Mercury-activated SERS nanoprobe and its preparation method and application

Abstract

The present invention discloses a mercury-activated SERS nanoprobe and its preparation method and application. The mercury-activated SERS nanoprobe is a Raman signal molecule modified on the surface of a gold nanosphere. The Raman signal molecule is b-(s)-EPBA, which is obtained by amidation reaction of chlorinated 4-carboxylphenylboronic acid and cystamine dihydrochloride. The Raman signal molecule b-(s)-EPBA is bonded to the gold nanosphere via a disulfide bond, resulting in a large number of aromatic rings on the probe. The aromatic rings can serve as Raman active sites to ensure the intensity of the Raman signal; the boric acid group can be used as a recognition group to undergo electrophilic substitution, thereby improving the detection sensitivity, selectivity, and detection speed of the probe. Raman detection can be performed after only 5-10 minutes of incubation, achieving rapid quantitative detection of inorganic mercury and methylmercury.

Description

Technical Field

[0001] The present invention relates to the field of quantitative detection of mercury, in particular to a mercury-activated SERS nanoprobe, a method for preparing the mercury-activated SERS nanoprobe, and an application of the mercury-activated SERS nanoprobe in the quantitative detection of organic mercury and / or inorganic mercury. Background Art

[0002] Heavy metal pollution, such as mercury, arsenic, cadmium, and lead, can severely impact the environment and even ecosystems and human health. Currently, mercury pollution in the environment is steadily increasing, with mercury levels in various environmental carriers exceeding permitted levels, severely impacting the ecological environment. Furthermore, inorganic mercury can be converted into organic mercury forms such as methylmercury and ethylmercury by microorganisms. Methylmercury is the most toxic of all mercury forms, potentially harmful to human health through the food chain and causing irreversible damage to the central nervous system. Clinically, the maximum acceptable dose of methylmercury should not exceed 0.1 micrograms per kilogram of body weight. Given the toxicity of mercury, the national "Surface Water Environmental Quality Standards" stipulate that mercury levels in Class I and Class II surface waters should be ≤ 0.00005 mg / L (50 ng / L); and in Class III surface waters, ≤ 0.00001 mg / L (100 ng / L).

[0003] Currently, environmental and ecological concerns have made the detection of inorganic and organic mercury in environmental samples urgent. Traditional detection methods include fluorescence spectroscopy, electrochemical detection, enzyme-linked immunosorbent assay (ELISA), and inductively coupled plasma-mass spectrometry (ICP-MS). However, these methods often suffer from complex sample preparation procedures, high laboratory equipment costs, and long detection cycles, which limit their further application in mercury detection.

[0004] Surface enhanced Raman scattering technology (SERS technology) has become a powerful means for quantitative detection of organic pollutants, inorganic pollutants and pathogens in environmental samples due to its advantages such as high sensitivity, rapidity and lack of photobleaching. In addition, the low polarizability index of water means that it produces negligible signals relative to the more polarizable pollutant analytes dissolved or suspended in it, so that pollutant-containing wastewater can be detected by SERS without being interfered with by the water medium. The excellent performance of SERS technology makes it a promising candidate for the detection of mercury ions and organic mercury in the environment. Kuang et al. developed a ssDNA-modified nanostar dimer as a method for detecting Hg 2+ The study showed that in Hg 2+ In the presence of thymine (T)-Hg 2+The formation of -T is converted into a hairpin structure, which leads to the dimerization of nanostars. The huge enhancement of the electromagnetic field by the dimer structure increases the Raman intensity of 4-ATP. Li et al. developed a morphology-based transition-based dual-mode nanoprobe GNRs@PATP to simultaneously detect Hg using the unique surface chemical properties of mercury. 2+ and CH3Hg + GNRs@PATP nanoprobe for Hg 2+ showed significant SERS enhancement, but for CH3Hg + There is an immediate color response.

[0005] At present, although the SERS probe for detecting mercury has made certain progress, the following defects are common: First, other metal ions with similar electron shell structures (such as Ag + , Pb 2+ 、Cu 2+ ) can also be combined with the amino group, thiol group or I - response, low selectivity, which in turn affects Hg 2+ Secondly, DNA-modified SERS probes require time-consuming surface functionalization, complex incubation processes, and strict testing environments, which to some extent limit their application. Furthermore, most SERS probes respond to mercury with a single absolute signal intensity, and many environmental factors can cause signal intensity fluctuations, leading to inaccurate determination of heavy metal ion concentrations. Finally, due to the lack of suitable CH3Hg + Specific Raman probe, simultaneous detection of Hg using SERS nanoprobe 2+ and CH3Hg + , especially CH3Hg + Imaging in living organisms remains a significant challenge.

[0006] In summary, it is crucial to design a SERS nanoprobe with simple preparation steps, short incubation time, strong selectivity, high detection sensitivity, and the ability to simultaneously achieve quantitative detection of organic and inorganic mercury. Summary of the Invention

[0007] In view of this, the first object of the present invention is to provide a mercury-activated SERS nanoprobe, which not only has good selectivity but also has excellent stability and sensitivity, and can achieve simultaneous quantitative detection of organic mercury and inorganic mercury, which is of great significance for the detection of mercury ions or organic mercury in the environment.

[0008] The second purpose of the present invention is to provide a mercury-activated SERS nanoprobe. The method first synthesizes the Raman signal molecule b-(s)-EPBA and bonds it to the surface of gold nanospheres through Au-S bonds, providing a Raman identification tag for the detection of mercury and methylmercury.

[0009] The third object of the present invention is to provide the use of mercury-activated SERS nanoprobes in the quantitative detection of organic mercury and / or inorganic mercury, Hg 2+ The detection limit is as low as 10 nM; the detection limit for methylmercury is as low as 25 nM.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The mercury-activated SERS nanoprobe of the present invention is characterized in that the nanoprobe is a Raman signal molecule modified on the surface of a gold nanosphere, and the Raman signal molecule is b-(s)-EPBA, with the following structural formula:

[0012] .

[0013] In the present invention, the Raman signal molecule is bonded to the gold nanosphere through a disulfide bond, so that the probe has a large number of aromatic rings. The aromatic rings can serve as Raman active sites to ensure the intensity of the Raman signal; the boronic acid group can act as a recognition group to undergo electrophilic substitution, thereby improving the detection sensitivity, selectivity and detection speed of the probe. Raman detection only requires incubation for 5-10 minutes, realizing rapid quantitative detection of inorganic mercury and methylmercury.

[0014] Preferably, the diameter of the gold nanospheres is 50-70 nm, and the b-(s)-EPBA on the surface of the gold nanospheres is a monolayer. More preferably, the diameter of the gold nanospheres is 60 nm, which is beneficial for the excitation and detection of analytes.

[0015] The present invention also provides a method for preparing a mercury-activated SERS nanoprobe, comprising the following steps:

[0016] In the first step, the carboxyl group in 4-carboxyphenylboronic acid is activated to form an acyl chloride to obtain an acyl chloride product; the amino group in cystamine dihydrochloride is subjected to an amidation reaction with the acyl chloride product to obtain a Raman probe molecule b-(s)-EPBA;

[0017] In the second step, the gold nanospheres with surface plasmon resonance effect were prepared by seed-mediated growth method;

[0018] In the third step, a layer of b-(s)-EPBA synthesized in the first step is modified on the surface of the gold nanospheres. After the reaction is completed, the precipitate is dispersed in a buffer solution to obtain a mercury-activated SERS nanoprobe.

[0019] In the present invention, the first step specifically includes the following:

[0020] S11, 4-carboxyphenylboronic acid and thionyl chloride are subjected to an acyl chloride reaction under heating and reflux conditions to activate the carboxyl group in the 4-carboxyphenylboronic acid to form an acyl chloride, the reaction temperature is 80°C-95°C, and the reflux time is 12-36 h;

[0021] S12, dissolving the acyl chloride product in a mixed solution of sodium hydroxide and tetrahydrofuran, mixing well, and slowly adding dropwise to an aqueous solution of cystamine dihydrochloride to carry out an amide reaction under nitrogen protection; after the reaction, removing tetrahydrofuran, adjusting the pH to 2-3 with concentrated hydrochloric acid, filtering, and drying to obtain b-(s)-EPBA as a white powder.

[0022] More preferably, in S12, the amide reaction is first maintained in an ice bath for 30 min-60 min (preferably 60 min), then heated to 20°C-30°C (preferably 25°C) and maintained for 10-24 h; the drying condition in S12 is drying at 50°C-80°C under vacuum overnight.

[0023] Preferably, the buffer in the third step is HEPES buffer with a concentration of 10 mM and a pH of 7.4, that is, the probe prepared in the present invention is dispersed in HEPES buffer.

[0024] The present invention also provides the use of mercury-activated SERS nanoprobes in detecting organic mercury and / or inorganic mercury. Organic mercury includes methylmercury, ethylmercury and phenylmercury; the mercury ions in inorganic mercury are Hg 2+ , which can be a divalent mercury salt soluble in water, such as mercuric chloride, mercuric nitrate, and mercuric sulfate; the probe of the present invention can detect Hg in the sample. 2+ The method can be used for quantitative detection of mercury and organic mercury, and the detection steps are simple and quick.

[0025] The present invention also provides a method for detecting organic mercury and inorganic mercury in a sample based on a mercury-activated SERS nanoprobe and surface-enhanced Raman scattering technology, which specifically includes the following steps:

[0026] S1, centrifuge the sample at a high speed of 5000 rpm or higher and filter it through a 0.25 μm filter;

[0027] S2: Add mercury-activated SERS nanoprobes to the treated sample, incubate at 37°C for 5-10 min, and then perform Raman detection. The Raman test parameters are: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 .

[0028] During the test, a standard curve needs to be drawn, with the concentration as the horizontal axis and the ratio of the intensity signal as the vertical axis. Specifically, it includes the following contents:

[0029] To the known concentration of Hg 2+ and CH3Hg + Add the nanoprobe AuNPs@b-(s)-EPBA and incubate at constant temperature for 5-10 minutes; 2+ Hg in the mixed probe solution 2+ The final concentrations of the following were: 0.01 μM, 3 μM, 6 μM, 10 μM and 15 μM; containing CH3Hg + CH3Hg in the mixed probe solution + The final concentrations were: 0.05 μM, 2 μM, 5 μM, 7 μM, and 10 μM;

[0030] After the incubation, the probe was dropped onto tin foil for Raman characterization. The Raman spectroscopy parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600–1800 cm -1 ;

[0031] Take I 1002 / I 1303 or I 1080 / I 1303 The relative peak intensity of Hg is used as the vertical axis to reduce the error as much as possible and improve the reliability of the test results. 2+ The concentration is the horizontal axis, and I 1002 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=-0.08X+1.43,R 2 =0.994 ; or I 1080 / I 1303 Draw the standard curve as the vertical axis, and the fitting equation is Y=0.11X+0.17,R 2 =0.993 , Hg 2+ The minimum detection limit can reach 10 nM;

[0032] CH3Hg + The concentration value is the horizontal axis, with I 1002 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=-0.12X+1.3,R 2 =0.999 ; or I 1080 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=0.11X+0.15,R 2 =0.997 The minimum detection limit can reach 25 nM.

[0033] Compared with the prior art, the advantages of the present invention are:

[0034] The present invention synthesizes a novel functionalized Raman signal molecule b-(s)-EPBA, and bonds b-(s)-EPBA to gold nanospheres of approximately 60 nm in size via Au-S bonds, thereby synthesizing a SERS nanoprobe with high plasmon resonance activity. The preparation method of the Raman signal molecule is simple, and the Raman signal molecule has two benzene rings, which means that the probe of the present invention has a large number of aromatic rings that can serve as Raman active sites, thereby ensuring the intensity of the Raman signal.

[0035] The SERS nanoprobe of the present invention has a large number of phenylboronic acid groups, which can react with Hg through mercury-induced metal transfer. 2+ and CH3Hg + It exhibits a specific activation effect, through the cleavage of the C-B bond and the formation of the C-Hg bond, the boronic acid group can be activated by Hg 2+ and CH3Hg + Specific substitution to avoid other metal ions Fe 2+ 、Al 3+ 、Cd 2+ 、Ag + Cr 3+ Mg 2+ GSH, L-Cys, active biothiols, reactive oxygen species (H2O2), L-AA, CO3 2- 、NO2 - 、H2PO4 - It has good selectivity and no interference.

[0036] The present invention also establishes a Raman detection method based on b-(s)-EPBA for the simultaneous detection of organic and inorganic mercury. Raman detection can be performed by incubating the probe and the analyte for 5-10 minutes, with low sample pretreatment requirements. It also achieves simultaneous and rapid detection of organic and inorganic mercury with high detection efficiency.

[0037] After testing, the present invention is effective in detecting Hg 2+ and CH3Hg + When the method is used, it can not only realize the simultaneous detection of the two, but also has lower detection limit and quantification limit, and has higher sensitivity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the synthesis route of AuNPs@b-(s)-EPBA in the present invention.

[0039] Figure 2 It is the mass spectrum of the Raman signal molecule b-(s)-EPBA in the present invention.

[0040] Figure 3 It is the nuclear magnetic hydrogen spectrum of the Raman signal molecule b-(s)-EPBA in the present invention.

[0041] Figure 4 It is the nuclear magnetic carbon spectrum of the Raman signal molecule b-(s)-EPBA in the present invention.

[0042] Figure 5 This is the synthesis route and specific adsorption diagram of AuNPs@b-(s)-EPBA of the present invention.

[0043] Figure 6 This is the TEM image of AuNPs@b-(s)-EPBA of the present invention.

[0044] Figure 7 This is the high-angle annular dark field scanning and elemental analysis spectrum of AuNPs@b-(s)-EPBA of the present invention.

[0045] Figure 8 It is the ultraviolet absorption spectrum of AuNPs and AuNPs@b-(s)-EPBA in the present invention.

[0046] Figure 9 This is the Raman characterization spectrum of AuNPs and AuNPs@b-(s)-EPBA of the present invention.

[0047] Figure 10 This is a selective analysis diagram of AuNPs@b-(s)-EPBA of the present invention for HgCl2.

[0048] Figure 10 Middle: The unfilled histogram represents the SERS intensity ratio of HgCl2 and interfering substances after incubation with the probe (the vertical axis is I 1080 / I 1303 The histogram filled with oblique lines represents the histogram of the Raman intensity ratio after the interfering substances containing HgCl2 are incubated with the probe (the ordinate is I 1080 / I 1303 ).

[0049] Figure 11 The ratiometric SERS nanoprobe of the present invention is CH3Hg + Selectivity analysis diagram.

[0050] Figure 11 Middle: Unfilled histogram represents CH3Hg + , and the SERS intensity ratio after the interfering substances were incubated with the probe (the vertical axis is I1080 / I 1303 ); The histogram filled with slash lines indicates the presence of CH3Hg + The histogram of the Raman intensity ratio of the interfering substances after incubation with the probe (the vertical axis is I 1080 / I 1303 ).

[0051] Figure 12 The AuNPs@b-(s)-EPBA and HgCl2, CH3Hg + Raman spectra after incubation.

[0052] Figure 13 This is the high-angle annular dark field scanning TEM and elemental analysis spectrum of AuNPs@b-(s)-EPBA after adding HgCl2.

[0053] Figure 14 The AuNPs@b-(s)-EPBA of the present invention is added with CH3Hg + The subsequent high-angle annular dark field scanning TEM and elemental analysis spectra.

[0054] Figure 15 The standard curve of HgCl2 in the present invention (with the concentration of HgCl2 as the horizontal axis; with the relative peak intensity as the vertical axis, with I 1002 / I 1303 or I 1080 / I 1303 is the vertical axis). DETAILED DESCRIPTION

[0055] The following is a detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. In addition, it should be pointed out that, unless otherwise specified, the reagents used in the present invention are all existing commercial reagents, and the detection equipment used are all conventional laboratory equipment. In the following embodiments, CH3Hg + The stock solution is a standard solution with a concentration of 46 μM.

[0056] Example 1 Preparation and Characterization of the Mercury-Activated SERS Nanoprobe of the Present Invention

[0057] 1. The mercury-activated SERS nanoprobe of the present invention is a Raman signal molecule modified on the surface of gold nanospheres, wherein the Raman signal molecule is b-(s)-EPBA, and specifically comprises the following preparation steps:

[0058] The first step is to synthesize the Raman signal molecule b-(s)-EPBA (synthesis process see Figure 1 )

[0059] S11: 500 mg of 4-carboxyphenylboronic acid and 30 mL of thionyl chloride were heated under reflux for 24 h to perform an acyl chloride reaction to activate the carboxyl group in the 4-carboxyphenylboronic acid to form an acyl chloride. The heating temperature was 88°C. After the reaction, the thionyl chloride was removed from the reaction system to obtain chlorinated 4-carboxyphenylboronic acid.

[0060] S12, dissolving the acyl chloride product in a mixed solution of sodium hydroxide solution and tetrahydrofuran, mixing well, and slowly adding the mixture dropwise to an aqueous solution of cystamine dihydrochloride (450 mg) in an ice bath, during which a large amount of white mist is generated. After the addition is completed, the mixture is kept in an ice bath for 1 hour, then heated to 25°C and held for 12 hours. N2 protection is required throughout the reaction process.

[0061] After the reaction is complete, tetrahydrofuran is removed and the pH is adjusted to 2-3 with dilute hydrochloric acid. A white precipitate is obtained after filtration. The white precipitate is dried overnight in a vacuum (generally at 60°C) to obtain a white powdery Raman signal molecule b-(s)-EPBA. To identify the structural formula of b-(s)-EPBA, mass spectrometry, H-NMR, and C-NMR analyses were performed. The results are shown in the table below. Figure 2-4 , the specific data are as follows:

[0062] 1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (t, J = 5.6 Hz, 0H), 8.15 (s, 1H), 7.82– 7.69 (m, 2H), 3.54 – 3.42 (m, 1H), 2.91 –2.83 (m, 1H); 1 H NMR (400 MHz, DMSO) δ 8.70, 8.69, 8.67, 8.22, 7.86, 7.86,7.85, 7.84, 7.80, 7.79, 7.78,3.59, 3.57, 3.57, 3.56, 3.55, 3.54, 2.95, 2.94,2.93, 2.92;

[0063] 13 C NMR (101 MHz, CDCl3) δ 171.65, 171.65, 142.65, 142.65, 140.72,140.72, 139.17, 139.17, 131.29, 131.29, 43.96, 43.96, 42.25,42.25;

[0064] The mass spectrometry results showed a bright main peak at 471.1, which was consistent with the sodium addition peak [M + Na] of the Raman signal molecule b-(s)-EPBA. + The molecular weight is consistent.

[0065] The characterization results of mass spectrometry, H-NMR spectrum and C-NMR spectrum show that the Raman signal molecule b-(s)-EPBA synthesized in the present invention has the structural formula:

[0066] ;

[0067] In the second step, gold nanospheres were prepared using the seed-mediated growth method.

[0068] S21: Add 50 mL of ultrapure water (preferably Milli-Q water) to 2.5 mL of 5 mM HAuCl4·3H2O solution, heat to boiling, and quickly add 2 mL of sodium citrate solution (1% w / v, containing 0.05% citric acid) with vigorous stirring. Keep boiling for 10 minutes to obtain an orange-red gold seed solution with a gold seed particle size of approximately 13 nm.

[0069] S22, dilute 2 mL of HAuCl4·3H2O (5 mM) solution to 10 mL to obtain growth solution A1, and mix 0.5 mL of L-AA (i.e., ascorbic acid, 1% w / v) and 0.25 mL of sodium citrate solution (1% w / v, containing 0.05% citric acid) and dilute to 10 mL to obtain reduction solution B1;

[0070] Growth solution A1 and reduction solution B1 were slowly and simultaneously added dropwise to the gold seed solution (volume 3 mL) obtained in S21. The addition time was controlled within 60 min. The mixed solution was then heated to boiling and maintained for 30 min to obtain a wine-red solution of 30 nm gold nanocores.

[0071] S23, dilute 1.18 mL of HAuCl4·3H2O (5 mM) solution to 10 mL to obtain growth solution A2, and mix 0.295 mL of L-AA (1% w / v) and 0.148 mL of sodium citrate solution (1% w / v, containing 0.05% citric acid) and dilute to 10 mL to obtain reduction solution B2;

[0072] The wine-red solution obtained in S22 (volume 8 mL) was mixed with 8 mL of 50 mM hexadecyltrimethylammonium chloride solution. Growth solution A2 and reduction solution B2 were then slowly added dropwise to the mixture, with the addition time controlled within 60 min. After the addition, the mixture was heated to boiling and maintained for 30 min, and then cooled to room temperature to obtain a pink AuNPs solution with a particle size of approximately 60 nm.

[0073] The third step is to modify a layer of Raman signal molecule b-(s)-EPBA on the surface of the gold nanospheres.

[0074] S31, centrifuging the AuNPs solution obtained in step S23 at 8000 rpm for 6 min, and washing with deionized water 2 to 4 times to obtain gold nanospheres;

[0075] S32, freshly prepared b-(s)-EPBA (final concentration 50 μM) was added to the washed gold nanospheres and stirred at room temperature for 3 h. After the reaction, the mixture was centrifuged at 8000 rpm for 6 min to obtain the precipitate.

[0076] S33, the precipitate was dispersed in HEPES buffer (10 mM, pH = 7.4) to obtain mercury-activated SERS nanoprobes (hereafter referred to as AuNPs@b-(s)-EPBA) at a concentration of 0.05 nM.

[0077] Among them, the bonding path between b-(s)-EPBA and gold nanospheres, and the bonding path between AuNPs@b-(s)-EPBA and Hg 2+ and CH3Hg + The identification path is shown in Figure 5 .

[0078] In the present invention, the mixed solution of sodium hydroxide solution and tetrahydrofuran is prepared by mixing sodium hydroxide solution and tetrahydrofuran in a volume ratio of 1:2 (in this embodiment, the volumes of sodium hydroxide solution and tetrahydrofuran are 10 mL and 20 mL, respectively), and the concentration of the sodium hydroxide solution is 2 M.

[0079] 2. Characterization of the prepared AuNPs@b-(s)-EPBA and gold nanospheres

[0080] 1. AuNPs@b-(s)-EPBA was characterized by transmission electron microscopy (TEM). Figure 6 The characterization results show that the AuNPs@b-(s)-EPBA of the present invention is a nano-spherical structure with a particle size of about 60 nm.

[0081] 2. AuNPs@b-(s)-EPBA were characterized by high-angle annular dark-field scanning TEM and energy dispersive X-ray spectrometry, respectively. Figure 7 .

[0082] Depend on Figure 7 It can be seen that the elemental maps of Au and S match well with the bright areas in the HAADF-STEM image, indicating the presence of Au and S elements in AuNPs@b-(s)-EPBA.

[0083] 3. The AuNPs solution and AuNPs@b-(s)-EPBA were characterized by UV absorption spectrometer. The results are as follows: Figure 8 As shown. Combined Figure 8 It can be seen that the ultraviolet absorption peak of the AuNPs solution is located at 532 nm; the absorption peak of the AuNPs@b-(s)-EPBA probe shifts to 533 nm.

[0084] 4. Raman characterization was performed on the AuNPs solution and AuNPs@b-(s)-EPBA of the present invention. The Raman spectra are shown in Figure 9 .Depend on Figure 9 It can be seen that at 1002 cm -1 There is an in-plane deformation of BOH with boric acid molecules at 1601 cm -1 There is a strong SERS peak at the Raman shift, which is the stretching vibration of the C=C double bond in the aromatic ring, indicating that the b-(s)-EPBA molecules are successfully modified on the surface of the AuNPs nanosubstrate.

[0085] Example 2 The effect of AuNPs@b-(s)-EPBA on Hg2 + and CH3Hg + Identification analysis

[0086] Prepare HgCl2 mother liquor and CH3Hg with deionized water + The concentration of HgCl2 mother solution is 100 μM, CH3Hg + The concentration of the stock solution was 46 μM; the HgCl2 stock solution was diluted to 20 μM and CH3Hg + The stock solution was diluted to 20 μM; 180 μL of AuNPs@b-(s)-EPBA was added to 20 μL of HgCl2 solution and 20 μL of CH3HgCl2 solution respectively; the final concentration of AuNPs@b-(s)-EPBA was 0.05 nM, the final concentration of HgCl2 was 10 μM, and the + The final concentration was 7 μM;

[0087] Each mixed probe was incubated at 37°C for 10 min. After incubation, the probe was dropped onto tin foil for Raman characterization. The Raman spectroscopy parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 .

[0088] AuNPs@b-(s)-EPBA were respectively + The Raman spectrum after mixed incubation is shown in Figure 12 .Depend on Figure 12 It can be seen that AuNPs@b-(s)-EPBA and HgCl2, CH3Hg + After mixed incubation, the -1 A new characteristic peak appeared at 1002 cm -1 The Raman characteristic peak at 1303 cm-1 decreased, indicating that the probe has good recognition performance for divalent mercury ions and methylmercury. In addition, the AuNPs@b-(s)-EPBA probe and the mixed probe after incubation showed a strong Raman peak at 1303 cm-1. -1 The peak intensity at 1 remains basically unchanged, so the present invention uses I 1002 / I 1303 or I 1080 / I 1303 The relative peak intensity of was used as a reference for the subsequent selective analysis and quantitative detection analysis.

[0089] The mixed probe solutions after incubation were characterized by high-angle annular dark field scanning TEM and energy dispersive X-ray spectroscopy. Figure 13 and Figure 14 .Depend on Figure 13-14 It can be seen that there is Hg element around AuNPs@b-(s)-EPBA-HgCl2 and AuNPs@b-(s)-EPBA-CH3Hg, indicating that the probe and HgCl2, CH3Hg + A reaction occurred, further proving the reaction mechanism of the present invention.

[0090] Example 3 Selectivity of AuNPs@b-(s)-EPBA of the present invention

[0091] The first step is to prepare HgCl2 mother liquor and CH3Hg with deionized water. + The concentration of HgCl2 mother solution is 100 μM, CH3Hg + The concentration of the mother solution was 46 μM; heavy metal interfering ions Fe 2+ 、Al 3+ 、Cd 2+ 、Ag + Cr 3+ and Mg 2+ ; Use deionized water to prepare interferences related to biological analysis, including GSH, L-Cys, reactive oxygen (H2O2), L-AA, CO3 2- 、NO2 - 、H2PO4 - , where all three anions are sodium salts;

[0092] The above solutions were prepared and used immediately. The heavy metal interfering ions were prepared using FeCl2, AlCl3, CdCl2, AgNO3, CrCl3 and MgCl2, respectively. These metal salts were all analytically pure.

[0093] In the second step, the HgCl2 stock solution was diluted to 20 μM and CH3Hg + The stock solution was diluted to 20 μM; 20 μL HgCl2, 20 μL CH3Hg + and 20 μL of each interfering substance, respectively, to HgCl2, CH3Hg + 180 μL of AuNPs@b-(s)-EPBA was added to each interfering substance. The final concentration of the probe in each mixed probe solution was 0.05 nM, HgCl2, CH3Hg + The final concentration of the interfering substances was 20 μM, and the final concentration of the interfering substances was 50 μM;

[0094] The mixed probe solution was incubated at 37°C for 30 min. After the incubation, each mixed probe solution was dropped onto aluminum foil for SERS characterization (Raman parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 ), the results are shown in Figure 10 The histogram without padding and Figure 11 The unfilled histogram in ;

[0095] In the third step, HgCl2 was added to each interfering substance to obtain a mixture containing HgCl2 and the interfering substance; 20 μL of each mixed solution was taken and 180 μL of AuNPs@b-(s)-EPBA was added.

[0096] The final concentration of HgCl2 in each mixed probe solution was 20 μM, the final concentration of AuNPs@b-(s)-EPBA probe was 0.05 nM, and the final concentration of other interfering substances was 50 μM.

[0097] Each mixed probe solution was incubated at 37°C for 30 min. After the incubation, the mixed probe solution was dropped onto aluminum foil for SERS characterization (Raman parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 ), the results are shown in Figure 10 A histogram filled with a medium slash;

[0098] Similarly, CH3Hg + Added to each interfering substance, respectively, to obtain CH3Hg+ and interfering substances; 20 μL of each mixture was taken and 180 μL of AuNPs@b-(s)-EPBA was added respectively;

[0099] Where: CH3Hg in each mixed probe solution + The final concentration of AuNPs@b-(s)-EPBA was 20 μM, the final concentration of each probe was 0.05 nM, and the final concentration of other interfering substances was 50 μM;

[0100] The mixed probe solution was incubated at 37°C for 30 min. After the incubation, the mixed probe was dropped onto aluminum foil for SERS characterization (Raman parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 ), the results are shown in Figure 11 A histogram with a medium diagonal fill.

[0101] Depend on Figure 10 and Figure 11 It can be seen that HgCl2, CH3Hg + After incubation with AuNPs@b-(s)-EPBA, the Raman peak intensity ratio (I 1080 / I 1303 ) was significantly enhanced compared to AuNPs@b-(s)-EPBA, while heavy metal interfering substances (Fe 2+ 、Al 3+ 、Cd 2+ 、Ag + Cr 3+ Mg 2+ ), GSH, L-Cys, H2O2, L-AA, CO3 2- 、NO2 - 、H2PO4 - The Raman peak intensity ratios of AuNPs@b-(s)-EPBA after incubation (I 1080 / I 1303 ) has no obvious change, indicating that only HgCl2 and CH3Hg + At 1080 cm -1 The strong SERS signal response is caused at 1080 cm -1 No obvious response was triggered at the place, which further showed that the AuNPs@b-(s)-EPBA of the present invention had no significant effect on HgCl2, CH3Hg + Shows high selectivity.

[0102] When HgCl2 and CH3Hg +After mixing with the interference, they were incubated with the AuNPs@b-(s)-EPBA of the present invention. + Each interfering substance after I 1080 / I 1303 The ratio peak intensity increased significantly, indicating that other interferences did not affect the detection of mercury and methylmercury.

[0103] In summary, the AuNPs@b-(s)-EPBA of the present invention is basically unresponsive to heavy metal interferents and other biological interferents, indicating that the nanoprobe of the present invention has good selectivity for mercury and methylmercury.

[0104] Example 4 Raman standard curve and sensitivity of AuNPs@b-(s)-EPBA to mercury and methylmercury

[0105] The first step is to prepare HgCl2 mother liquor and CH3Hg with deionized water. + The concentration of HgCl2 mother solution is 100 μM; CH3Hg + The concentration of the stock solution was 46 μM; HgCl2 and CH3Hg + The mother liquor was diluted to different concentrations;

[0106] In the second step, 20 μL of HgCl2 was taken and 180 μL of AuNPs@b-(s)-EPBA was added to the HgCl2 to obtain mixed probe solutions. The final concentration of the probe in each mixed probe solution was 0.05 nM, and the final concentrations of HgCl2 were 0.01 μM, 3 μM, 6 μM, 10 μM, and 15 μM, respectively.

[0107] Similarly, take 20 μL CH3Hg + , per CH3Hg + 180 μL of AuNPs@b-(s)-EPBA was added to obtain multiple mixed probe solutions, where the final concentration of the probe in the mixed probe solution was 0.05 nM, CH3Hg + The final concentrations were: 0.05 μM, 2 μM, 5 μM, 7 μM, and 10 μM;

[0108] Each mixed probe solution was incubated at 37°C for 10 min. After incubation, the probe was dropped onto tin foil for Raman characterization. The Raman spectroscopy parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 ;

[0109] To ensure the reliability of the test results, HgCl2 and CH3Hg + Each concentration of the solution was tested in parallel 3 times, and the standard curve was drawn with the average value and relative deviation value at each concentration and the concentration value:

[0110] Since the probe is at 1080 cm -1 The characteristic peak signal at 1002 cm -1 The Raman characteristic peak at 1303 cm -1 The peak at the position is not affected by the external environment, and the intensity remains basically unchanged. 1002 / I 1303 or I 1080 / I 1303 The relative peak intensity is used as the vertical coordinate to reduce the error as much as possible and improve the reliability of the detection results.

[0111] The HgCl2 concentration is the horizontal axis, and I 1002 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=-0.08X+1.43,R 2 =0.994 ; when using I 1080 / I 1303 When is the vertical coordinate, the fitted equation is Y=0.11X+ 0.17，R 2 =0.993 , the results are shown in Figure 15 and Table 1.

[0112] CH3Hg + The concentration value is the horizontal axis, when I 1002 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=-0.12X+1.3,R 2 =0.999 ; when using I 1080 / I 1303 Draw the standard curve for the vertical axis, and the fitting equation is Y=0.11X+0.15,R 2 =0.997， The results are shown in Table 1.

[0113] Table 1 HgCl2 and CH3Hg of the present invention + Standard curve

[0114]

[0115] As can be seen from Table 1, whether it is I 1080 / I 1303 As the vertical axis, or I 1002 / I 1303 As the vertical axis, HgCl2 and CH3Hg + The correlation coefficient of the fitting equation R 2 All of them are greater than 0.99, indicating a good linear relationship. + The minimum detection limits of the AuNPs@b-(s)-EPBA nanoprobes were 10 nM and 25 nM, respectively, and the quantitative limits were 10 nM and 50 nM, respectively, indicating that the AuNPs@b-(s)-EPBA nanoprobes of the present invention are effective in the quantitative detection of Hg 2+ and CH3Hg + It has high sensitivity and accuracy, which further proves that Raman detection of Hg 2+ and CH3Hg + reliability.

[0116] Example 5 Detection of AuNPs@b-(s)-EPBA of the present invention in actual environmental samples

[0117] The first step is sample pretreatment

[0118] Four samples (tap water, drinking water, Meihu Lake water, and normal human urine) were placed in centrifuge tubes, allowed to stand for 24 hours, and then centrifuged at 8000 rpm for 5 minutes to obtain the supernatant. The supernatant was further filtered through a 0.25 μm filter to remove impurities, achieving sample pretreatment.

[0119] Step 2: Spiking

[0120] HgCl2 solution was added to each pretreated sample to obtain mercury-containing test mixtures with concentrations of 3 μM and 10 μM, respectively. CH3Hg was added to each pretreated sample to obtain mercury-containing test mixtures with concentrations of 3 μM and 10 μM, respectively. + solution, and obtained the test mixed solution containing methylmercury with the concentration of 2 μM and 10 μM respectively;

[0121] Step 3: Probe incubation

[0122] 20 μL of the spiked test solution and the unspiked pretreated sample were taken, and 180 μL of AuNPs@b-(s)-EPBA was added to the spiked test solution and the pretreated sample, respectively, to obtain a mixed probe. The final concentration of the probe in each mixed probe solution was 0.05 nM. The mixed probe solution was incubated at 37°C for 5 min before Raman detection. Raman signals were collected at room temperature (Raman test parameters: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm). -1 );

[0123] Raman test results showed that no Hg was detected in the four water samples without spike addition. 2+ and CH3Hg + , drinking water, tap water and lake water samples are all safe. In addition, the Hg 2+ The spiked test data of CH3Hg are shown in Table 2. + The spiked test data are shown in Table 3.

[0124] Table 2 Spiked recovery of four actual samples (spiked solution is HgCl2)

[0125]

[0126] As shown in Table 2, the spiked recoveries after adding HgCl₂ solution ranged from 84.5% to 118%, with RSDs between 0.11 and 0.18, both within the acceptable range. These results demonstrate that the AuNPs@b-(s)-EPBA of the present invention can be used for the specific detection of mercury in water samples and exhibits good stability.

[0127] Table 3 Spiked recovery of four actual samples (spiked solution is CH3Hg + )

[0128]

[0129] As can be seen from Table 3, adding CH3Hg + The spiked recovery after solution was in the range of 81.6-130%, and the RSD was in the range of 0.04-0.17. The results show that the AuNPs@b-(s)-EPBA of the present invention can be used for the specific detection of methylmercury in water samples and has good stability.

[0130] In summary, the nanoprobe AuNPs@b-(s)-EPBA of the present invention can not only be used for Hg 2+ It can also be used for quantitative detection of CH3Hg in water samples + It can be used for quantitative detection, and has good selectivity, effectively avoiding interference from other heavy metal ions, bioactive molecules and various anions, and has high detection sensitivity and stability, which is a good choice for the detection of Hg in water samples. 2+ and CH3Hg + It provides a new method for quantitative detection.

Claims

1. A mercury-activated SERS nanoprobe, characterized by: The nanoprobe is a Raman signal molecule modified on the surface of a gold nanosphere. The Raman signal molecule is b-(s)-EPBA, and its structural formula is as follows: 。 2. The mercury-activated SERS nanoprobe according to claim 1, characterized in that: The diameter of the gold nanospheres is 50-70 nm, and the b-(s)-EPBA on the surface of the gold nanospheres is a single layer.

3. A method for preparing the mercury-activated SERS nanoprobe according to claim 1, characterized in that: The preparation method comprises the following steps: In the first step, the carboxyl group in 4-carboxyphenylboronic acid is activated to form an acyl chloride to obtain an acyl chloride product; the amino group in cystamine dihydrochloride is subjected to an amidation reaction with the acyl chloride product to obtain a Raman probe molecule b-(s)-EPBA; In the second step, the gold nanospheres with surface plasmon resonance effect were prepared by seed-mediated growth method; In the third step, a layer of b-(s)-EPBA synthesized in the first step is modified on the surface of the gold nanospheres. After the reaction is completed, the precipitate is dispersed in a buffer solution to obtain a mercury-activated SERS nanoprobe.

4. The method for preparing a mercury-activated SERS nanoprobe according to claim 3, wherein: The first step specifically includes the following contents: S11, 4-carboxyphenylboronic acid and thionyl chloride are subjected to an acyl chloride reaction under heating and reflux conditions to activate the carboxyl group in the 4-carboxyphenylboronic acid to form an acyl chloride, the reaction temperature is 80°C-95°C, and the reflux time is 12-36 h; S12, dissolving the acyl chloride product in a mixed solution of sodium hydroxide and tetrahydrofuran, mixing well, and slowly adding dropwise to an aqueous solution of cystamine dihydrochloride to carry out an amide reaction under nitrogen protection; after the reaction, removing tetrahydrofuran, adjusting the pH to 2-3 with concentrated hydrochloric acid, filtering, and drying to obtain b-(s)-EPBA as a white powder.

5. The method for preparing a mercury-activated SERS nanoprobe according to claim 4, wherein: In the S12, the amide reaction is first maintained in an ice bath for 30 min-60 min, then heated to 20°C-30°C and maintained for 10-24 h; the drying condition in S12 is drying at 50°C-80°C under vacuum overnight.

6. The method for preparing a mercury-activated SERS nanoprobe according to claim 4, wherein: The buffer in the third step is HEPES buffer with a concentration of 10 mM and a pH of 7.

4.

7. Use of the mercury-activated SERS nanoprobe according to any one of claims 1 to 2, or the mercury-activated SERS nanoprobe prepared by the preparation method according to any one of claims 3 to 6, in detecting organic mercury and / or inorganic mercury.

8. The use according to claim 7, characterized in that: The organic mercury is methylmercury, ethylmercury and phenylmercury, and the inorganic mercury is a divalent mercury salt soluble in water.

9. The use according to claim 7, characterized in that: When detecting organic mercury or inorganic mercury, surface-enhanced Raman scattering technology is used for detection, which specifically includes the following steps: S1, centrifuge the sample at a high speed of 5000 rpm or higher and filter it through a 0.25 μm filter; S2: Add mercury-activated SERS nanoprobes to the treated sample, incubate at 37°C for 5-10 min, and then perform Raman detection. The Raman test parameters are: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 600-1800 cm -1 .

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