1. A method for detecting heavy metal ions by combining a complexing colorimetric array microfluidic paper chip with a smart phone is characterized by comprising the following steps:

(1) preparing a paper chip device: the paper chip comprises a central sample adding area, six channels respectively communicated with the central sample adding area and a waste liquid area communicated with the six channels; the channels are used for embedding complexing reagents, wherein the central sample adding area, the six channels and the waste liquid area are channels with hydrophilic properties, and the rest areas of the paper chip are channels with hydrophobic properties;

(2) obtaining a Linear Discriminant Analysis (LDA) profile of a known type of metal ion: respectively dripping six complexing reagent solutions into the passage areas on the paper chip obtained in the step (1), and drying for later use after the six passages are fully diffused; respectively dropwise adding solutions containing heavy metal ions with different concentrations into the central sample adding area to react with the complexing agent array on the paper chip obtained in the step (1) to generate a color development reaction, collecting RGB value data of color development photos before and after the reaction, and analyzing the collected map data to obtain an LDA map of the known metal ions;

(3) detecting heavy metal ions in a water sample to be detected: respectively dripping six complexing reagent solutions at the passages on the paper chip obtained in the step (1), drying for later use after the six passages are fully diffused, dripping a filtered water sample to be detected to a central sample adding area, standing to enable the water sample to be reacted with the complexing agent array on the paper chip obtained in the step (1), photographing the paper chip before and after the reaction by using a smart phone, taking colors of the photos by using software to obtain RGB values, carrying out LDA analysis on the RGB values, and comparing the RGB values with the LDA atlas of the known type metal ions obtained in the step (2), so that whether the water sample to be detected contains the heavy metal ions can be judged.

2. The method according to claim 1, wherein in step (1), the complexing reagent comprises six, namely, a thiomizone solution, a 4- (2-pyridylazo) -1, 3-benzenediol solution, a bathocuproine solution, a cadmium reagent alkaline solution, a xylenol orange solution and a dibenzoyl dihydrazide solution.

3. The method according to claim 2, wherein the concentration of the thiomicone solution is 1 to 5mmol/L, and the solvent is 95% ethanol water; the concentration of the 4- (2-pyridylazo) -1, 3-benzenediol solution is 1-5 mmol/L, and the solvent is 95% ethanol water; the concentration of the bathocuproine solution is 1-5 mmol/L, and the solvent is 95% of ethanol water; the concentration of the cadmium reagent alkaline solution is 1-5 mmol/L, and the solvent is 0.2mol/L potassium hydroxide-ethanol; the concentration of the xylenol orange solution is 1-5 mmol/L, and the solvent is water; the concentration of the diphenyl carbonyl dihydrazide solution is 1-20 mmol/L, and the solvent is water.

4. The method according to any one of claims 1 to 3, wherein in the step (2), the heavy metal ions comprise Pb²⁺，Cd²⁺，Hg²⁺And Cu²⁺One or more of (a).

5. The method according to any one of claims 1 to 4, wherein in the step (3), the software for color extraction is Photoshop software, and the software for LDA analysis is IBM SPSS Statistics 22.

6. Use of the method of any one of claims 1 to 5 for detecting heavy metal ions in soil, wastewater, lake water and tap water.

7. A device for detecting heavy metal ions in a water sample is characterized by comprising a paper chip and six complexing reagents, wherein the paper chip comprises a central sample adding area, six passages respectively communicated with the central sample adding area and a waste liquid area communicated with the six passages, the passages are used for embedding the complexing reagents, the central sample adding area, the six passages and the waste liquid area are channels with hydrophilic properties, and the rest areas of the paper chip are channels with hydrophobic properties; the six complexing reagents are a thiomicotin solution, a 4- (2-pyridylazo) -1, 3-benzenediol solution, a bathocuproine solution, a cadmium reagent alkaline solution, a xylenol orange solution and a dibenzoyl dihydrazide solution respectively, and the six complexing reagents are expanded into six paths respectively.

8. The device for detecting the heavy metal ions in the water sample according to claim 7, wherein the concentration of the thiomizone solution is 1-5 mmol/L, and the solvent is 95% ethanol water; the concentration of the 4- (2-pyridylazo) -1, 3-benzenediol solution is 1-5 mmol/L, and the solvent is 95% ethanol water; the concentration of the bathocuproine solution is 1-5 mmol/L, and the solvent is 95% of ethanol water; the concentration of the cadmium reagent alkaline solution is 1-5 mmol/L, and the solvent is 0.2mol/L potassium hydroxide-ethanol; the concentration of the xylenol orange solution is 1-5 mmol/L, and the solvent is water; the concentration of the diphenyl carbonyl dihydrazide solution is 1-20 mmol/L, and the solvent is water.

9. The apparatus of claim 7, wherein the heavy metal ions comprise Pb²⁺，Cd²⁺，Hg²⁺And Cu²⁺One or more of (a).

10. The application of the device for detecting heavy metal ions in water sample according to any one of claims 7 to 9 in the aspect of detecting heavy metal ions in soil, wastewater, lake water and tap water.

Background

In the 50 s of the 20 th century, Japan, due to Hg²⁺And Cd²⁺The water caused by pollution preferably causes diseases, osteodynia and other events, and heavy metal pollution is more and more concerned. All in oneIn the 50 s, due to environmental problems, the new subject of environmental science emerges and develops gradually, and the research on heavy metals is the first place of the research on pollutants of environmental ecology regardless of foreign environmental protection ecology or domestic pollution ecology. The presence of heavy metals in various ecosystems has serious effects on organisms, and the heavy metals are difficult to degrade and are extremely dangerous along with the characteristic of food chain enrichment. Therefore, the research of heavy metals is very important for the environmental ecology and the health of organisms.

At present, heavy metal detection methods can be mainly divided into two categories of traditional detection and rapid detection, wherein the content of heavy metal in a sample can be accurately known by means of detection of a precision instrument, and the method has the advantages of good sensitivity, high accuracy and the like, is very suitable for trace detection, and simultaneously needs expensive instruments, complex pretreatment and professional technicians. The rapid detection methods are concerned in recent years, can realize rapid field detection of heavy metal ions, have the advantages of rapidness, simple operation, uncomplicated sample pretreatment and the like, but cannot achieve the sensitivity and accuracy of detection of a precision instrument, and can only realize qualitative semi-quantitative detection in most cases.

With the improvement of the economy, development and industrialization level of China, especially in underdeveloped areas, a large amount of factory wastewater is directly or detected to be discharged into environmental water, and the areas do not have the condition of laboratory detection. In recent years, too many serious metal pollution events are developed in China, such as the event that the blood lead of children in Cambodia county in south of lake exceeds standard, the soil pollution events of cadmium rice, sunshine and the like, and the like. Therefore, the situation of heavy metal pollution in China still needs to be prevented and remedied, so that the rapid monitoring of the heavy metal ion pollution situation is very necessary, and the establishment of a method for rapidly detecting the heavy metal ions on site is very necessary.

The primary difference between array detection and conventional detection methods is that arrays utilize multiple sensing elements to interact with multiple analytes simultaneously to identify bulk changes in complex mixtures, rather than specific elements thereof. And thus are particularly powerful for identification in complex analytes. In addition, due to the developments in chemical analysis of statistics and chemometrics, and the recognition that many complex sensors cannot be addressed by traditional methods, sensors based on rich data output of arrays are being widely accepted and used by the analytical community. In particular, in a colorimetric array, the sensor relates to mutual identification of an analyte and a dye, and generates color change visible to the naked eye, the method is quick, simple and convenient, and the signal acquisition is visual. In previous researches, the complex colorimetric array is found to be capable of distinguishing multiple heavy metal ions simultaneously (application number 202011387904.6), however, the detection depends on related professional instruments and laboratory conditions, and the portable field detection cannot be realized.

Paper-based microfluidics has developed rapidly in recent decades, particularly in the field of life sciences. The microfluidic paper-based detection platform can realize low-cost, portable and simple analysis and detection and integrates enrichment, separation and detection into a whole. The micro-fluidic construction on the filter paper mainly distinguishes hydrophilic and hydrophobic channels, and different hydrophobic materials are solidified on the paper. There are several significant advantages to testing via paper-based: 1) the liquid self-conveying at a certain distance can be realized without external power; 2) the filter paper is made of cellulose, has high cellulose content and is compact, and can store trace substances; 3) low cost, and easy transportation and storage. The incorporation of paper-based into microfluidics is very advantageous for the presentation of colorimetric arrays. Therefore, the complexing array is combined with paper-based microfluidics to explore the possibility of realizing the on-site detection of heavy metal ions.

Disclosure of Invention

In order to realize the purpose, the invention screens a proper complexing reagent to form an array, embeds the complexing array in a paper-based micro-fluidic chip and constructs the micro-fluidic paper chip to detect Hg²⁺、Cd²⁺、Pb²⁺And Cu²⁺The four heavy metal ions are photographed by using a common smart phone and combined with an RGB (red, green and blue) standardized color system to realize signal acquisition and data analysis, a database and a model are established by using LDA (Linear discriminant analysis) to a standard solution (training set) of the heavy metal ions, and the distinguishing capability of an array in an actual water sample to the heavy metal ions is compared with the database and the model, so that the existence of the heavy metal ions can be rapidly judged in an off-line manner.

The invention aims to provide a method for detecting heavy metal ions by combining a complex colorimetric array microfluidic paper chip with a smart phone, which comprises the following steps:

(1) preparing a paper chip device: the paper chip comprises a central sample adding area, six passages which are respectively communicated with the central sample adding area and a waste liquid area which is communicated with the six passages, wherein the passages are used for embedding complexing reagents, the central sample adding area, the six passages and the waste liquid area are channels with hydrophilic properties, and the rest areas of the paper chip are channels with hydrophobic properties;

(2) obtaining a Linear Discriminant Analysis (LDA) profile of a known type of metal ion: respectively dripping six complexing reagent solutions into the passage area on the paper chip obtained in the step (1), drying for later use after the six passages are fully diffused, respectively dripping solutions containing heavy metal ions with different concentrations into a central sample adding area to react with the complexing agent array on the paper chip obtained in the step (1) to generate a color reaction, collecting RGB (red, green and blue) value data of color photos before and after the reaction, and analyzing the collected map data to obtain an LDA (laser direct absorption) map of the known type metal ions;

(3) detecting heavy metal ions in a water sample to be detected: respectively dripping six complexing reagent solutions at the passages on the paper chip obtained in the step (1), drying for later use after the six passages are fully diffused, dripping a filtered water sample to be detected to a central sample adding area, standing to enable the water sample to be reacted with the complexing agent array on the paper chip obtained in the step (1), photographing the paper chip before and after the reaction by using a smart phone, taking colors of the photos by using software to obtain RGB values, carrying out LDA analysis on the RGB values, and comparing the RGB values with the LDA atlas of the known type metal ions obtained in the step (2), so that whether the water sample to be detected contains the heavy metal ions can be judged.

In one embodiment of the present invention, in step (1), the paper material for the paper chip includes filter paper, nitrocellulose membrane, Polydimethylsiloxane (PDMS), etc., preferably Whatman filter paper.

In one embodiment of the present invention, in step (1), the hydrophilic pathway is used to ensure the flow of liquid, and the hydrophobic pathway may be made hydrophobic by using hydrophobic wax, so as to block the flow of liquid and control the flow.

In one embodiment of the present invention, in step (1), the complexing reagent comprises six, which are Thiomicotin (TMK) solution, 4- (2-pyridylazo) -1, 3-benzenediol (PAR) solution, Bathocuproine (BCP) solution, cadmium reagent (CDI) alkaline solution, Xylenol Orange (XO) solution, and dibenzoyl Dihydrazide (DPC) solution.

In one embodiment of the invention, the concentration of the Thiomicotin (TMK) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the 4- (2-pyridylazo) -1, 3-benzenediol (PAR) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the Bathocuproine (BCP) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the cadmium reagent (CDI) alkaline solution is 1 to 5mmol/L, preferably 2mmol/L, and the solvent is 0.2mol/L potassium hydroxide-ethanol.

In one embodiment of the invention, the concentration of the Xylenol Orange (XO) solution is 1 to 5mmol/L, preferably 3mmol/L, and the solvent is water.

In one embodiment of the invention, the concentration of the dibenzoyl Dihydrazide (DPC) solution is 1-20 mmol/L, preferably 15mmol/L, and the solvent is water.

In one embodiment of the present invention, in step (1), the paper chip preparation apparatus may be implemented by: firstly heating and melting beeswax into liquid, fully immersing one piece of filter paper in the liquid for several seconds, taking out the filter paper and airing the filter paper for later use, stacking the filter paper soaked with the wax and blank filter paper, then heating a high-temperature metal module with a pattern corresponding to the paper chip to 150 ℃, quickly pressing the two pieces of filter paper for several seconds and then removing the filter paper, and separating the two pieces of filter paper to obtain a corresponding passage with hydrophilic and hydrophobic properties on the lower layer of filter paper.

In one embodiment of the present invention, in the step (2), the heavy metal ion includes Pb²⁺，Cd²⁺，Hg²⁺And Cu²⁺The concentration of the solution containing heavy metal ions with different concentrations can be 2, 10, 20, 50, 100 and 500 mu mol/L.

In one embodiment of the present invention, in step (3), the software for color extraction may be Photoshop software; preferably, the RGB values are acquired, and the RGB values of 5 x 5 pixel points in the center of the reaction area are acquired; the software used for LDA analysis was IBM SPSS Statistics 22.

The invention also provides a device for detecting heavy metal ions in a water sample, which comprises a paper chip and six complexing reagents, wherein the paper chip comprises a central sample adding area, six passages respectively communicated with the central sample adding area and a waste liquid area communicated with the six passages, the passages are used for embedding the complexing reagents, the central sample adding area, the six passages and the waste liquid area are channels with hydrophilic property, and the rest areas of the paper chip are channels with hydrophobic property; the six complexing reagents are a Thiomicotin (TMK) solution, a 4- (2-pyridylazo) -1, 3-benzenediol (PAR) solution, a Bathocuproine (BCP) solution, a cadmium reagent (CDI) alkaline solution, a Xylenol Orange (XO) solution and a dibenzoyl Dihydrazide (DPC) solution respectively, and the six complexing reagents are respectively expanded into six paths.

In one embodiment of the invention, the concentration of the Thiomicotin (TMK) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the 4- (2-pyridylazo) -1, 3-benzenediol (PAR) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the Bathocuproine (BCP) solution is 1-5 mmol/L, preferably 4mmol/L, and the solvent is 95% ethanol water.

In one embodiment of the invention, the concentration of the cadmium reagent (CDI) alkaline solution is 1 to 5mmol/L, preferably 2mmol/L, and the solvent is 0.2mol/L potassium hydroxide-ethanol.

In one embodiment of the invention, the concentration of the Xylenol Orange (XO) solution is 1 to 5mmol/L, preferably 3mmol/L, and the solvent is water.

In one embodiment of the invention, the concentration of the dibenzoyl Dihydrazide (DPC) solution is 1-20 mmol/L, preferably 15mmol/L, and the solvent is water.

The invention also provides application of the water sample detection device in detection of heavy metal ions in soil, wastewater, lake water and tap water.

Compared with the prior art, the invention has the following advantages and effects:

the invention constructs a micro-fluidic paper chip based on a complex colorimetric array, is used for identifying four single heavy metal ions in a water sample by combining with a smart phone for photographing, has the accuracy rate of 100 percent and has the detection limit of 2 mu mol/L. Meanwhile, the distinguishing and sensing of the multi-component mixture of the four heavy metal ions are realized, and the anti-interference performance is better. Based on the method, the actual water sample is further sensed to discover the Cd2+, Cu2+, Hg2+ and Pb2+ in the lake water and the tap water, and the method has important significance for realizing the field detection of the heavy metal ions. The method greatly reduces the operation difficulty, can finish signal acquisition by using a mobile phone, breaks away from the environmental restriction of a laboratory, and is expected to realize field detection. In addition, the amount of the array reagent and the sample reagent is in the microliter level, so that the cost and the related pollution are effectively reduced.

Drawings

FIG. 1 is a schematic view of a microfluidic paper-based detection process.

Fig. 2 is a schematic diagram of a metal module design (a) in three views and (B) in 3D.

Fig. 3 is a schematic view of microfluidic paper chip device fabrication.

FIG. 4(A) CDI 20. mu. mol/LCd at different concentrations²⁺Euclidean distance after reaction; (B) different concentrations of DPC and 50. mu. mol/LHg²⁺Euclidean distance after reaction (inset is the photo after complexing reagent and reaction with ion).

FIG. 5(A) different concentrations of TMK and 50. mu. mol/LHg²⁺Euclidean distance after reaction; (B) different concentrations of PAR and 50. mu. mol/LCu²⁺And 50. mu. mol/LPb²⁺After reactionThe distance from the probe (inset is the photograph of the complexing reagent and the ion after reaction).

FIG. 6 different concentrations (A) of XO with 50. mu. mol/LPb²⁺And (B) BCP with 50. mu. mol/LCu²⁺Euclidean distance after reaction (inset is the photo after complexing reagent and reaction with ion).

FIG. 7(A) schematic of paper-based array, (B) microfluidic paper-based array and 500. mu. mol/LCd²⁺，Cu²⁺，Hg²⁺，Pb²⁺Photograph after reaction.

FIG. 82-500. mu. mol/L of LDA analysis chart after reaction of heavy metal ions with the array.

Figure 9 LDA graph of response of paper-based complexing array to different concentrations of multi-metal ion mixture.

FIG. 10 is a photograph of a paper-based complexing array reacted with 13 ions that may be present in water at 500. mu. mol/L.

FIG. 11 is a graph based on the LDA paper-based array and the identification of the reaction of four concentrations (A) 1.5. mu. mol/L, (B) 2. mu. mol/L, (C) 2.5. mu. mol/L, (D) 5. mu. mol/L of heavy metal ions.

FIG. 12 is a graph of LDA analysis in the presence of 1. mu. mol/L of four heavy metal ions in lake water and tap water, respectively.

FIG. 13(A) Complex array with 200. mu. mol/LCd²⁺，Cu²⁺，Hg²⁺，Pb²⁺Pictures after reaction, wherein 1-6 are six complexing reagent detection solutions of CDI, DPC, TMK, VBB, XO and BCP respectively; (B) complex arrays of the invention with 200. mu. mol/LCd²⁺，Cu²⁺，Hg²⁺，Pb²⁺Photograph after reaction.

FIG. 14 LDA graph of six complexing reagents for detecting single heavy metal ion in comparative example 1

FIG. 15 other complexing agents (A) dithizone and (B) chrome Black T with 200. mu. mol/LCd²⁺，Cu²⁺，Hg²⁺，Pb²⁺Photograph after reaction.

Detailed Description

White beeswax, pigment for powdered wax, Whatman filter paper Grade 1.

Preparing a metal ion stock solution with the concentration of 10mmol/L, wherein the solvent is ethanol water (1: 1) which is four solutions of cadmium nitrate, lead nitrate, copper nitrate and mercury chloride respectively.

Fig. 1 is a schematic view of a microfluidic paper-based detection process according to the present invention, which is further described below with reference to examples, but embodiments of the present invention are not limited thereto.

Example 1 apparatus for preparing paper chips

(1) Designing a paper chip device: the complexing array is composed of six reagents, so that a six-channel embedding complexing reagent needs to be designed except for a central sample adding region, and a waste liquid region is arranged at the channel terminal so as to bear excessive uncomplexed sample liquid. The sample adding area, the channel and the waste liquid area are hydrophilic, so that the flow of liquid can be ensured, and the rest areas are covered with hydrophobic wax to block the flow direction of the liquid and control the flow direction.

(2) Design of the stamped metal module: and in the area covered by the hydrophobic wax, stamping a piece of wax paper by using a stamping method, namely stamping a metal module with a corresponding pattern at a high temperature, and printing the wax on the lower blank filter paper after the wax is melted at the high temperature, thereby obtaining the microfluidic paper chip with a corresponding channel. Therefore need design the metal module that has corresponding passageway pattern, metal module bottom sunk part is hydrophilic region, and the bulge is the region that the wax covered, can melt wax when the punching press and press to below filter paper on, increases the handle in addition in order conveniently to take, because the pattern is special, this metal module of 3D printing preparation, the design is as shown in fig. 2.

(3) Manufacturing of paper chip: firstly, heating and melting beeswax into liquid, fully immersing a piece of filter paper in the liquid for several seconds, clamping the filter paper by using tweezers, and airing the filter paper for later use. The filter paper soaked with the wax and the blank filter paper are stacked, then the metal module is heated to about 150 ℃, the two filter papers are quickly pressed for a plurality of seconds and then removed, the two filter papers are separated, and corresponding hydrophilic and hydrophobic channels can be obtained on the lower layer of filter paper (figure 3).

EXAMPLE 2 selection of reagent concentration for paper-based Complex arrays

In order to ensure that the paper-based complex array has obvious color change in an adjustable range so as to be beneficial to further analyzing the color development effect, the embodiment optimizes the color development condition of the paper-based complex array.

The euclidean distance is selected as a main index for condition optimization in this embodiment (the euclidean distance, also called as euclidean distance, defined as distance, refers to the true distance between two points in the m-dimensional space). The Euclidean distance in the experiment is that an RGB color mode is used as a three-dimensional space, different colors before and after reaction can be regarded as two different points in the three-dimensional space, the Euclidean distance reflects the real distance between the two points, the larger the distance is, the larger the color difference is, the more sufficient the corresponding reaction is carried out, and the better the corresponding visual observation effect is.

The paper-based array reacts with heavy metal ions for color development, wherein the most important influencing factor is the concentration of six reagents forming the array, and since the complexing reagent has a color, if the concentration is too high, even if the reaction is fully carried out, the color of a compound after the reaction is covered and can not be identified, and if the concentration is too low, the color of the compound is too light, so that the detection of the array on the low-concentration heavy metal ions is not facilitated, and therefore, the selection of the actual concentration of the proper composition array is of great importance for the distinguishing and detection of the heavy metal ions.

(1) Optimizing color development concentration on CDI reagent paper: due to Cd²⁺The reaction with CDI was most pronounced, therefore, at 20. mu. mol/LCd²⁺The reaction was optimized for CDI reagent concentration.

Respectively dripping 20 mu L of CDI solution (1, 2, 3, 4, 5mmol/L, solvent is 0.2mol/L potassium hydroxide-ethanol) with different concentrations on Whatman Grade 1 filter paper, standing until the diffusion is complete and the solvent is volatilized, forming a plurality of rings with uniform colors on the paper, and dripping 10 mu L of Cd with the concentration of 50 mu mol/L in the center of the ring formed by the complex diffusion²⁺And (5) standing the ionic solution for five minutes, photographing, and calculating the Euclidean distance before and after reaction. As shown in fig. 4A, as the CDI concentration increases, the euclidean distance value gradually decreases, and the color that appears after the reaction becomes darker and darker visually. This is because the color of the CDI on the paper changes to light yellow for several seconds and then begins to change to pink purple of the compound when reacting with the ionic solution, and because the newly formed color becomes darker and darker, and the CDI itself is darker and darker, the Euclidean distance becomes smaller and smaller as the concentration of the reagent increases, and the CDI can be combined with the visual observationFinally 2mmol/L was chosen as the optimal concentration of CDI reagent.

(2) Color development concentration optimization on DPC reagent paper

Preparation of DPC solutions (solvent is water) of 1, 5, 10, 15, 20mmol/L at various concentrations as described above with 50. mu. mol/LHg²⁺And (4) reacting for five minutes, wherein the two are completely reacted and the color is stable, and calculating the Euclidean distance before and after the reaction. The DPC appeared pale pink when dropped on Whatman filter paper, with a high concentration of Hg²⁺The color is purple after the reaction, and the concentration of heavy metal ions used in the experiment is low, so that the DPC and the ions are combined to be a less obvious light purple color, as shown in FIG. 4B, with the increase of the concentration of the DPC, Euclidean distance values before and after the reaction are gradually increased, the color difference is larger and larger, and when the concentration of the DPC reaches 15mmol/L, the numerical value is stable and does not increase any more, so that 15mmol/L is selected as the optimal concentration of the DPC for subsequent detection.

(1) Color development concentration optimization on TMK reagent paper

Preparing 1, 2, 3, 4, 5mmol/L TMK solution (solvent is 95% ethanol water), dripping on paper according to the above method, and dripping 50 μmol/LHg²⁺And (4) reacting with the above-mentioned reaction solution, and calculating the Euclidean distance between two points before and after the reaction. As shown in FIG. 5A, with the increase of the TMK concentration, the Euclidean distance between the front and the back of the reaction gradually increases, the increase amplitude after 3mmol/L is gradually gentle, and the color is gradually stable, so that 4mmol/L is selected as the final TMK concentration for subsequent detection.

(4) Optimization of color development concentration on PAR reagent paper

Due to PAR to Cu²⁺，Pb²⁺Both ions react significantly, so the selection of both ions is optimized for PAR concentration. PAR solutions (95% ethanol water as solvent) of 1, 2, 3, 4, 5mmol/L were prepared in different concentrations and applied dropwise to paper as described above, after which 50. mu. mol/LCu were added dropwise²⁺And 50. mu. mol/LPb²⁺And (5) reacting with the array, and calculating Euclidean distances between two points before and after the reaction. As shown in FIG. 5B, the Euclidean distance between PAR and two ions before and after reaction reaches the maximum value at 4mmol/L, which indicates that the difference between the colors before and after reaction is the greatest at the above concentration, and is more advantageousFor observation and subsequent analysis, a concentration of 4mmol/L was chosen. When the concentration exceeds 4mmol/L, the Euclidean distance rather shows a tendency to decrease because the RAR reagent itself at a higher concentration has a darker color, resulting in a case where the difference in color between before and after the reaction is not sufficiently significant and the value of the Euclidean distance becomes small.

(5) Colour development concentration optimization on XO reagent paper

XO solutions (solvent is water) with different concentrations of 1, 2, 3, 4, 5mmol/L are prepared, and are dripped on paper according to the method, and then 50 mu mol/LPb is respectively dripped²⁺And (5) reacting with the array, and calculating Euclidean distances between two points before and after the reaction. As can be seen from fig. 6A, the euclidean distance of XO shows a trend of increasing in a zigzag manner with the increase of the concentration, and the euclidean distance value is larger and the color difference is more obvious when the concentration is 3mmol/L, which is equivalent to that of the higher concentration, so that the optimal concentration of XO of 3mmol/L is selected to realize the color development of the heavy metal ions.

(6) Color development concentration optimization on BCP reagent paper

Preparing BCP solution (solvent is 95% ethanol water) with different concentrations of 1, 2, 3, 4, 5mmol/L, dripping on paper according to the method, and dripping 50 μmol/LCu respectively²⁺And (5) reacting with the array, and calculating Euclidean distances between two points before and after the reaction. BCP itself is colorless, and the color of the complex after the reaction is light, so that the euclidean distance as a whole tends to be relatively gentle, and when the concentration thereof reaches 4mmol/L, the euclidean distance peaks, so that 4mmol/L is selected as the optimum concentration of BCP (fig. 6B).

Example 3 LDA profiles of known types of metal ions were obtained

The detection array obtained by dropping six complexing reagents with the concentrations determined in example 2 on a paper substrate and fully drying is sequentially CDI, DPC, TMK, PAR, XO and BCP from the right top in a clockwise rotation manner, and the color presented on the paper is the color of the complexing reagents after drying itself at this time, as shown in FIG. 7A. Blank reagent and 500 mu mol/L Cd²⁺，Cu²⁺，Hg²⁺，Pb²⁺The four ionic solutions were reacted with the paper chips, respectively, and the results are schematically shown in fig. 7B. It can be seen that each ion can react with multiple reagents of the arrayThe color change which can be seen by naked eyes is generated, and the reagents and colors of each ion which react with the array are different, so that the characteristic spectrum is provided, and the possibility of detecting a plurality of heavy metal ions by using the array spectrum is provided.

The colors before and after reaction are collected and are specifically represented in a numerical manner by an RGB color mode. Counting the RGB values of the color of the paper chip after the reaction of a plurality of concentrations of different ions, and respectively marking the RGB values of six reagents in the whole array as R1, G1 and B1; r2, G2, B2; … …, R6, G6, B6; the RGB values of six dimensions corresponding to the whole array jointly form a fingerprint spectrum corresponding to a sample, the collected data are sufficient through the reaction of different ions with a plurality of concentrations from small to large, and a plurality of experiments in each group are parallel to ensure the accuracy of the reaction data. And performing LDA analysis on the collected data, namely obtaining an LDA map of the known type of metal ions.

The obtained LDA atlas is shown in FIG. 8, and it can be found that after LDA dimensionality reduction clustering, various ions with different concentrations are divided into four non-overlapping areas which respectively represent Cd²⁺，Cu²⁺，Hg²⁺，Pb²⁺The paper-based complex array prepared by the method not only can detect single heavy metal ions, but also can effectively realize the distinguishing detection of multiple heavy metal ions.

Example 4 detection of Single heavy Metal ions Using paper-based Complex array

And respectively dripping 0.4 mu L of six complexing reagent solutions on six passages, and drying for later use after the passages are fully filled by the complexing reagent solutions. 2, 10, 20, 50, 100 and 500 mu mol/L Pb are randomly and respectively added²⁺，Cd²⁺，Hg²⁺And Cu²⁺React with the paper-based detection array. And (3) photographing the paper chip before and after the reaction by using the smart phone, taking color of the photo by using Adobe Photoshop CC 2015.5, and taking RGB value of a 5 multiplied by 5 pixel point in the center of the reaction area. Data is input into an IBM SPSS Statistics 22 software to carry out LDA analysis, four ion reaction maps under the data model are observed, and the feasibility of the paper-based detection array for distinguishing and detecting various heavy metal ions is analyzed. As shown in FIG. 8, the four ions were distinguished wellGood results are obtained. The model is further verified by using the unknown sample, the result is shown in table 1, and it can be seen that for single heavy metal ions, the accuracy of the method for predicting the unknown sample is 100%.

TABLE 1 prediction of LDA model for unknown sample detection

Example 5 detection of various heavy metal ions Using paper-based complexing arrays

When two or more heavy metal ions exist in the solution at the same time, the concentration of the heavy metal ions is equal, and the embodiment verifies the accuracy rate of distinguishing and detecting the existence of multiple ions by the paper-based complexing array.

Preparing a mixed ion solution with each ion concentration of 100 mu mol/L, wherein the mixed ion solution totally comprises Cd²⁺And Cu²⁺，Cd²⁺And Hg²⁺，Hg²⁺And Cu²⁺，Cd²⁺And Pb²⁺，Pb²⁺And Cu²⁺，Pb²⁺And Hg²⁺，Cd²⁺、Hg²⁺And Cu²⁺，Cu²⁺、Cd²⁺And Pb²⁺，Cd²⁺、Pb²⁺And Hg²⁺，Pb²⁺、Cd²⁺、Hg²⁺And Cu²⁺And ten ion mixed solutions, wherein 20 mu L of each ion solution is dripped into a prepared paper-based microfluidic central sample adding area, the paper-based microfluidic central sample adding area is placed on a horizontal plane to be completely diffused, a picture is taken by a mobile phone after five minutes, and three of each ion mixed solution are parallel.

After the reaction data of ten possible ion mixing conditions are processed by LDA, the result is shown in FIG. 9, and it can be seen that the reaction data are clearly divided into ten regions, which proves that different ion mixing conditions in the paper-based complexing array solution have different reaction maps, and the reaction data can be clearly divided into different regions in LDA analysis, thus the method of the present invention can realize the distinguishing detection of the mixed solution of ions.

The accuracy of the LDA model is also verified, the result is shown in Table 2, the accuracy can reach 93.3%, and the method provided by the invention is proved to have good distinguishing effect on the mixed ions.

TABLE 2 prediction of unknown samples by mixed ion LDA model

Note: marked as prediction error samples

EXAMPLE 6 interference rejection testing of paper-based Complex arrays of the present invention

Selection of Na⁺，Cl^-，K⁺，SO₄ ^2-，NO₂ ^-，Fe³⁺，Zn²⁺，Mg²⁺，Ca²⁺Respectively preparing 500 mu mol/L of interference ion solution for common metal ions and non-metal ions in various environmental water samples, respectively dropwise adding 20 mu L of interference ion solution into a paper-based microfluidic central sample adding area prepared in advance, placing the paper-based microfluidic central sample adding area on a horizontal plane until the interference ion solution is completely diffused, taking pictures by a mobile phone after five minutes, and enabling each ion mixed solution to be three-parallel.

The LDA treatment of the reaction data showed that the array showed almost no response to interfering ions at concentrations as high as 500. mu. mol/L, only Zn, as shown in FIG. 10²⁺Some color reaction occurs to the XO in the array, but the effect is small. The paper-based complexing array has strong anti-interference capability and high selectivity.

Example 7 comparison of paper-based Complex arrays of the present invention with other detection methods

In order to test the detection limit of the constructed paper-based complex detection array for heavy metal ions, the reaction condition of the heavy metal ions with the paper-based array at different concentrations is further analyzed, as shown in fig. 11, the reaction condition is the distinguishing condition of the heavy metal ions at four different concentrations of 1.5, 2, 2.5 and 5 μmol/L, and it can be seen that when the concentration is 1.5 μmol/L, certain overlap occurs in the aggregation areas of two ions, namely Cu2+ and Hg2+, and when the concentration of four heavy metal ions is higher than 2 μmol/L, the same ions are aggregated in the same area and can be completely distinguished from other ions without overlapping with each other, so that sufficient identification is obtained. The paper-based detection array can realize distinguishing detection on four heavy metal ions with the concentration not lower than 2 mu mol/L.

In addition, because the experiment is carried out in the presence of low-concentration heavy metal ions, a control group is added to prevent the situation that the color is not obvious due to low concentration and cannot be distinguished from the blank. It can be seen from FIG. 11A that even when the concentration of all heavy metals was as low as 1.5. mu. mol/L, there was a very clear difference from the blank group.

The detection limit of the paper-based complex array is compared with other similar methods, and the results are shown in Table 3, so that the detection limit of the paper-based complex array is only 1/25 of the detection limit of the paper-based pyridine array, the sensitivity of the paper-based complex array is basically equivalent to that of LDHs test strips and paper-based AuNPs array methods, and the synthesis modification of materials and the construction of a complex method are not needed. Therefore, compared with the similar method, the method has relatively higher sensitivity and is simpler.

TABLE 3 comparison of the sensitivity and specificity of the present method with other methods

LDHs: anion-intercalated layered double hydroxides.

[1]Wang N，Sun J，Fan H，et al.Anion-intercalated layered double hydroxides modified test strips for detection of heavy metal ions[J].Talanta，2016，148:301-307.

[2]Wang H，Li Y J，Wei J F，et al.Paper-based three-dimensional microfluidic device for monitoring of heavy metals with a camera cell phone[J].Analytical and Bioanalytical Chemistry，2014，406(12):2799-2807.

[3]Feng L，Li X，Li H，et al.Enhancement of sensitivity of paper-based sensor array for the identification of heavy-metal ions[J].Analytica Chimica Acta，2013，780:74-80.

Example 8 detection of actual samples by paper-based complexing arrays of the invention

Taking running water of a collaborative innovation building in campus (Jiangnan university) and lake water of Lihu, filtering the two water samples with 0.22 μm filter membrane to obtain a liquid to be detected of an actual sample, and storing in a refrigerator at 4 ℃ for later use. And (3) taking two water samples, adding 2 mu mol/L heavy metal ions, respectively dripping the heavy metal ions on prepared paper-based complexing array detection equipment, and taking a picture by a mobile phone after waiting for 5 min. The data is treated with LDA as shown in fig. 12, where the actual sample water sample shows a clear difference from the control group, which may be due to the presence of some other metal ions and anions, etc. in the actual sample water itself. Various ions and heavy metal ions in the water sample usually exist in trace amounts, so that the rapid and convenient detection of the various heavy metal ions in the actual water sample is difficult to realize under the condition. The experiment utilizes the multi-dimensional sensing colorimetric paper-based portable detection equipment, and can effectively distinguish various target heavy metal ions existing in an actual water sample, namely a tap water sample with a simpler matrix or slightly complex environmental lake water. The paper-based microfluidic complex array is proved to have great potential in practical detection application.

Comparative example

Comparative example 1:

preparing six solutions of 30 mu M cadmium reagent, 2 percent TritonX-100 mixed solution, 0.5mM DPC solution, 50 mu M TMMK solution, 100mM KI, 0.8mM ascorbic acid and 70 mu MVBB mixed solution, 50 mu M XO and 5mM phenanthroline mixed solution and 0.5mM BCP mixed 60mM hydroxylamine hydrochloride solution, dripping the six solutions on the micro-fluidic paper chip, drying, and dripping 200 mu mol/LPb²⁺,Cd²⁺,Hg²⁺And Cu²⁺Mixing the ionic solution, stabilizing the color after 5min, and taking the color by taking a picture with a mobile phone.

The photographs before and after the reaction are shown in FIG. 13(A), 1-6 are the detection solutions of six complexing reagents of CDI, DPC, TMK, VBB, XO and BCP, respectively, and the color development effect is obviously inferior to that of the complexing array optimized by the adjustment of the invention (FIG. 13B).

According to the mode of example 4, the paper-based complexing array prepared by the comparative example is used for detecting single heavy metal ions, and as shown in the result of fig. 14, accurate distinguishing detection of four heavy metal ions cannot be realized, and a detection model capable of accurately distinguishing four single heavy metal ions cannot be established.

Comparative example 2: dithizone system

Preparing 1mM dithizone solution, dripping the solution at six passages of a paper chip, and dripping 200 mu mol/LPb on a sample adding area²⁺,Cd²⁺,Hg²⁺And Cu²⁺Mixing the ionic solution, stabilizing the color after 5min, and taking the color by taking a picture with a mobile phone. As shown in fig. 15, no significant color change was found.

Comparative example 3: chromium black T system

Preparing 1mM chrome black T solution, dripping the solution at six passages of a paper chip, and dripping 200 mu mol/LPb on a sample adding area²⁺,Cd²⁺,Hg²⁺And Cu²⁺Mixing the ionic solution, stabilizing the color after 5min, and taking the color by taking a picture with a mobile phone. As shown in fig. 15, no significant color change was found.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.