TWI536009B - Method and system for detecting metal ion concentration - Google Patents

Description

Method and system for detecting metal ion concentration

The present invention relates to a method and system for detecting the concentration of metal ions.

With the economic development of developing countries and the rise of industry, leading to serious air pollution, the heavy metal ions emitted by these industries, such as mercury ions, may eventually accumulate in the human body through the transmission of the biological food chain. Among them, according to the World Health Organization report, mercury can seriously threaten human health, especially for the development of the fetus in the womb. In addition, mercury can affect human nerves, immunity, and the digestive system, and can cause damage to the human brain, kidneys, and lungs. In view of this, the World Health Organization and the US Environmental Protection Agency have separately set the maximum allowable amounts of mercury in drinking water to 30 nM and 10 nM. However, most of the reservoirs and water quality monitoring points are located in the suburbs. Due to the limited resources in the suburbs, it is time-consuming and labor-intensive to collect samples, and the samples may be partially lost or contaminated during transportation. Therefore, there is a need for a simple, fast, light, and sensitive detection platform in areas with limited resources to improve pollution control and environmental monitoring effectiveness.

Although the prior art can utilize ICP-MS and electrochemical detection To detect mercury ions, the detection limit can reach 0.1nM, but it requires sophisticated analysis, well-trained personnel or complicated processes, making this method difficult to use for on-site inspection. In recent years, with the advancement of various nanomaterial synthesis and biochemical analysis platforms, multifunctional biochemical sensors provide large-area high-density biochemical interactions as well as their opto-physical and sensitive, specific, high surface and volume nanomaterials. Quick analysis of chemical properties. Among these attractive nanomaterials, the gold nanoparticles have been widely used as colorimetric sensors, becoming a new alternative as a metal ion detection. The synthesis of gold nanoparticles can be modified by surface modification. Particles with different functions have good biocompatibility and unique photoelectrochemical properties.

In view of the above, the present invention utilizes the various performance characteristics described above for the gold nanoparticles to produce a method and system for detecting the metal ion content in a solution.

It is an object of the present invention to provide a method for detecting the concentration of metal ions in a sample by which the color change produced by the gold nanoparticles and the metal ions in the sample is transferred to a microfluidic test paper for numerical output. Different from the traditional colorimetric method, the present invention can achieve the effect of quantitative analysis of the detected concentration value by the ratio of the three primary colors of red, green and blue.

Another object of the present invention is to provide a detection system for detecting the concentration of metal ions in a sample, and more particularly to a novel microfluidic test paper, which eliminates the use of complicated equipment and shortens the efficiency required for data analysis and transmission, and is provided by the present invention. The detection system is capable of highly sensitive detection of mercury ions in the environment in a resource-poor environment.

In order to achieve the above object, the present invention provides a method for detecting a concentration of a metal ion in a sample, comprising: providing a sample containing a metal ion and a detection reagent, wherein the detection reagent comprises a plurality of biomolecules and a plurality of gold nanoparticles, and The biomolecules are attached to the surface of the gold nanoparticles; the sample and the detection reagent are mixed to obtain a mixture, wherein the metal ions in the sample are combined with the biomolecules to make the gold The nanoparticles produce different degrees of aggregation to produce a color change; the mixture is transferred to a microfluidic test strip; and the microfluidic test paper to which the mixture is transferred is detected by a closed working platform, wherein the closed working platform comprises a light source, a detecting unit and an output unit, wherein the light source illuminates the microfluid test strip, the detecting unit detects a color signal of the microfluid test strip after being irradiated by the light source, and the output unit uses the color signal to A numerical output.

The invention also provides a detection system for detecting the concentration of metal ions in a sample, comprising: a detection reagent comprising a plurality of biomolecules and a plurality of gold nanoparticles, wherein the biomolecules are attached to the surface of the gold nanoparticles, Wherein a metal ion-containing sample is mixed with the detection reagent to obtain a mixture, the metal ions in the sample are combined with the biomolecules to cause the gold nanoparticles to aggregate to produce a color change; a flow test paper carrying the mixture of the sample and the test reagent; and a closed working platform comprising a light source, a detecting unit and an output unit, wherein the light source illuminates the microfluid test strip, the detecting unit Detecting a color signal of the microfluid test paper after being irradiated by the light source, and the output unit outputs the color signal in a numerical manner.

In the present invention, the color change caused by the change of the distance of the gold nanoparticles by the surface plasmon resonance (SPR) of the gold nanoparticles. Specifically, the gold nanoparticles dispersed in the solution dissociate and neutralize the negative charge on the surface of the gold nanoparticles when the salt is contained in the solution, so that the gold nanoparticles are aggregated, and the color changes from red to purple. .

In such a situation, the sensing mechanism created by the present invention is a combination of a plurality of biomolecules, which are DNA, a protein, or a mixture thereof. Preferably, the plurality of biomolecules are DNA, and the DNA can be A DNA with a mismatched sequence. For example, an unlabeled oligonucleotide sequence is attached to the surface of a gold nanoparticle by a hydrophobic interaction, and combines with metal ions in water to cause aggregation, thereby causing a color change in the solution. Taking the unlabeled oligonucleotide sequence as a complex biomolecule as an example, when the T-base of the single-stranded deoxyribonucleic acid attached to the surface is added with mercury ions, the sequence T on the single-stranded DNA forms a T-mercury ion with the mercury ion- The hairpin structure of T. The results show that the electrostatic repulsion between the gold nanoparticles is reduced. Therefore, the gold nanoparticles have different degrees of aggregation due to different degrees of surface charge in the salt solution. In order to obtain quantitative results, the ratio of blue/red is analyzed from RGB. Since the surface potential of the gold nanoparticles decreases, the electrostatic force between adjacent gold nanoparticles decreases. Therefore, the gold nanoparticles are in the salt solution. Different degrees of aggregation occur due to the concentration of different mercury ions. The results of each mixed liquid are calculated from the degree of color change, and are related to the different degree of aggregation of the gold nanoparticles and the increase in the concentration of mercury ions.

After that, the gold nanoparticles are gathered to produce a mixture of color changes. The compound is transferred to a microfluid test paper, for example, a mixture of the color reaction mixture is dropped on a hydrophilic microfluid test paper surrounded by a hydrophobic wax. Among them, preferably, the microfluid test paper of the present invention is a hydrophilic chromatographic test paper.

In the present invention, a closed work platform is created for detecting the microfluidic test paper to which the mixture is transferred. The closed working platform includes a light source, a detecting unit and an output unit. The light source illuminates the microfluid test strip. For example, the light source is a light source of a light-emitting diode, and the light source generates a uniform visible light source or an ultraviolet light source, so that the visible color standard color strip is used before each detection. The fluorescent standard brightness test paper is subjected to standard calibration in order to achieve automatic color or intensity comparison interpretation of the test paper or related colorimetric or fluorescent biomedical test elements to achieve accurate and objective test results. Then, the detecting unit can be a device that includes an image capturing unit, such as a scanner or a smart phone, and then generates a color signal generated by the microfluidic paper after being irradiated by the light source. The output unit outputs the color signal in a numerical manner. In an embodiment of the present invention, the output unit can be a smart phone. In addition, in the present invention, in order to eliminate the use of complicated equipment and shorten the efficiency required for data analysis and transmission, the output information can be stored and read through cloud computing.

In the present invention, the metal ion in the test sample includes at least one element selected from the group consisting of mercury (Hg ²⁺ ), cobalt (Co ²⁺ ), manganese (Mn ²⁺ ), lead (Pb ²⁺ ), and calcium (Ca). Group of ²⁺ ), cadmium (Cd ²⁺ ), copper (Cu ²⁺ ), nickel (Ni ²⁺ ), zinc (Zn ²⁺ ), and chromium (Cr ³⁺ ).

Therefore, in order to avoid the use of complicated equipment and reduce the required Resources, microfluidic test strips and clouds are used for data storage and reading. Microfluidic test strips are used in biomedical sensing and analysis in resource-poor environments because of their cost-effective, processable, simple manufacturing, low sample size, and wettability (this helps reduce external flow) Control system), as well as easy to store and transport. In addition, the microfluidic test paper disclosed in the present invention requires only a trace amount (μL) of the sample amount to be detected, and the conventional colorimetric test method requires a reaction time of several hours or more. However, the total microfluidic test paper disclosed by the present invention It takes 40 minutes to complete. In addition, the detection method disclosed by the present invention can simultaneously detect a plurality of analytes extracted from a sample and obtain qualitative results from a scanner or a smart phone. In the present invention, the smart phone is used in a resource-constrained environment based on its portability, light weight, timely image recording and data transmission functions.

Therefore, the present invention is a simple, rapid, light, and sensitive detection method and system for improving pollution control and environmental monitoring performance.

1‧‧‧ssDNA-gold nanoparticle solution

11‧‧‧ssDNA of gold nanoparticles

2‧‧‧T-mercury ion-T hairpin structure

12‧‧‧Ginnel particles

3‧‧‧Microfluid test strips

4‧‧‧Closed work platform

41‧‧‧Light source

42‧‧‧Signal Processing Area

5‧‧‧Cloud processor

Figure 1 is a schematic diagram of a metal ion detection system.

Figure 2 is a schematic image of an electron microscopy (SEM) analysis of samples before and after transfer to a microfluidic test strip.

Fig. 3 is a graph showing the results of discoloration after mixing the colorimetric tube with the sample of the microfluidic test paper of the present invention and the test reagent.

Fig. 4 is a view showing a comparison of the color change time of the colorimetric tube and the sample of the microfluidic test paper of the present invention and the detection reagent.

Figure 5 is a diagram showing the selectivity analysis of mercury ions in the detection system of the present invention. intention.

The above and other objects, features, and advantages of the present invention will become more apparent from the description of the accompanying claims.

Hereinafter, the present invention will be described in detail by way of examples and with reference to the drawings. It is worth noting that these embodiments provide many possible creative concepts and can be implemented in a variety of specific situations. However, the specific embodiments discussed herein are merely illustrative of the fabrication and use of the present invention, but are not intended to limit the scope of the invention. Therefore, the description and drawings of the present invention are intended to It is to be understood that the embodiments of the present invention may be utilized in various other combinations and environments, and may be modified and modified without departing from the spirit and scope of the invention.

Preparation of gold nanoparticles

The average diameter of the citric acid-coated gold nanoparticles obtained by reduction from HAuCl ₄ using citric acid as a medium was 13 nm. That is, a 25 mL solution containing 38.8 mM trisodium citrate was added to a 250 mL solution containing 1 mM HAuCl ₄ , and stirring was continued for 15 minutes until the color of the mixture changed from yellow to dark red, and then cooled at room temperature. Obtained, wherein the concentration of the golden nanometer is calculated by analyzing the absorption spectrum (Beer-Lambert law) by Bayer's law.

Preparation of detection reagents - taking mercury ions as an example

The detection reagent disclosed in the present invention comprises a plurality of biomolecules and a plurality of gold nanoparticles, and the plurality of biomolecules in the present embodiment Taking ssDNA as an example, and the metal ions detected in this embodiment are exemplified by mercury ions; wherein ssDNA is dissolved in triborate buffer (100 mM, pH 9.0) and mixed with the gold nanoparticles. Then, the gold nanoparticles and ssDNA prepared by ultrapure water were brought to a concentration of 1 nM and 10 nM, respectively, and the mixtures were heated for 10 minutes to 90 ° C and stored at 4 ° C before the test, so that ssDNA was attached to the gold. On the surface of the nanoparticle.

Figure 1 is a schematic diagram of a metal ion detection system. In Fig. 1, the mercury ion is detected as an example, firstly added to a pre-formed ssDNA-gold nanoparticle solution 1, which contains gold nanoparticles containing ssDNA 11 Then, if the solution contains mercury ions, the sequence T on the ssDNA forms a T-mercury ion-T hairpin structure 2 with the mercury ions and forms a different proportion of the gold nanoparticles 12 according to the concentration of the mercury ions; The degree of aggregation of the gold nanoparticles 12 will have varying degrees of color change, for example, from red to purple and then to blue. After the color change is completed, the mixture is transferred to a microfluid test strip 3. Preferably, the center of the microfluidic test strip is surrounded by a barrier formed by wax, thereby limiting the reagent to a certain area, and then obtaining the reagent concentration. As a result, the color development effect is thus improved. The microfluidic test strip 3 is then placed in a closed working platform 4 having a light source 41 and a signal processing area 42. The signal processing area 42 includes a detecting unit and an output unit. The 41 system forms a uniform light field to improve the detection accuracy. The detecting unit can be a smart phone, collecting the color signal of the microfluid test paper after being irradiated by the light source, and storing the color signal in a numerical form. The foregoing signal processing area 42 further includes an image capturing unit, an algorithm computing unit, a system calibration device, and a The signal transmission and display device, for example, the color signal is calculated by the algorithm and corrected by the system correction device, and is outputted by an output unit, for example, a smart phone, and then the processed color signal is output, for example, uploading Stored in the cloud processor 5.

It is easy to have errors due to the color of the human eye, and the results are also likely to vary from person to person. The present invention will be to construct a closed working platform 4 in which a stable light source 41 will be provided, wherein the light source is a light emitting diode (LED), or an ultraviolet light. In the present invention, by supplying the white light and the ultraviolet light emitting diode as a light source, a uniform visible light source or an ultraviolet light source is generated, and then, by the computer vision method in the signal processing area 42, before each detection, Standard calibration using visible light color standard color bars or fluorescent standard brightness test papers, in order to achieve automatic color or intensity comparison interpretation of test paper or related colorimetric or fluorescent biomedical test elements to achieve accurate and objective test results .

On the other hand, the detection software of the present invention can be integrated with a lower cost visual soft and hardware system, for example, the image capturing unit, the algorithm computing unit, the system calibration device, the signal transmission and display of the present invention. The device and the output unit may all comprise the software or the hardware. More specifically, the function of the software includes: sampling area and correction area capable of manual/automatic positioning, visible color and fluorescence intensity correction for standard sheets Function; test paper sampling area color automatic alignment interpretation, output red (R), green (G), blue (B), blue to red (B / R), red than green (R / G) and green than blue Color (G/B) and other values, and has the function of converting the image into a grayscale image for grayscale value analysis; detecting the original data file can be stored Stored in memory or in the cloud for repeated access and analysis; can report test results in HTML or EXCEL format; output test results archive in text file; expandability of wireless transmission function.

Regarding the hardware part, for example, if the camera level is insufficient, there will be no color calibration function of the CCD or CMOS chip, and the light temperature white balance compensation adjustment, which will limit the computer visual color measurement accuracy. However, if the measurement accuracy required for the color comparison of the test paper does not need to be as high, the camera without the above adjustment function may not affect the color discrimination result of the computer vision; if the level of the Webcam USB camera is insufficient, the stability is not enough. Causes image color drift to cause the computer color to be misaligned, or the specification is incompatible with the final actual system integration. At this point, you need to replace the camera that is more suitable for his card. In terms of lighting problems, when the light source provided by the Webcam USB camera itself is not suitable for the image processing required by this project, the analysis-based samples will need to use software to correct their RGB values or grayscale values, and finally allow different samples. Test error <5%; white LED and UV LED power supply must be built-in, and provide stability and low power consumption requirements.

In this embodiment, preferably, the ssDNA may be a 24 base oligonucleotide sequence, 5'-TTT-GTT-TGT-TGG-GGT-TCT-TTC-TTT-3'.

The microfluid test paper disclosed in this embodiment uses a solid ink printing method, and the detection area size is about 3 mm. The size of the detection area was controlled by a printing machine (Xerox 8570, Fuji, Japan) to print solid wax on the surface of the test paper and then heated at 160 ° C for 2 minutes to allow the solid wax to penetrate into the test paper.

After the sample produced a color change, 50 μL of the mixture was placed in the center of the printing area. In order to allow the sample to dry quickly, preferably, a dry part can be placed under the test paper. When the mixture is completely dried and filtered by the micro-test paper, the image is captured by the smart phone under the illumination of the light source and recorded. And the images are synchronized to the cloud storage device.

Figure 2 is a schematic diagram of an electron microscopy (SEM) analysis of the sample before and after transfer of the microfluidic test paper. Figure 2a shows the image of the sample solution containing mercury ions and transferred to the microfluidic test paper. Figure 2b shows the image. The image after the sample solution is transferred to the microfluid test strip. It can be seen from Fig. 2 that after the sample is transferred to the microfluidic test paper, no aggregation occurs because of the dryness of the sample. In addition, in addition, a small amount of sample volume requires less diffusion time, so the total analysis time can be reduced from a few hours to 40 minutes, for example, the detection area in this embodiment ranges only 50 μL.

Fig. 3 is a graph showing the results of discoloration after mixing the colorimetric tube with the sample of the microfluidic test paper of the present invention and the test reagent. It can be seen from Fig. 3 that in the conventional colorimetric tube, since the chemical reaction continues in the liquid environment, there is an unstable color change. On the contrary, the present invention proposes the detection of a dry sample. As a result, there is a stable color change performance.

Fig. 4 is a view showing a comparison of the color change time of the colorimetric tube and the sample of the microfluidic test paper of the present invention and the detection reagent. It is further pointed out from FIG. 4 that the test sample required by the present invention not only requires less sample detection than the conventional colorimetric detection (for example, only 50 μL), but also shortens the required color change time. In the example, the detection time can be determined by the traditional colorimetric tube reaction, which is only 40 minutes, which is effectively improved. Its detection efficiency. In addition, as can be seen from Fig. 4, the microfluidic test paper has a more sensitive colorimetric result as compared with the colorimetric tube.

Figure 5 is a schematic illustration of the selectivity analysis of mercury ions in the detection system of the present invention. In Figure 5, mercury ions and other metal ions: cobalt (Co ²⁺ ), manganese (Mn ²⁺ ), lead (Pb ²⁺ ), calcium (Ca ²⁺ ), cadmium (Cd ²⁺ ), copper (Cu ²⁺ ), nickel (Ni ²⁺ ), zinc (Zn ²⁺ ), and chromium (Cr ³⁺ ) are placed together in the sample, and the ratio of mercury ions to other metal ions added to the gold nano-solution is 1:10 (Examples: 1 μM and 10 μM), such a composition is to test the selective analysis of mercury ions in the present invention, to see if the detection of mercury ions is interfered by other metal ions and affect the detection results. The test results are shown in Fig. 5. The results show that the detection system of the present invention has good selectivity for mercury ions. Even in high concentrations of other metal ions, the other metal ion color solutions are still red, and the average blue/red ratio is <1 (0.96 to 0.98) after analysis on microfluidic test paper. In contrast, the mixture of gold nanoparticles containing 1 μM mercury ions is blue in color with a blue/red ratio >1 (~1.03). The results show that the microfluidic test paper disclosed by the present invention has a more sensitive analysis result.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

1‧‧‧ssDNA-gold nanoparticle solution

11‧‧‧ssDNA of gold nanoparticles

2‧‧‧T-mercury ion-T hairpin structure

12‧‧‧Ginnel particles

3‧‧‧Microfluid test strips

4‧‧‧Closed work platform

41‧‧‧Light source

42‧‧‧Signal Processing Area

5‧‧‧Cloud processor

Claims (14)

A method for detecting a concentration of a metal ion in a sample, comprising: providing a sample containing a metal ion and a detection reagent, wherein the detection reagent comprises a plurality of biomolecules and a plurality of gold nanoparticles, and the biomolecules are attached thereto On the surface of the gold nanoparticles; mixing the sample and the detection reagent to obtain a mixture, wherein the metal ions in the sample are combined with the biomolecules to aggregate the gold nanoparticles to produce a color change Transferring the mixture to a microfluidic test strip; and detecting the microfluidic test paper with the mixture transferred by a closed working platform, wherein the closed working platform comprises a light source, a detecting unit and an output unit, the light source Irradiating the microfluid test strip, the detecting unit detects a color signal of the microfluid test paper after being irradiated by the light source, and the output unit outputs the color signal as a numerical value; wherein the metal ion is a mercury ion . The method for detecting a metal ion concentration in a sample according to the first aspect of the invention, wherein the plurality of biomolecules are DNA, protein, or a mixture thereof. The method of detecting a metal ion concentration in a sample according to claim 1, wherein the light source is a light emitting diode (LED) or an ultraviolet lamp. A method of detecting a metal ion concentration in a sample according to claim 3, wherein the light source forms a uniform light field. The method for detecting a metal ion concentration in a sample according to the first aspect of the invention, wherein the microfluid test paper is a hydrophilic wax chromatography test paper. The method for detecting a metal ion concentration in a sample according to claim 1, wherein the plurality of biomolecules are DNA, and the DNA is a DNA having a mismatched sequence. The method for detecting a metal ion concentration in a sample according to claim 1, wherein the detecting unit is a device including an image capturing unit. A detection system for detecting a concentration of a metal ion in a sample, comprising: a detection reagent comprising a plurality of biomolecules and a plurality of gold nanoparticles, wherein the biomolecules are attached to the surface of the gold nanoparticles, wherein When a sample of metal ions is mixed with the detection reagent to obtain a mixture, the metal ions in the sample are combined with the biomolecules to aggregate the gold nanoparticles to produce a color change; a microfluid test paper a mixture of the sample and the test reagent; and a closed working platform comprising a light source, a detecting unit and an output unit, wherein the light source illuminates the microfluid test strip, and the detecting unit detects the a color signal of the microfluid test paper after the light source is irradiated, and the output unit outputs the color signal as a numerical value; wherein the metal ion is a mercury ion. A detection system for a metal ion concentration in a test sample according to claim 8, wherein the plurality of biomolecules are DNA, protein, or a mixture thereof. A detection system for measuring a metal ion concentration in a sample according to the invention of claim 8, wherein the light source is a light emitting diode (LED) or an ultraviolet lamp. A detection system for detecting a metal ion concentration in a sample according to the invention of claim 8, wherein the light source forms a uniform light field. A detection system for a metal ion concentration in a test sample according to the invention of claim 8, wherein the microfluid test paper is a hydrophilic chromatography test paper surrounded by a hydrophobic wax. A detection system for a metal ion concentration in a test sample according to claim 8, wherein the plurality of biomolecules are DNA, and the DNA is a DNA having a mismatched sequence. The detection system for detecting a metal ion concentration in a sample according to claim 8 of the patent application, wherein the detecting unit is a device including an image capturing unit.