A method for detecting heavy metals in tea based on a ratiometric SERS sensing platform

Abstract

The present invention discloses a method for detecting heavy metals in tea leaves based on a ratiometric SERS sensing platform. The method comprises preparing gold and silver core-shell nanomaterials and amino-modified magnetic nanospheres respectively; mixing the amino-modified magnetic nanospheres and the gold and silver core-shell nanomaterials to prepare FAA; modifying the surface of the FAA with a DNA double-strand functionalized with a SERS signal molecule to achieve functional modification of the FAA; utilizing the functionally modified FAA to perform SERS signal detection on solutions containing heavy metal elements of different concentrations, and establishing a relationship curve between concentration and signal. When the sensor designed in the present application detects the presence of a heavy metal to be detected in the system, the signal molecule falls off the surface of the signal element, providing a binding site for another signal molecule, thereby achieving ratiometric rapid detection of heavy metals in tea leaves. In addition, the sensor after the reaction returns to its original state through a chain displacement reaction, thereby achieving regeneration and recycling of the sensor.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and in particular relates to a method for rapid detection of cadmium metal in tea leaves based on a ratiometric SERS biosensor platform based on chain displacement. Background Art

[0002] China was the first country in the world to discover and utilize tea trees, fostering a long-standing tea culture. However, tea trees can be contaminated by heavy metals during their growth process. Heavy metal content is a key indicator of tea's hygienic quality, so testing and monitoring heavy metal levels in commercially available tea is crucial for consumer safety.

[0003] Traditional methods for detecting heavy metal content include graphite furnace atomic absorption spectrometry, inductively coupled plasma mass spectrometry, flame atomic absorption spectrometry, and disulfide hydrazone colorimetry. However, these traditional methods suffer from expensive equipment, high testing costs, and cumbersome procedures. Furthermore, they cannot meet the requirements for rapid, real-time detection of heavy metal content. Therefore, a rapid method for detecting heavy metals in tea leaves is urgently needed that can overcome the shortcomings of these traditional methods and improve the speed, specificity, and sensitivity of heavy metal content detection in tea leaves. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of conventional detection technologies, such as long detection times, expensive equipment, and complex detection procedures. This invention provides a method for detecting heavy metals in tea leaves based on a ratiometric SERS sensing platform. Gold and silver core-shell-coated magnetic nanoparticles are prepared as SERS signal elements, and double-stranded DNA functionalized with SERS signal molecules is surface-modified. When the heavy metal to be detected is present in the system, the signal molecule detaches from the surface of the signal element, providing binding sites for another signal molecule, thereby achieving rapid ratiometric detection of heavy metals in tea leaves. Furthermore, the sensor returns to its original state through a strand displacement reaction after reaction, enabling sensor regeneration and recycling.

[0005] The technical solutions adopted in the present invention are as follows:

[0006] The technical solution adopted by the present invention is: a method for detecting heavy metals in tea based on a ratiometric SERS sensing platform, comprising the following steps:

[0007] Step 1, preparing gold and silver core-shell nanomaterials;

[0008] Step 2: preparing gold-silver core-shell modified magnetic nanoparticles FAA;

[0009] The ferroferric oxide microspheres are mixed with polyethyleneimine, and the mixture is ultrasonically dissolved and magnetically separated to obtain amino-modified magnetic nanospheres; the amino-modified magnetic nanospheres are nano-mixed with the gold-silver core-shell material prepared in step 1, and the mixture is mixed, magnetically separated, washed, and vacuum-dried to obtain FAA particles;

[0010] Step 3, functional modification of FAA;

[0011] The FAA particles were mixed with a streptavidin solution to obtain streptavidin-coated magnetic nanoparticles;

[0012] A single-stranded DNA H1 containing a DNA enzyme and modified with avidin at one end is prepared; a single-stranded DNA H2 complementary to H1 and containing an RNA fragment is prepared, and the end of H2 is modified with a SERS signal molecule A; H1 and H2 are linked by PCR to form a double strand; the RNA fragment in H2 is complementary to the DNA enzyme in H1;

[0013] The double strands were mixed with streptavidin-coated magnetic nanoparticles and incubated to obtain the magnetic nanobiosensor FAA-cDNA1;

[0014] Step 4: Establishment of identification method for heavy metal elements;

[0015] Prepare different concentrations of heavy metal solutions to be tested. First, add the different concentrations of heavy metal solutions to be tested to FAA-cDNA1 for incubation. The H2 on the surface of FAA-cDNA1 breaks in the presence of the target heavy metal, and then perform magnetic separation.

[0016] H3 is added to the magnetically separated FAA-cDNA1, wherein H3 is a DNA single strand with a terminal modified SERS signal molecule B; H3 replaces the broken H2 on the surface of FAA-cDNA2, and the replaced FAA-cDNA2 is obtained after incubation and magnetic separation. The SERS signal of the replaced FAA-cDNA2 is collected to establish the concentration and ratio signal I B / I A The relationship curve between

[0017] Step 5: Regeneration and reuse of sensors;

[0018] The intact H2 is added to the FAA-cDNA2 solution after H3 replacement. Based on the chain displacement reaction, H2 replaces H3. After incubation and magnetic separation, the magnetic nanobiosensor FAA-cDNA1 is recovered. The magnetic nanobiosensor is re-processed for heavy metal element identification in step four to achieve the regeneration and reuse of the sensor.

[0019] Furthermore, in step 1, 1% chloroauric acid solution was heated to 100° C., 1% trisodium citrate solution was added within 5 seconds to continue the reaction, and AgNO3 solution was added dropwise at a rate of 30 μL / min after 30 minutes. After the addition was completed, the mixture was stirred for another 30 minutes, the reaction was stopped, and the gold-silver core-shell nanomaterial was obtained after cooling.

[0020] Furthermore, the volume ratio of chloroauric acid, trisodium citrate and silver nitrate is 100:1.5:2, and the concentration of silver nitrate is 2-8 mM.

[0021] Furthermore, in step 2, the volume ratio of the ferroferric oxide microspheres, polyethyleneimine and gold-silver core-shell material is 1000:0.2:200, the size of the ferroferric oxide microspheres is 150-250 nm, the ferroferric oxide concentration is 1 mg / mL, the polyethyleneimine concentration is 10 g / mL, and the shaking table temperature is 25°C.

[0022] Furthermore, in step 3, the volume ratio of the FAA particles to streptavidin is 1:1, the concentration of the FAA particles is 1 mg / mL, the concentration of the streptavidin is 0.1-0.2 mg / mL, and the shaking temperature is 37°C.

[0023] Furthermore, the concentration ratio of H1, H2 and streptavidin-coated magnetic nanoparticles was 1 μM:1 μM:1 g / mL, and the incubation temperature was 37°C.

[0024] Furthermore, in step 4, the concentration of heavy metal ions is in the range of 1.0×10 -11 ~1.0×10 -5 M; the volume ratio of heavy metal solution, FAA-cDNA1 and H1 was 200:10:(0.2-0.5), the concentration of FAA-cDNA1 was 1 mg / mL, the concentration of H1 was 100 ng / mL, and the incubation temperature was 37°C.

[0025] Furthermore, in step 4, the SERS signal molecules in H3 and H2 are different, and H3 can be connected to H1.

[0026] Furthermore, in step five, the volume ratio of H2 to FAA-cDNA2 was (0.2-0.5):1, the concentration of FAA-cDNA1 was 1 mg / mL, the concentration of H1 was 100 ng / mL, and the incubation temperature was 37°C.

[0027] Furthermore, in step five, H2 replaces H3 and connects to H1 through strand displacement.

[0028] Compared with existing detection technologies, the present invention has the following beneficial effects:

[0029] 1. The present invention provides a method for detecting heavy metals in tea leaves based on a ratiometric SERS sensing platform. A gold-silver core-shell-coated magnetic nanoparticle is prepared as a SERS signal element, and a double-stranded DNA functionalized with a SERS signal molecule is modified on the surface. When the heavy metal to be detected is present in the system, the signal molecule falls off the surface of the signal element, thereby realizing a rapid detection method for heavy metals in tea leaves.

[0030] 2. The present invention provides a method for detecting heavy metals in tea based on a ratiometric SERS sensing platform. The specifically designed sensor has a magnetic separation function, which has the advantages of fast separation speed and simple operation steps.

[0031] 3. The present invention provides a method for detecting heavy metals in tea based on a ratiometric SERS sensing platform. The heavy metal DNA enzyme specifically designed on the surface of FAA-cDNA1 can specifically recognize heavy metals, causing H2 to detach from the surface of H1, providing a site for the attachment of H3, and realizing a ratiometric change in the signal.

[0032] 4. The present invention provides a method for detecting heavy metals in tea leaves based on a ratiometric SERS sensing platform. Specifically, H2 can replace H3 through chain displacement, enabling the recycling of the sensor.

[0033] 5. The present invention provides a method for detecting heavy metals in tea based on a ratiometric SERS sensing platform. By replacing the DNA enzyme on the surface of the material, ratiometric detection of different analytes can be achieved, which has a certain universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only an implementation example of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Detection principle diagram

[0036] Figure 2 Electron microscope images of FAA prepared by the present invention; in the figure, A is a transmission electron microscope image of FAA; B is a scanning electron microscope image of FAA; DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the following will be clearly described in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0038] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0039] A method for detecting heavy metals in tea based on a ratiometric SERS sensing platform, comprising the following steps:

[0040] Step 1: Preparation of gold and silver core-shell nanomaterials:

[0041] Heat 50 ml of 1% chloroauric acid solution to 100°C, add 750 μL of 1% trisodium citrate solution within 5 seconds to continue the reaction, and after 30 minutes, add 1 mL of 4 mM AgNO3 solution dropwise at a rate of 30 μL / min. After the addition is complete, stir for another 30 minutes, stop the reaction, and cool to obtain gold-silver core-shell nanomaterials.

[0042] Step 2: Preparation of gold-silver core-shell modified magnetic nanoparticles (FAA):

[0043] 10 ml of 1 mg / mL ferrosoferric oxide microsphere aqueous solution was mixed with 200 μL of 10 g / mL polyethyleneimine, and dissolved by ultrasonication for 2 hours. Amino-modified magnetic nanoparticles were obtained by magnetic separation.

[0044] 80 ml of amino-modified magnetic nanospheres were mixed with 200 ml of gold-silver core-shell nanomaterials prepared in step 1, and the mixture was shaken at 25°C for 3 hours, magnetically separated and washed three times with water, and vacuum dried to obtain FAA;

[0045] Step 3, functional modification of FAA:

[0046] Heavy metal DNA enzymes consist of a single-stranded DNA sequence that binds to target mRNA at a single programmable site through complementary base pairing. Metal ions act as cofactors, leading to partial enzymatic cleavage of the bound RNA. For this application, DNA fragments ordered from Shanghai Sangon Biotech Co., Ltd. were selected, as follows:

[0047] H1:5'-biotin-ACAGACATCATCTCTGAAGTAGCGCCGCCGTATAGTAG

[0048] H2:5'-CTACTATrAGGAAGAGATGATGTC-Cy3-3'

[0049] H3:5'-TCAGAGATGATGTC-MB-3'

[0050] The specific process of functional modification of FAA is as follows:

[0051] 10 ml of 1 mg / mL FAA particles were mixed with 10 ml of 0.1 g / mL streptavidin solution, shaken on a shaker at 37 degrees for 4 h, and then magnetically separated. After washing with water, the mixture was dissolved in 0.1 M PBS solution to 1 mg / mL to obtain streptavidin-coated magnetic nanoparticles.

[0052] A single-stranded DNA enzyme, H1, is prepared, with one end modified with avidin and containing a DNA enzyme. A single-stranded DNA enzyme, H2, complementary to H1 and containing an RNA fragment, is prepared. The end of H2 is modified with a SERS signal molecule, A. The RNA fragment in H2 is complementary to the DNA enzyme in H1 and can be decomposed by the DNA enzyme in the presence of the target heavy metal, causing cleavage. The DNA enzyme is selected based on the type of heavy metal to be detected.

[0053] 100 μL of 10 μM H1 and 100 μL of 10 μM H2 were linked by PCR to form a double strand.

[0054] The double strands were mixed with 100 μL of 10 mg / mL streptavidin-coated magnetic nanoparticles, incubated at 37°C for 8 h, and then removed to obtain the magnetic nanobiosensor FAA-cDNA1;

[0055] Step 4: Establishment of identification method for heavy metal elements:

[0056] In this embodiment, lead is used as an example to illustrate the preparation concentration range of 1.0×10 -11 ~1.0×10 -5 First, 2 mL of each lead metal solution was added to 100 μL of 1 mg / mL FAA-cDNA1 and incubated for 20 min. Since the heavy metal DNA enzyme on the surface of FAA-cDNA1 can specifically recognize lead, H2 is broken. Then, magnetic separation is performed and the supernatant is discarded.

[0057] Then, 30 μL of 100 ng / mL H3 was added to the magnetically separated FAA-cDNA1. H3 is a single-stranded DNA with a terminal modified SERS signal molecule B. Based on the strand displacement effect, H3 can replace the broken H2 in FAA-cDNA1. After incubation on a shaker for 20 minutes, magnetic separation was performed to obtain FAA-cDNA2 after H3 replacement. The SERS signal of the obtained product (i.e., FAA-cDNA2 after H3 replacement) was collected, and the signals corresponding to SERS signal molecules A and B were obtained simultaneously. Therefore, the relationship between this type of heavy metal and ratio signal I can be established. B / I A The relationship curve between .

[0058] Step 5: Sensor regeneration and reuse: 30 μL of 100 ng / mL H2 was added to 100 μL of 1 mg / mL FAA-cDNA2 solution. The mixture was incubated on a shaker at 37°C for 1 hour. FAA-cDNA1 was obtained after magnetic separation. The resulting product was then subjected to step 4 again to verify sensor regeneration. In the sensor designed in this application, since H2 can replace H3 through strand displacement, sensor regeneration and reuse are possible.

[0059] In this embodiment, the SERS signal molecules A and B of H2 and H3 can be Cy3, Cy5MB or rhodamine, but A and B must be of different species.

[0060] In summary, the present invention provides a method for rapid detection of heavy metals in tea leaves based on a strand-displacement ratiometric SERS biosensing platform. Gold and silver core-shell-coated magnetic nanoparticles are prepared as SERS signal elements, and double-stranded DNA functionalized with SERS signal molecules is surface-modified. When the heavy metal to be detected is present in the system, the signal molecule detaches from the surface of the signal element, providing binding sites for another signal molecule, thereby achieving a ratiometric rapid detection method for heavy metals in tea leaves. Furthermore, after the reaction, the sensor returns to its original state through a strand-displacement reaction, enabling sensor regeneration and recycling.

[0061] Although the embodiments of the present invention have been described with reference to the accompanying drawings, those skilled in the art may make various modifications and variations without departing from the spirit and scope of the present invention. Such modifications and variations are all within the scope defined by the appended claims.

Claims

1. A method for rapid detection of heavy metals in tea leaves based on a strand displacement ratiometric SERS biosensor platform, characterized by: The following steps are involved: Step 1, preparing gold and silver core-shell nanomaterials; Step 2: preparing gold-silver core-shell modified magnetic nanoparticles FAA; The ferroferric oxide microspheres are mixed with polyethyleneimine, and the mixture is ultrasonically dissolved and magnetically separated to obtain amino-modified magnetic nanospheres; the amino-modified magnetic nanospheres are nano-mixed with the gold-silver core-shell material prepared in step 1, and the mixture is mixed, magnetically separated, washed, and vacuum-dried to obtain FAA particles; Step 3, functional modification of FAA; The FAA particles were mixed with a streptavidin solution to obtain streptavidin-coated magnetic nanoparticles; A single-stranded DNA H1 containing a DNA enzyme and modified with avidin at one end is prepared; a single-stranded DNA H2 complementary to H1 and containing an RNA fragment is prepared, and the end of H2 is modified with a SERS signal molecule A; H1 and H2 are linked by PCR to form a double strand; the RNA fragment in H2 is complementary to the DNA enzyme in H1; The double strands were mixed with streptavidin-coated magnetic nanoparticles and incubated to obtain the magnetic nanobiosensor FAA-cDNA1; Step 4: Establishment of identification method for heavy metal elements; Prepare different concentrations of heavy metal solutions to be tested. First, add the different concentrations of heavy metal solutions to be tested to FAA-cDNA1 for incubation. The H2 on the surface of FAA-cDNA1 breaks in the presence of the target heavy metal, and then perform magnetic separation. H3 is added to the magnetically separated FAA-cDNA1, wherein H3 is a DNA single strand with a terminal modified SERS signal molecule B; H3 replaces the broken H2 on the surface of FAA-cDNA2, and the replaced FAA-cDNA2 is obtained after incubation and magnetic separation. The SERS signal of the replaced FAA-cDNA2 is collected to establish the concentration and ratio signal I B / I A The relationship curve between Step 5: Regeneration and reuse of sensors; The intact H2 is added to the FAA-cDNA2 solution after H3 replacement. Based on the chain displacement reaction, H2 replaces H3. After incubation and magnetic separation, the magnetic nanobiosensor FAA-cDNA1 is recovered. The magnetic nanobiosensor is re-processed for heavy metal element identification in step 4 to achieve the regeneration and reuse of the sensor.

2. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 1, 1% chloroauric acid solution was heated to 100°C, 1% trisodium citrate solution was added within 5 seconds to continue the reaction, and AgNO3 solution was added dropwise at a rate of 30 μL / min after 30 minutes. After the addition was completed, the mixture was stirred for another 30 minutes, the reaction was stopped, and the gold-silver core-shell nanomaterial was obtained after cooling.

3. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 2, characterized in that: The volume ratio of the chloroauric acid, trisodium citrate and silver nitrate is 100:1.5:2, and the concentration of silver nitrate is 2-8 mM.

4. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 2, the volume ratio of the ferroferric oxide microspheres, polyethyleneimine and gold-silver core-shell material is 1000:0.2:200, the size of the ferroferric oxide microspheres is 150-250 nm, the ferroferric oxide concentration is 1 mg / mL, the polyethyleneimine concentration is 10 g / mL, and the shaking table temperature is 25°C.

5. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 3, the volume ratio of the FAA particles to streptavidin was 1:1, the concentration of the FAA particles was 1 mg / mL, the concentration of the streptavidin was 0.1-0.2 mg / mL, and the shaking temperature was 37°C.

6. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: The concentration ratio of H1, H2 and streptavidin-coated magnetic nanoparticles was 1 μM:1 μM:1 g / mL, and the incubation temperature was 37°C.

7. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 4, the heavy metal ion concentration range is 1.0 × 10 −11 ~ 1.0 × 10 −5 M; the volume ratio of heavy metal solution, FAA-cDNA1 and H1 was 200:10:(0.2-0.5), the concentration of FAA-cDNA1 was 1 mg / mL, the concentration of H1 was 100 ng / mL, and the incubation temperature was 37°C.

8. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 4, the SERS signal molecules in H3 and H2 are different, and H3 can be connected to H1.

9. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 1, characterized in that: In step 5, the volume ratio of H2 to FAA-cDNA2 was (0.2-0.5):1, the concentration of FAA-cDNA1 was 1 mg / mL, the concentration of H1 was 100 ng / mL, and the incubation temperature was 37°C.

10. The method for rapid detection of heavy metals in tea leaves based on a strand displacement ratio SERS biosensor platform according to claim 9, characterized in that: In step five, H2 displaces H3 and is connected to H1 by strand displacement.

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