TWI445946B - Method and kit for detecting lead ions - Google Patents

Description

Method and kit for detecting lead ions

The invention relates to a method for detecting lead ions and a detection kit.

Lead is widely used in industry and is mainly used to manufacture lead storage batteries. With the development of the automobile industry, the amount of lead used has increased year by year, causing lead to be widely distributed in the global environment. However, the toxicity of lead can affect the human body system such as reproduction, immunity, nerves, cardiovascular, etc., especially the physical development and intellectual development of children. Once lead enters the human body, it will form a neurotoxin, slowing down the speed of brain and nerve conduction, which leads to Behavior can not be controlled and so on.

The U.S. Environmental Protection Agency has a safe value of 15 ppb for lead in general drinking water. Since the lead concentration of human urine and blood can be used as a sampling source for lead pollution in the workplace or the environment, the US Department of Diseases has published a urine lead level of 23 ppb, and the World Health Organization has also established lead in adult blood. The safety range should be less than 300 ppb, especially when the blood lead level reaches 600 ppb, which requires chelation therapy.

Conventionally, the lead ion detection method for detecting an environmental or biological sample may be an atomic absorption spectrometry, a graphite furnace atomic absorption spectrometry, an anode stripping voltammetry, or an induction. Coupled plasma atomic emission spectrometer (ICP-AES), inductively coupled plasma mass spectrometer (ICP-MS), x-ray fluorescence spectroscopy, neutron activation analysis, differential Differential pulse anode stripping voltammetry or isotope dilution mass spectrometry. Although the above detection methods or instruments can reach the ppb level, the operation of the instrument is cumbersome, highly specialized, expensive, and difficult to maintain.

In recent years, a variety of relatively simple optical techniques have been developed to replace instruments such as small molecules, DNAzymes, oligonucleotides, polymers, and proteins. Or nanomaterials, etc., which can be used to detect the concentration of lead ions in aqueous solution, but with many restrictions, poor water solubility, easy interference with other metal ions, matrix interference, high cost, complicated detection process, poor stability or linearity. Shortcomings such as narrow range.

In summary, although the precision instruments and equipment have high sensitivity, they are not easy to operate and expensive, and the relatively simple optical technology has poor precision and limited conditions. Therefore, there is an urgent need to create a detection method to achieve a sensitive, but simple, low-cost lead ion detection.

The present invention is the first to apply protein-modified gold nanoparticles to a test paper, and to add a reaction containing sodium thiosulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution. When the reagent and the sample to be tested contain lead ions, the color intensity of the test paper is weakened, and therefore, it can be used to detect lead ions of the sample to be tested. The detection according to the present invention has a high degree of specificity and sensitivity to lead ions, and can be applied to various samples such as high-concentration salts, biological urine or blood, and the detection limit is picomolar grade.

Accordingly, in one aspect, the present invention provides a method of detecting lead ions comprising the steps of:

(a) providing a test strip comprising a substrate and protein-modified gold nanoparticles attached to the substrate, the test strip exhibiting a first intensity of a color;

(b) providing a reaction reagent comprising sodium thiosulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution;

(c) reacting the sample to be tested with the test strip and the reagent to cause the test strip to exhibit a second intensity of the color;

(d) analyzing and comparing the first intensity and the second intensity of the color, wherein the second intensity being less than the first intensity indicates that the sample to be tested contains lead ions.

Preferably, the method of the present invention further comprises treating the test strip with microwaves after reacting the sample to be tested with the test strip and the reagent. This microwave treatment can be used to accelerate the reaction time.

In another aspect, the present invention provides a kit for detecting lead ions, comprising: a substrate and protein-modified gold nanoparticles attached to the substrate; and a reagent comprising sodium thiosulfate (Na) ₂ S ₂ O ₃ ), 2-mercaptoethanol and a buffer solution.

Detailed descriptions of various specific examples of the invention are given below. Other features of the present invention will be apparent from the following detailed description and claims.

Without further elaboration, it is believed that those of ordinary skill in the art of Therefore, it is to be understood that the following description is for illustrative purposes only and is not intended to limit the disclosure.

The words used in the description of the invention generally have their original meaning in the art, in the context of the invention, Certain terms used to describe the invention are discussed below or in the description of the invention to provide additional guidance to the skilled person in the description of the invention. For convenience, certain words may be highlighted, such as using a collaborator and/or quotation marks. The use of the awake target does not affect the scope and meaning of a vocabulary; the scope and meaning of a vocabulary are the same in the same content, whether or not it is shown by a awake target. It can be understood that the same thing can be explained in more than one way. Thus, other language and synonyms may be used for any one or more of the vocabulary discussed herein, and whether a vocabulary is elaborated or discussed herein does not confer any particular meaning. Provide synonyms for certain words. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples, including examples of any vocabulary discussed herein, is intended to be illustrative only and not limiting the scope and meaning of the invention. As such, the invention is not limited to the specific embodiments disclosed herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art. In case of conflicts, this document (including definitions) will prevail.

The term "a" as used herein, unless otherwise specified, refers to the quantity of at least one (one or more).

In one aspect, the invention provides a method of detecting lead ions comprising the steps of:

(a) providing a test strip comprising a substrate and protein-modified gold nanoparticles attached to the substrate, the test strip exhibiting a first intensity of a color;

(b) providing a reaction reagent comprising sodium thiosulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution;

(c) reacting the sample to be tested with the test strip and the reagent to cause the test strip to exhibit a second intensity of the color;

(d) analyzing and comparing the first intensity and the second intensity of the color, wherein the second intensity being less than the first intensity indicates that the sample to be tested contains lead ions.

In another aspect, the present invention provides a detection kit for lead ions, comprising: a test strip comprising a substrate and protein-modified gold nanoparticles attached to the substrate; and a reagent comprising sulfur Sodium sulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution.

The "substrate" of the test strip described herein can be any material that can be adsorbed to proteins, including but not limited to nitrocellulose membrane, nylon spray film, polyvinylidene fluoride film (PVDF film), cellulose acetate film. , a hybrid fiber membrane, an alumina filter membrane, and a polycarbonate resin membrane. Among them, because of the high hydrophobic interaction between the nitrocellulose membrane and the protein, which can be used as the main adsorption force, and the nitrocellulose membrane has been widely used for transferring proteins, in a specific embodiment, the nitrocellulose membrane is selected as the nitrocellulose membrane. Test the substrate of the test paper.

The "golden nanoparticle" described herein can be any nanometer size gold particle. The shape of the gold nanoparticles may be, for example, a dot, a sphere, a rod or a disk; the average diameter may range from 5 to 25 nm, specifically 10 to 20 nm, and more specifically about 13 nm; And the absorption wavelength can range from 400 nm to 600 nm, specifically from about 450 nm to 550 nm, and more specifically about 520 nm. Specifically, the gold nanoparticles used in the present invention are spherical and have an average diameter of 13 nm and a maximum absorption wavelength of 520 nm (presenting red) in the ultraviolet-visible absorption spectrum. The gold nanoparticle used in the present invention can be obtained by reducing a trivalent gold ion complex into a monovalent gold ion in a conventional manner, and then reducing it to a zero-valent gold atom and collecting it into a nanometer scale. For example, after placing the sodium citrate solution in a round bottom double-mouth bottle, heating and stirring to a boiling state, adding a tetrachloroauric acid solution, heating and stirring continuously, the solution changes from the original yellow trivalent gold ion complex to a colorless one. The price of gold ions, from the colorless to black gold nanocrystal seeds, and finally converted into wine red gold nanoparticles, followed by rapid cooling to room temperature in an ice bath, stored in a refrigerator at 4 ° C.

The gold nanoparticles according to the present invention are protein-modified and thus adsorbable on the substrate of the test strip. Proteins that can be used to modify the gold nanoparticles include, but are not limited to, bovine serum albumin, albumin, myoglobin, fibrin, and casein.

The test strip according to the present invention comprises a substrate and protein-modified gold nanoparticles attached to the substrate. The test strip of the present invention can be obtained by, for example, first mixing a gold nanoparticle with a protein solution having the protein to form a gold nanoparticle protein mixture, and then coating the gold nanoparticle protein mixture on the base. Drying and/or washing, etc., on the material and as needed. Figure 1 shows the manner in which the test strips are prepared in accordance with a particular example of the invention.

The reagents used in the present invention include sodium thiosulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution. They can be formulated with appropriate concentrations as needed. For example, the sodium thiosulfate can be adjusted to a concentration ranging from 50 to 150 mM, preferably about 100 mM; the 2-thioglycol can be adjusted to a concentration ranging from 200 to 300 mM, preferably about 250 mM; and the buffer solution can be The sodium glycinate buffer solution has a concentration of about 1-10 mM, preferably 5 mM. The buffer solution used in the present invention may have a pH in the range of 9-11, preferably about pH 10. The buffer solution used in the present invention may also be other kinds of buffer solutions including, but not limited to, phosphate buffer solution, trisethanol buffer (Tris) buffer solution and citrate buffer solution. The thiosulfate ion in the reagent passes through the bovine serum albumin on the surface of the test strip to form a complex (Au(S ₂ O ₃ ) ₂ ^{3 -} ) on the surface of the gold nanoparticles. Lead ions are contained, and lead ions are deposited on the surface of the gold nanoparticles, thereby accelerating the precipitation of 2-thioglycol and gold nanoparticles in the form of Au ⁺ -2-ME. In a specific example, before the gold nanoparticles are precipitated, the absorption peak of Surface Plasmon Resonance (SPR) falls at 520 nm. Once the gold nanoparticles are precipitated, the absorption intensity at 520 nm is It will be reduced, which can be measured by an instrument that generally analyzes the color intensity.

As used herein, "intensity of color" refers to the intensity (or depth) of a particular color and can be quantified by a variety of known measurement methods. For example, an image of the object to be tested can be scanned to analyze the color intensity in a soft body, which mainly analyzes three values of red, blue, and green (RGB), and the intensity is represented by a value of 0 to 255. In a specific example, the present invention measures the G value (defined as G _abs ) of the green light absorption of the test strip to determine the intensity of red, wherein when the value of G _abs is large, the intensity of red is small.

The "first intensity" as used herein refers to the intensity of the color of the test strip before the reaction between the test reagent and the sample to be tested; and the "second intensity" refers to the color of the test strip after reacting with the test reagent and the sample to be tested. Strength. According to the present invention, the first intensity and the second intensity of the color of the test strip are analyzed and compared, and if the second intensity is less than the first intensity, the sample to be tested contains lead ions. Specifically, the "second strength is less than the first intensity" as described herein may be such that the second intensity is 95% or less of the first intensity, preferably 85% or less, more preferably 75% or less.

The sample to be tested suitable for use in the present invention is preferably a liquid sample, for example, a liquid sample from the environment, such as sea water or rain water; or a liquid sample from a living body such as urine or blood.

According to the present invention, after the sample to be tested is reacted with the test strip and the reaction reagent, the test strip may have a change in the intensity of the color due to the presence or absence of lead ions in the sample to be tested, and the lead ion concentration may be calculated according to the change in the intensity. Specifically, when the detection is performed, the reaction reagent is first mixed with the sample to be tested, and then the mixture is applied to the test paper. The reaction time of the sample to be tested and the test strip and the reaction reagent is preferably 30 minutes or more, more preferably 1 hour or more, still more preferably 2 hours or more. In a particular embodiment, to speed up the reaction, the test strip can be further processed by microwave. As used herein, "microwave" refers to electromagnetic radiation having a wavelength between infrared and ultra-high frequencies, having a wavelength in the range of about 1 m to 0.1 mm and a frequency in the range of 300 MHz to 300 GHz. In one embodiment, the test strip is microwaved using a microwave oven having a frequency of 2-3 GHz at a power of about 450 W. After microwave treatment, the reaction time can be shortened to 2 hours or less, more preferably 1 hour or less, still more preferably 30 minutes or less, and most preferably up to about 10 minutes.

The following examples are illustrative only and not intended to be limiting of the invention.

Experimental Materials

Hydrogen tetrachloroaurate (III) trihydrate (HAuCl ₄ ), 2-mercaptoethanol (C ₂ H ₆ OS), sodium thiosulfate (Na ₂ S ₂ O ₃ ), glycine ( Glycine), cysteine, sodium hydroxide (NaOH), bovine serum albumin (BSA), lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl) Calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), ferrous chloride (FeCl ₂ ), ferric chloride (FeCl ₃ ), nickel nitrate (Ni(NO ₃ ) ₂ ‧6H ₂ O), copper nitrate ( Cu(NO ₃ ) ₂ ), cobalt nitrate (Co(NO ₃ ) ₂ ‧6H ₂ O), zinc chloride (ZnCl ₂ ), manganese chloride (MnCl ₂ ), cadmium chloride (CdCl ₂ ), platinum chloride (PtCl ₂ ), lead chloride (PbCl ₂ ), lead nitrate (Pb(NO ₃ ) ₂ ), strontium chloride (SrCl ₂ ), chromium chloride (CrCl ₃ ), silver nitrate (AgNO ₃ ) and mercuric chloride ( HgCl ₂ ) was purchased from Sigma-Aldrich, USA. Sodium dihydrogen phosphate (NaH ₂ PO ₄ ‧H ₂ O), disodium hydrogen phosphate (Na ₂ HPO ₄ ‧2H ₂ O), trisodium phosphate (Na ₃ PO ₄ ‧12H ₂ O) and trisodium citrate ( Trisodium citrate dehydrate) was purchased from JT Baker, USA. Urine sample (SRM 2672a) was purchased from the National Institute of Standards and Technology (NIST, Maryland). Blood samples (BCR 636, 635, 634) were purchased from the Institute for Reference Materials and Measurement (IRMM, Geel, Belgium).

Equipment

Disc Absorber SynergyTM 4 Multi-Mode Microplate Reader (Biotek Instruments, USA); Transmission Electron Microscope H-7100 (Hitachi, Tokyo); Inductively Coupled Plasma Mass Spectrometer E-6000 (Perkin-Elmer, Germany); Heating Mixer PC-420 (Corning, USA); Ultrasonic Cleaner D9NX-DC200H (Delta, Taiwan); Oscillating Mixer G-560 (Scientific-Industries, USA); Rotary Mixer S-101 (Firstek, Taiwan) Pure water machine Milli-Q system QGARD00R1 (MA, USA); electronic analytical balance HR-200 (Adapter, Japan); high speed centrifuge 3K-30 (Sigma, Germany); pH pH meter SP-701 (Switzerland, UK) ) desktop scanner Epson Perfection 1660 Photo Scanner (Epson, USA); flight mass spectrometer Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

Example 1: Synthesis of gold nanoparticles

100 mL of 4.0 mM sodium citrate solution was placed in a round bottom double-mouth bottle. After heating and stirring to a boiling state under a condensing reflux device, 1.0 mL of 0.1 M tetrachloroauric acid solution was quickly added and heating was continued for about 8 minutes. The solution changes from the original yellow trivalent gold ion complex to a colorless monovalent gold ion, and then turns from a colorless to black gold nanocrystal, and finally turns into a wine red, which is then rapidly cooled to room temperature in an ice bath and stored. Refrigerator at 4 ° C. The wine red liquid was observed by transmission electron microscopy (TEM). The diameter of the gold nanoparticles was 13 nm, and the maximum absorption wavelength of the gold nanoparticles in the ultraviolet-visible absorption spectrum was 520 nm.

Example 2: Preparation of test strips

Before preparing the test strip, prepare the gold nanoparticles modified with bovine serum albumin, which is to mix 20 mL of 3 μM bovine serum albumin solution with 20 mL of 15 nM gold nanoparticle solution, and equilibrate at room temperature. It is 30 minutes. Using flocculation analysis, it was estimated that about 20 bovine serum albumin was bound to the surface of each gold nanoparticle.

According to the measurement results of dynamic light scattering (DLS), the gold nanoparticles of unmodified bovine serum albumin and the modified gold nanoparticles were estimated to have hydration radii of 28.3±6.2 and 45.2±7.5 nm, respectively. . The gold nanoparticles modified with bovine serum albumin can be dispersed in a liquid due to steric hindrance and electrostatic repulsion, and can be stably stored in a 500 mM aqueous solution of sodium chloride, and can also be stored in a solid state.

Next, a nitrocellulose membrane of about 0.5 square centimeter is provided, and 1 μL of a 15 nM bovine serum albumin-gold nanoparticle solution is applied thereto, dried for 15 minutes, and then rinsed with about 20 mL of deionized water for 30 seconds. Remove any weakly bound bovine serum albumin-gold nanoparticles and dust on the nitrocellulose membrane and let stand at room temperature for about 1 hour before use. The test strips are stored in a refrigerator at 4 ° C for 6 months.

2(A), 2(B), and 2(C) are image diagrams of a gold nanoparticle on a scanning electron microscope (SEM), and FIG. 2(D) and FIG. 2(E). And Fig. 2(F) is an image representation of a high magnification SEM.

As shown in Fig. 2 (A) and Fig. 2 (D), it was observed by SEM that the gold nanoparticles modified with bovine serum albumin did not aggregate on the nitrocellulose membrane. Further, as shown in Fig. 2 (B) and Fig. 2 (E), the same arrangement of the gold nanoparticles after the addition of the reaction reagent showed no aggregation. Further, as shown in Fig. 2(C) and Fig. 2(F), the gold nanoparticles after the addition of the reaction reagent and the lead ions also showed no aggregation of the gold nanoparticles, but it is apparent that the gold nanoparticles are The diameter is much smaller after the lead ion reaction.

As shown in Fig. 3, in the ultraviolet-visible absorption spectrum, (a) aqueous solution of citrate-coated gold nanoparticles; (b) aqueous solution of gold nanoparticles coated with bovine serum albumin; (c) detection For the test paper, the absorption peaks of the three were consistent at 520 nm, indicating that the gold nanoparticles did not aggregate in all three states.

Furthermore, as shown in FIG. 4, the measurement results of the X-ray Energy Dispersive Spectrometer (EDS) indicate that the signal of the gold nanoparticles is detected on the test paper. As shown in FIG. 5(A) and FIG. 5(B), the test paper is further analyzed by a color analyzer and a surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). It was observed that the color of the test strip was uniform red (see Fig. 5(A)), and the gold nanoparticles modified with bovine serum albumin were uniformly adsorbed on the nitrocellulose membrane (as shown in Fig. 5(B)). . Among them, the relative standard deviation values (RSD values) obtained by color analyzer and mass spectrometer analysis were 1.8% and 4.1%, respectively. In addition, ICP-MS can also be used to determine the amount of gold nanoparticles contained in each test strip (0.5 cm × 0.5 cm) is 2.95 × 10 ^-8 mg.

The test strip can be scanned by a desktop scanner, and the color analysis is performed by Image J software, mainly red, blue, and green (RGB). Among them, the G value ( G _abs ) that absorbs green light is used as an indicator. This method quantifies the test strips and serves as a reference for determining whether or not to contain lead ions.

Example 3: Preparation of reagents and detection limits

The reagents were optimized to 100 mM sodium thiosulfate (Na ₂ S ₂ O ₃ ), 250 mM 2-thioglycol (2-ME) at 5 mM, pH 10 glycine-sodium hydroxide buffer solution ( Glycine-NaOH). The buffer solution was prepared by weighing 1.5014 g of glycine, adding it to secondary ionized water and adjusting to 100 mL to prepare a glycine acid solution. Next, take 50 mL, 200 mM glycine acid solution, and 1.0 M sodium hydroxide solution to prepare glycine-sodium hydroxide buffer with pH values of 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. The solution was stored in a refrigerator at 4 ° C with a buffer solution concentration of 100 mM.

As shown in Figure 1, the lead-containing standard is mixed with the reaction reagent (the final concentration of each component of the reagent is as above), and the test paper is immersed in the mixed solution, reacted at room temperature for 2 hours, and then rinsed twice with water (each Approximately 5 mL), and after 30 seconds, the test paper was dried using an air compressor (60 lb/in ² ). In the presence of lead ions, the gold nanoparticles are leached, so that the red color of the test strip after the reaction disappears. Therefore, when the sample to be tested contains lead ions, the red intensity of the test strip is weakened.

As shown in Figure 6, the logarithm of G _abs and lead ion concentration is a linear relationship ( R ² =0.98), especially when the lead ion concentration ranges from 10 nM to 1 μM. Among them, the lead ion concentration detection limit (LOD) is 5 nM when the noise ratio (signal/noise (S/N) ratio) is 3.

Example 4: Test condition test

As shown in Fig. 7(A), the absorption value of the protein-modified gold nanoparticles aqueous solution ( Abs ₅₂₀ ), the change of Abs ₅₂₀ over time in the reaction time of 2 hours, wherein (a) is 1.5 nM modified with cattle Gold albumin particles of serum albumin; (b) 1.5 nM gold nanoparticles modified with bovine serum albumin, 1.0 mM sodium thiosulfate, 100 mM 2-thioglycol; (c) 1.5 nM modified Gold nanoparticles of bovine serum albumin, 1.0 mM sodium thiosulfate, 100 mM 2-thioglycol, 1.0 μM lead chloride; (d) 1.5 nM modified gold nanoparticles with bovine serum albumin and 1.0 μM lead chloride. Among them, only the reaction reagent and lead ions exist at the same time, and its absorbance at 520 nm will decrease with time.

And, FIG. 7 at (B), the preferred reaction conditions (100 mM sodium thiosulfate and 250 mM 2- thioglycolate), a reaction time of 2 hours, with the chromaticity change of the test strip G _abs correspond, (c) containing 1.0 μM lead ion and reagent, the test strip G _{abs is} significantly increased, indicating that the gold nanoparticles have been leached, and it can be judged by the naked eye that the red chromaticity is weakened, and it is confirmed that it has bovine serum white. The result of leaching of protein-modified gold nanoparticles.

Further, as shown in FIG. 8, it is also possible to demonstrate by SALDI-MS that the surface of the gold nanoparticles after leaching does have an Au-Pb alloy. Further, as shown in FIG. 9, G _abs test strip was observed over time, changes easing after 2 hours, the detection reagents in the test strip is preferred reaction time is 2 hours.

Example 5: Detection of lead ions under microwave

The invention shortens the reaction time required for detecting lead ions, and further heats the reaction by microwave to accelerate the leaching efficiency of the gold nanoparticles. As shown in Fig. 10(A), under the preferred reaction conditions, lead ions were detected in the case of experimental microwaves (power: 400 W, 450 W and 500 W) and no microwave (power: 0 W), and G _{abs was} monitored. corresponds to the degree of leaching of gold nanoparticles, the lead ion concentration and the corresponding relationship of G _abs can indeed increase the gold nanoparticles described microwave leaching efficiency.

The microwave power is preferably 450 W, as shown in Fig. 10(B), 450W microwave irradiation, lead ion concentration in the range of 100 pM to 1.5 nM, [( G _abs - G ⁰ _abs ) / G ⁰ _abs ] It has a linear relationship with the concentration of lead ions in the sample to be tested, where R ² = 0.96.

As shown in Fig. 11, under the microwave irradiation condition of 450 W, the change of G _abs with time in about 10 minutes has slowed down, indicating that the reaction time is reduced from 2 hours to 10 minutes under microwave irradiation.

Further discussion, the temperature changes generated by the microwave also increase the sensitivity of the detection. As shown in Fig. 12, when the microwave power is 400 W, 450 W and 500 W, the corresponding reaction temperatures are 50 ° C, 75 ° C and 90 ° C, respectively, and the reaction has a higher G _abs than the room temperature. Under microwave conditions, the detection limit of lead ion concentration can be increased to 50 pM, and the noise ratio (signal/noise (S/N) ratio) is 3.

Example 6: Selectivity of lead ions

As shown in Figure 13, under the microwave reaction conditions (450 W), lead ions and single metal ions (such as Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , Ba) are detected. ²⁺ , Mn ²⁺ , Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Pt ²⁺ , Cd ²⁺ , Pd ²⁺ , Cr ³⁺ , Fe ³⁺ , Ag ⁺ , Au ³⁺ and Hg ²⁺ ), and the change of G _abs is used as a criterion for judging selectivity. Furthermore, the concentration of a single metal ion is 100 nM, and the concentration of lead ion is only 10 nM, which is still highly selective, and the selectivity is at least 100 times higher than that of other metal ions. The change of red chromaticity is more visible to the naked eye. Observed easily.

Example 7: Detecting a sample to be tested

In order to confirm the high tolerance of the test strips (such as salt tolerance, thiol resistance and protein resistance), there is no aggregation when detecting the actual sample to be tested, so the simulated salt environment and the simulated protein environment are respectively The lead ion is detected. As shown in Figure 14 (A), in a salt environment (150 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, and 1 mM calcium chloride), or as shown in Figure 14 (B), the protein environment ( In the 25 μM bovine serum albumin solution, the detection limit is still up to 100 pM.

In addition, seawater, urine and blood samples are also tested.

The seawater sample was taken from the East China Sea and filtered with a 0.2 μm filter to remove impurities. The ion content was analyzed by ICP-MS. First, after the seawater sample is diluted 10 times, additional lead ions and reaction reagents are added, and the prepared lead solution has a lead ion concentration of 0-10 nM, and 1 ml of the mixed solution is added to the test strip for microwave irradiation (450 W). The reaction was carried out for 10 minutes and was judged.

The urine test sample was prepared using the standard method (1180B/05-OD) provided by the National Center for Environment Health. The lead ion content in the urine sample (SRM 2672a) is 1.20 μM, and the urine side sample is diluted 200 times, then lead ions and reagents are added thereto, and the mixed solution lead ion concentration is 0- 1.0 nM, 1 ml mixed solution was added to the test strip, and reacted under microwave irradiation (450 W) for 10 minutes to determine.

The blood samples BCR 636, 635 and 634 were all powdered and dissolved in 0.6 mL of 3 mL deionized water to make lead ion concentrations of 2.51 ± 0.24 μM, 1.2 ± 0.12 μM and 0.22 ± 0.02 μM, respectively. Blood sample to be tested. The samples to be tested for blood were separately digested by nitrification. After taking 1 mL of the blood sample to be tested, 1 mL of the nitrate solution and 8 mL of deionized water for two hours, the sample was placed in a centrifuge at a speed of 4000 rpm. After 2 minutes of rotation, the liquid was taken out, and BCR 636, 635, and 634 were diluted to a lead ion concentration of 0.5 nM, and then lead ions and a reagent were added thereto, and the final concentration of lead ions in the mixed solution prepared was 0-0.5 nM. The test paper was added to 1 ml of the mixed solution, and reacted under microwave irradiation (450 W) for 10 minutes to determine.

As shown in Fig. 15, lead ions are additionally added to the seawater sample solution, and the concentration of lead ions in the range of 100 pM to 10 nM is added, and the concentration of lead ions is [( G _abs - G ⁰ _abs ) / G ⁰ _Abs ] still has a linear relationship, where R ² = 0.96.

Continue to test the concentration of lead ions in urine and blood samples. Please refer to Table 1 for data.

As shown in Fig. 16(A), the standard urine sample SRM 2672a (the theoretical value of lead ion concentration 1.20 μM) was first detected by the standard addition method. The lead ion concentration contained in the urine sample was 1.17±0.071 μM (n= 5) When the confidence level is 95% and the t-test value is less than 2.776, the SRM 2672a has a confidence range of 1.08 μM to 1.26 μM. As shown in Fig. 16(B), the blood sample was detected in a similar manner, as shown in Table 1, and it was confirmed that the test paper was usable for the detection of a real sample. Further, referring to Fig. 17, the red chromaticity change of the test paper is checked and determined by the naked eye.

In summary, the detection method of the present invention has high specificity and sensitivity for lead ions, and can also use microwave irradiation to shorten the analysis time. Furthermore, the method can be applied to real samples, and the detection set is detected. The test paper and the reaction reagent are therefore simple to operate, portable and inexpensive.

Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the present invention, and those skilled in the art should be able to make some modifications and refinements without departing from the spirit and scope of the present invention. It belongs to the scope of protection defined by the scope of patent application of the present invention.

FIG. 1 is a schematic diagram of a detection kit for lead ions and a detection method thereof.

Figure 2 (A) and Figure 2 (D) are SEM images and high-magnification SEM images of gold nanoparticles modified with bovine serum albumin, the particle size of which is 13.3 ± 0.6 nm; Figure 2 (B) and Figure 2 (E) SEM image and high-magnification SEM image of the gold nanoparticles after the addition of the reaction reagent, the particle size of which is 12.1±0.8 nm; FIG. 2(C) and FIG. 2(F) are the reagents and lead ions added. The SEM image and the high-magnification SEM image of the posterior gold nanoparticles have a particle size of 3.2±1.2 nm.

3 is an ultraviolet-visible absorption spectrum diagram (a) is an aqueous solution of citrate-coated gold nanoparticles; (b) an aqueous solution of gold nanoparticles coated with bovine serum albumin; (c) is a test strip. Abs . is an arbitrary unit.

Figure 4 illustrates the relative EDS intensity signal of the gold nanoparticles of the test strip.

Figure 5 illustrates the detection of test paper on (A) Image J image software and (B) surface assisted laser desorption mass spectrometer to analyze test paper color uniformity. The value of Abs . is an arbitrary unit.

Figure 6 is a graph showing the relationship between the concentration of 2-thioglycol and the concentration of G _abs on the concentration of lead ions. Among them, under the preferred reaction conditions, the lead ion concentration is 10 nM to 1 μM, and G _abs has a linear relationship with the lead ion concentration ( R ² =0.98). G _abs is an arbitrary unit.

Fig. 7(A) is a graph showing the change of the absorption value ( Abs ₅₂₀ ) of an aqueous solution of a gold nanoparticle modified with bovine serum albumin over time, wherein (a) is a gold nanoparticle modified with bovine serum albumin (1.5 nM). (b) modified with gold serum albumin (1.5 nM), sodium thiosulfate (1.0 mM), 2-thioglycol (100 mM); (c) modified bovine serum albumin Gold nanoparticles (1.5 nM), sodium thiosulfate (1.0 mM), 2-thioglycol (100 mM), lead chloride (1.0 μM); (d) Chennai modified with bovine serum albumin Rice particles (1.5 mM), lead chloride (1.0 μM). The value of Abs ₅₂₀ is an arbitrary unit, and the error value is the result of four analyses. FIG. 7 (B) G _abs system reaction after 2 hours test strip. Wherein (a) no reagent was added; (b) sodium thiosulfate (100 mM), 2-thioglycol (250 mM); (c) sodium thiosulfate (100 mM), 2-thioglycol added Alcohol (250 mM), lead chloride (1.0 μM); (d) lead chloride (1.0 μM). G _abs is an arbitrary unit, and the error value is the result of four analyses.

Figure 8 is an analysis of SALDI-MS mass spectrometry using a test strip as a substrate. In the enlarged view, the left side is the database map, and the right side is the detection map. The map signals of lead ions and AuPb alloys are m/z 205.974, 206.976, 207.973, 402.941, 403.942 and 404.943, respectively [ ₂₀₆ Pb] ⁺ , [ ₂₀₇ Pb] ⁺ , [ ₂₀₈ Pb] ⁺ , [Au ₂₀₆ Pb] ⁺ , [Au ₂₀₇ Pb] ⁺ and [Au ₂₀₈ Pb] ⁺ ion signals.

9 illustrates the preferred reaction conditions, G _abs correspondence relationship versus time.

FIG 10 (A) are described in microwave power 0 W, 400 W, 450 W and 500 W, and the concentration of lead ions G _abs correspondence relationship of FIG. Fig. 11(B) is a view showing the correspondence relationship between the microwave power of 450 W, [( G _abs - G ⁰ _abs ) / G ⁰ _abs ] and the lead ion concentration.

Figure 11 is a graph showing the correspondence between G _{abs and} time for microwave power of 450 W under the preferred reaction conditions.

Figure 12 is a graph showing the relationship between G _{abs and} lead ion concentration under the preferred reaction bars for reaction temperatures of 25 ° C, 50 ° C, 75 ° C and 90 ° C, respectively.

Figure 13 illustrates the selectivity of test strips for lead ions compared to other single metal ions under preferred microwave reaction conditions.

Figure 14 is a graph showing the correlation between the detection of lead ions, [( G _abs - G ⁰ _abs ) / G ⁰ _abs ] and the concentration of lead ions in a salt environment and a protein environment, and the reaction conditions are preferably microwave reaction conditions. Wherein (A) comprises 150 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride and 1 mM calcium chloride; (B) comprises 250 μM cysteine; (C) comprises 25 μM bovine serum albumin solution .

Fig. 15 is a view showing the relationship between the detection of seawater sample, [( G _abs - G ⁰ _abs ) / G ⁰ _abs ] and the concentration of lead ions, and the reaction conditions are preferred microwave reaction conditions.

Fig. 16 is a view showing the relationship between the detection of urine and blood samples, [( G _abs - G ⁰ _abs ) / G ⁰ _abs ] and the concentration of lead ions, and the reaction conditions are preferred microwave reaction conditions. Among them, (A) standard urine sample SRM 2672a, externally added lead ion concentration from 0 to 1.0 nM; (B) blood sample BCR 636, externally added lead ion concentration from 0 to 0.5 nM.

Figure 17 is a blood sample tested to be diluted 50 times, and the reaction conditions are preferably microwave reaction conditions. Among them, (A) BCR 634, lead ion concentration 0.22 ± 0.02 μM; (B) BCR 635, lead ion concentration 1.01 ± 0.12 μM; (C) BCR 636, lead ion concentration 2.51 ± 0.24 μM.

Claims (7)

A method for detecting lead ions, comprising the steps of: (a) providing a test strip comprising a substrate and protein-modified gold nanoparticles attached to the substrate, the test strip exhibiting a first intensity of a color (b) providing a reagent comprising sodium thiosulfate (Na ₂ S ₂ O ₃ ), 2-mercaptoethanol, and a buffer solution; (c) the sample to be tested and the test strip and the Reacting the reagent, then treating the test strip with microwaves to exhibit a second intensity of the color; and (d) analyzing and comparing the first intensity and the second intensity of the color, wherein the second intensity is less than the first intensity The sample contains lead ions. The test method according to claim 1, wherein the test paper is obtained by mixing the gold nanoparticles with a protein solution having the protein to form a mixture of gold nanoparticle proteins, and A gold nanoparticle protein mixture is applied to the substrate. The test method of claim 1, wherein the gold nanoparticles have an average diameter of 5 to 25 nm. The detection method according to claim 1, wherein the substrate is selected from the group consisting of a nitrocellulose membrane, a nylon-resistant film, a polyvinylidene fluoride film (PVDF film), a cellulose acetate film, a mixed fiber membrane, and oxidation. A group of aluminum filter membranes and polycarbonate resin membranes. The method of detecting according to claim 1, wherein the protein is selected from the group consisting of bovine serum albumin, albumin, myoglobin, fibrin, and casein. The detection method according to claim 1, wherein the buffer solution has a pH range of 9-11. The method of claim 1, wherein the buffer solution is selected from the group consisting of sodium glycinate buffer solution, phosphate buffer solution, trisethanol buffer (Tris) buffer solution, and citrate buffer solution. The group of people.