatographic sensor and intelligent mobile phone imaging analysis system
As shown in fig. 7 and 8, the peak area obtained by using the smartphone imaging analysis system according to the present embodiment has a good correlation with the mercury ion concentration; the lateral flow chromatography sensor based on nucleic acid base mismatch and the smartphone imaging analysis system of the embodiment can realize the lowest detection line of 10nM mercury ions, and the sensitivity is high; the linear range is 10nM to 1mM, and the mercury ion in water can be quantitatively detected in the range.
In addition, fig. 9 shows a schematic structural diagram of a quantitative detection analysis system, which includes:
the system comprises an image acquisition module, an image interception module, a regional image processing module and a standard curve module, wherein the image acquisition module is used for calling a camera to shoot images or reading images from a mobile phone storage device, the image interception module is used for intercepting a part needing to be detected in the images, the regional image processing module is used for calculating the pixel gray value of the part, constructing a gray intensity function, selecting a peak surface according to the gray intensity function and calculating the area S of the peak surface; the standard curve module is used for inputting a standard curve and calculating and/or outputting the concentration of a to-be-detected product;
the calculation method of the gray intensity function and the peak area S comprises the following steps:
taking the flowing direction of a sample to be detected in the image on the flow measurement paper-based chromatographic sensor as the direction of an abscissa, wherein the ordinate is vertical to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the image is recorded as a gray intensity value Y of a column, and a gray intensity function curve is established by using the obtained gray intensity value Y of the column and the abscissa value x;
the gray value Y calculation method comprises the following steps: y is 0.299R +0.587G +0.114B, where R, G, B is the R, G, B value of the pixel;
the gray scale intensity function curve of the image with the resolution of mxn is:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the image;
and selecting a peak surface of the gray intensity function curve in a detection line area on the flow measurement paper-based chromatographic sensor in the image, and integrating and calculating the peak area to obtain a peak area S.
The method for obtaining the standard curve comprises the following steps:
1) providing a plurality of standard samples, wherein the concentration of the substance to be detected in the plurality of standard samples is diluted by the same multiple;
2) respectively detecting the plurality of standard sample samples by using a lateral flow paper-based chromatographic sensor, and respectively obtaining and/or displaying detection images of detection results of the lateral flow paper-based chromatographic sensor by using a mobile phone;
3) calculating and/or outputting a plurality of peak areas S formed by detection line regions of the flow measurement paper-based chromatographic sensor in the detection images of the plurality of standard samples;
4) and (3) taking the concentration value C of the object to be detected in a plurality of standard sample samples or the logarithm lgC of the concentration value C of the object to be detected as an abscissa, taking a plurality of peak areas S values corresponding to different concentration values of the object to be detected obtained in the step 3) as an ordinate to form a graph, obtaining a plurality of discrete points, connecting the plurality of discrete points into a straight line, wherein the slope of the straight line is the slope value a in a standard curve S (a x C + b) or S (a x lgC + b), and the intercept of the straight line and the abscissa is the intercept value b, wherein C is the concentration of the object to be detected and S is the peak area S.
Claims (8)
1. A composition for detecting mercury and/or mercury ions, comprising the sequence:
sequence 1: 5 '-ThioMC 6-GGTGGTGGTGGTGG-3';
sequence 2: 5 '-Biotin-CCCCCCCTCCTCCTCCTCC-3';
and (3) sequence: 5 '-Biotin-CCCCCCCACCACCACCACC-3'.
2. A lateral flow chromatographic sensor comprising the composition of claim 1; the lateral flow chromatographic sensor takes gold nanoparticles as signals, a thymine base-rich nucleic acid sequence capable of specifically identifying mercury ions and mercury ions in a water sample to be detected form a 'T-Hg (I) -T' structure, a red line which can be identified by naked eyes is presented on a detection line, and the depth of the line color is in positive correlation with the concentration of the mercury ions; a red line which can be identified by naked eyes appears on the detection line, and the larger the concentration of mercury ions in the sample is, the deeper the red color is; the sample does not contain mercury ions, and no red line which can be identified by naked eyes appears on a detection line;
the lateral flow chromatography sensor comprises a quality control line and a detection line;
the lateral flow chromatography sensor further comprises the following 1) -4):
1) the nucleotide sequence shown in the sequence 2 is positioned on a detection line;
2) the lateral flow chromatography sensor also comprises a binding pad, wherein the nucleotide sequence shown in the sequence 1 is coupled with the gold nanoparticles and is positioned on the binding pad;
3) the nucleotide sequence shown in the sequence 3 is positioned on a quality control line;
4) the lateral flow sensor also comprises a low liner, an NC membrane, a combined pad, a water absorption pad or paper and a sample pad; the NC film is positioned in the middle of the low liner; the combining pad is overlapped with one end part of the NC film, and the absorbent pad or paper is overlapped with the other end part of the NC film; the sample pad overlaps with an end portion of the conjugate pad away from the NC film; the quality control line and the detection line are both positioned on the NC membrane, wherein the quality control line is positioned at one end close to the water absorption pad or the paper, and the detection line is positioned at one end close to the combination pad.
3. A method of detecting mercury and/or mercury ions, comprising detecting with the composition of claim 1 or with the lateral flow chromatography sensor of claim 2.
4. A method for obtaining a concentration of an analyte from the lateral flow sensor of claim 2, wherein a sample to be measured is dropped on a sample pad area of the lateral flow sensor, and a quantitative analysis is performed to obtain the concentration of the analyte after a detection result is displayed on a detection line of the lateral flow sensor, the method further comprising:
1) acquiring and/or displaying a detection image of a detection result of the lateral flow chromatography sensor through a mobile phone;
2) calculating and/or outputting a gray intensity value and a peak area S formed by a detection line region of the lateral flow tomography sensor in the detection image;
3) inputting the quantitative detection standard curve S of the lateral flow chromatographic sensor into mobile phone software manually, wherein the curve S is 693.71lgC-1360.4, and R is20.9868, wherein lgC is a logarithm value of the concentration of the analyte, and S is the peak area S in the step 2);
4) substituting the peak area S value obtained in the step 2) into the quantitative detection standard curve in the step 3), calculating and outputting the concentration value of the object to be detected in the sample to be detected, and finishing the detection work;
wherein the method for calculating the gray-scale intensity value and the peak area S value formed by the detection line region of the lateral flow tomography sensor in the detection image comprises the following steps:
taking the flowing direction of a sample to be detected on the lateral flow chromatographic sensor in the detection image as the direction of an abscissa, wherein the ordinate is vertical to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the detection image is recorded as a column gray intensity value Y, and a gray intensity P (x) function curve is established by using the obtained column gray intensity value Y and the abscissa value x;
the gray value Y calculation method comprises the following steps: y is 0.299R +0.587G +0.114B, where R, G, B is the R, G, B value of the pixel;
wherein, the gray scale intensity function curve of the detected image with the resolution of mxn is:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the detected image;
and selecting the peak area of the gray intensity function curve in the detection line area, and calculating the peak area by integration to obtain the peak area S.
5. A storage medium comprising a stored program, wherein the method of claim 4 is performed by a processor when the program is run.
6. A quantitative detection analysis system, comprising:
the system comprises an image acquisition module, an image interception module, an area image processing module and a standard curve module, wherein the image acquisition module is used for calling a camera to shoot images or reading images from a mobile phone storage device, the image interception module is used for intercepting parts needing to be detected in the images, the area image processing module is used for calculating pixel gray values of the parts needing to be detected in the images, constructing a gray intensity function, selecting a peak surface according to the gray intensity function and calculating a peak area S; the standard curve module is used for inputting a standard curve and calculating and/or outputting the concentration of a to-be-detected product;
the calculation method of the gray intensity function and the peak area S comprises the following steps:
taking the flowing direction of a sample to be detected on the lateral flow chromatography sensor as claimed in claim 2 in the image as the direction of an abscissa, the ordinate is perpendicular to the abscissa, the average value of gray values Y at all ordinate positions with the same abscissa x in the image is recorded as a column gray intensity value Y, and a gray intensity function curve is established by using the obtained column gray intensity value Y and the abscissa value x;
wherein, the gray scale intensity function curve of the image with the resolution of m multiplied by n is as follows:
in the gray intensity function curve, Y is a gray value, x is an abscissa value, Y is an ordinate value, and m and n are the resolution of the image;
and selecting the peak area of the gray scale intensity function curve in the detection line area on the lateral flow tomography sensor in the image, and calculating the peak area through integration to obtain the peak area S.
7. The system of claim 6, wherein the standard curve is obtained by a method comprising:
1) providing a plurality of standard samples, wherein the concentration of the substance to be detected in the plurality of standard samples is diluted by the same multiple;
2) respectively detecting the plurality of standard samples by using a lateral flow chromatography sensor, and respectively acquiring and/or displaying detection images of detection results of the lateral flow chromatography sensor by using a mobile phone;
3) calculating and/or outputting a plurality of peak areas S formed by detection line regions of the lateral flow tomography sensor in the detection images of the plurality of standard samples;
4) and (3) taking the concentration value C of the object to be detected in a plurality of standard sample samples or the logarithm lgC of the concentration value C of the object to be detected as an abscissa, taking a plurality of peak areas S values corresponding to different concentration values of the object to be detected obtained in the step 3) as an ordinate to form a graph, obtaining a plurality of discrete points, connecting the plurality of discrete points into a straight line, wherein the slope of the straight line is the slope value a in a standard curve S (a x C + b) or S (a x lgC + b), and the intercept of the straight line and the abscissa is the intercept value b, wherein C is the concentration of the object to be detected and S is the peak area S.
8. Use of the composition of claim 1, the lateral flow chromatography sensor of claim 2, the method of claim 3 or 4, the storage medium of claim 5, the system of claim 6 or 7 for mercury ion detection.
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