An internal standard-assisted petal-shaped SERS nanoprobe, its preparation method, and its application.

Abstract

This invention discloses an internal standard-assisted petal-shaped SERS nanoprobe, its preparation method, and its applications. The internal standard-assisted petal-shaped SERS nanoprobe of this invention is a core-molecule-shell structure gold nanoprobe consisting of a gold nanosphere core, an internal standard modifying molecule, a petal-shaped gold nanoshell, and a recognition molecule, 4-nitrobenzenethiophenol. The internal standard modifying molecule is a Raman signal molecule with nitrile, alkyne, or azide functional groups. This invention uses a Raman signal molecule as an internal standard modifying molecule to modify the gold nanosphere core. The corrected working curve exhibits good reproducibility and small error, improving the reliability of the detection results. Growing a petal-shaped gold nanoshell on the molecular layer of the internal standard modifying molecule enhances the SERS activity both inside and outside the SERS substrate, and the petal-shaped shell increases the surface area of ​​the SERS substrate. Furthermore, the recognition molecule of this invention enables ratiometric Raman detection of hydrogen sulfide, effectively reducing background signal interference and improving detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of H2S detection, and in particular to an internal standard-assisted petal-shaped SERS nanoprobe, a method for preparing the internal standard-assisted petal-shaped SERS nanoprobe, and the application of the internal standard-assisted petal-shaped SERS nanoprobe. Background Technology

[0002] Hydrogen sulfide (H2S) is the third bioactive gaseous signaling molecule identified in living organisms, following NO and CO. H2S is widely distributed throughout the body. In mammals, endogenous H2S is typically generated from L-cysteine ​​via enzymatic reactions catalyzed by 3-mercaptopyruvate thiotransferase (3-MST), cystathionine-β-synthase (CBS), and cystathionine-γ-lyase (CSE). Studies have shown that H2S participates in a series of physiological processes in the body, including regulating vascular tone, myocardial contraction, nerve conduction, and insulin secretion. Furthermore, abnormal H2S concentrations are closely associated with a range of malignant diseases, such as Alzheimer's disease, Down syndrome, diabetes, and acute kidney injury. Therefore, quantitative detection of H2S is of significant clinical importance.

[0003] In recent years, gas chromatography, electrochemical methods, chemiluminescence, and fluorescence methods have been developed for the detection of H2S. However, most of these methods require complex sample pretreatment, which can even damage the sample and severely affect the detection results.

[0004] SERS, characterized by its real-time, rapid, non-invasive, highly sensitive, non-autofluorescent, and unaffected by photobleaching, has become one of the most effective tools for detecting biomolecules and for bioimaging. For H2S detection, developing SERS nanoprobes with high sensitivity, specificity, stability, and strong anti-interference capabilities is a key technology for applying SERS technology to the real-time, highly sensitive, and specific detection of H2S.

[0005] Currently, based on some special chemical reactions caused by H2S (such as the reduction of azide groups, the reaction between Ag and H2S, etc.), some SERS nanoprobes have been developed for the detection of H2S in living cells.

[0006] Li et al. reported the first SERS nanoprobe for detecting H2S—4-acetamidobenzenesulfonyl azide-functionalized gold nanoparticles (denoted as AuNPs / 4-AA). This probe is based on the reduction of the azide group to an amino group by H2S, rapidly responding to H2S through SERS spectral changes, and monitoring endogenously generated H2S in in vivo glioma cells with high sensitivity. However, although azides can react rapidly, azide derivatives are often unstable and prone to generating unwanted byproducts under laser irradiation, resulting in false positive signals and affecting the reliability of the detection results, thus limiting the further application of this probe to some extent.

[0007] Wang et al. designed a near-infrared activated SERS nanoprobe that monitors the distribution of H2S in cells and zebrafish embryos based on changes in the SERS spectrum caused by the reaction of H2S with Ag. However, although the nanoprobe exhibits high sensitivity to H2S, the Ag shell is easily destroyed by reactive oxygen species in the cells, resulting in poor selectivity and affecting the reliability of the detection results.

[0008] In addition, the applicant also discovered during the research process that most existing SERS nanoprobes use a single absolute signal intensity for quantitative detection of H2S. Due to the presence of many interfering factors in the environment that cause signal intensity fluctuations, the reliability of this detection method is low and the error is large.

[0009] In summary, designing a SERS nanoprobe that exhibits good selectivity for H2S, high detection sensitivity, and minimizes interference from external factors to improve the reliability of detection results is crucial for the study of H2S in vivo. Summary of the Invention

[0010] In view of this, the primary objective of the present invention is to provide an internal standard-assisted petal-shaped SERS nanoprobe that improves the detection sensitivity of H2S and has good laser stability and selectivity.

[0011] The second objective of this invention is to provide a method for preparing an internal standard-assisted petal-shaped SERS nanoprobe, which improves the SERS activity inside and outside the SERS substrate, provides more binding sites for recognition molecules, and further improves detection sensitivity.

[0012] The third objective of this invention is to provide the application of internal standard-assisted petal-shaped SERS nanoprobes in detecting H2S in samples, and in the imaging of H2S in live cells for non-disease diagnosis purposes.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] The internal standard-assisted petal-shaped SERS nanoprobe of this invention is a gold nanoflower probe with a core-molecule-shell structure, consisting of a gold nanosphere core, an internal standard modifying molecule, a petal-shaped gold nanoshell, and a recognition molecule; wherein, the internal standard modifying molecule is a Raman signal molecule with nitrile, alkyne, or azide functional groups; and the recognition molecule is 4-nitrobenzenethiophenol.

[0015] In this invention, Raman signal molecules are used as internal standard modifying molecules to modify the core of gold nanospheres. The corrected working curve has good reproducibility and small error, thus improving the reliability of the detection results.

[0016] A petal-shaped gold nanoshell is grown on the molecular layer of the internal standard modified molecule. On the one hand, it has a large number of "hot spots", which improves the SERS activity inside and outside the SERS substrate. On the other hand, the petal-shaped shell also increases the surface area of ​​the SERS substrate, providing more binding sites for recognition molecules.

[0017] This invention uses 4-nitrobenzenethiophenol (i.e., 4-NTP) as a sensitive recognition molecule for H2S. The nitro group in 4-NTP, as a recognition group, improves the detection sensitivity, selectivity and detection speed of the probe, enabling rapid quantitative detection of H2S in environmental or biological samples.

[0018] More preferably, the internal standard modification molecule is 4-mercaptobenzonitrile. Using 4-mercaptobenzonitrile as an internal standard, ratiometric Raman detection of hydrogen sulfide is achieved through the reduction reaction of hydrogen sulfide and 4-nitrobenzenethiophenol, effectively reducing background signal interference and improving detection sensitivity.

[0019] More preferably, the particle size of the gold nanosphere core is 20–40 nm; and the thickness of the petal-shaped gold nanoshell is 30–50 nm.

[0020] This invention also provides a method for preparing an internal standard-assisted petal-shaped SERS nanoprobe, the method comprising the following steps:

[0021] The first step is to prepare AuNPs cores of gold nanospheres with surface plasmon resonance effect;

[0022] The second step involves modifying the AuNPs core of gold nanospheres with an internal standard modifying molecule, which is a small molecule that has a Raman signal and possesses nitrile, alkyne or azide functional groups.

[0023] The third step is to grow a petal-shaped gold nanoshell outside the molecular layer of the internal standard modified molecule to obtain gold nanoflowers doped with the internal standard modified molecule as the SERS substrate.

[0024] The fourth step involves modifying the surface of the SERS substrate with a layer of 4-nitrobenzylthiophenol to obtain a core-molecule-shell structured internal standard-assisted petal-shaped SERS nanoprobe, in which 4-nitrobenzylthiophenol is used to recognize H2S.

[0025] In the above preparation method, the internal standard modification molecule in the second step is 4-mercaptobenzonitrile. During modification, the pH of the AuNPs solution is adjusted to 9.0, an ethanol solution of 4-mercaptobenzonitrile is added, the mixture is stirred at a constant temperature for 3 h, unbonded 4-mercaptobenzonitrile is removed by centrifugation, and the precipitate is dispersed with deionized water to obtain 4-mercaptobenzonitrile-modified AuNPs.

[0026] The third step in the above preparation method specifically includes the following: mixing the 4-mercaptobenzonitrile-modified AuNPs obtained in the second step with a hexadecyltrimethylammonium chloride solution, and then adding HAuCl4·3H2O and ascorbic acid in sequence. After the reaction is completed, a deep blue SERS substrate—Au@MPBN@Au NFs solution—is obtained.

[0027] The fourth step in the above preparation method includes the following specific contents: centrifuge the Au@MPBN@Au NFs solution, wash the precipitate with deionized water; after washing, add p-nitrobenzenethiophenol, stir the reaction at room temperature, centrifuge after the reaction is completed, and disperse the precipitate in HEPES buffer to obtain the 4-NTP modified internal standard-assisted petal-shaped SERS nanoprobe - Au@MPBN@Au@4-NTP.

[0028] The present invention also relates to the application of internal standard-assisted petal-shaped SERS probes in the detection of H2S in test samples, which can be used for the semi-quantitative detection of trace amounts of H2S in biological samples (especially cells).

[0029] This invention also provides an application of an internal standard-assisted petal-shaped SERS nanoprobe in live-cell H2S imaging for non-disease diagnostic purposes. Results show that the SERS nanoprobe of this invention has good cellular uptake capacity and intracellular stability. After incubating cells with the SERS nanoprobe, SERS signals of H2S can be observed, making it suitable for detecting H2S or endogenous H2S in live cells.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] This invention first modifies gold nanospheres with a layer of 4-mercaptobenzonitrile and synthesizes core-molecule-shell gold nanoflowers Au@MPBN@Au NFs with high plasmon resonance activity as an internal standard embedded SERS substrate. 4-NTPs are covalently bonded to the surface of Au@MPBN@Au NFs as H2S recognition molecules. The ratiometric Raman detection of hydrogen sulfide is achieved by utilizing the reduction reaction of H2S and 4-nitrobenzylthiophenol, effectively reducing background signal interference and improving detection sensitivity and anti-interference ability. Using 4-nitrobenzylthiophenol as the recognition molecule improves the probe's selectivity for H2S.

[0032] The 4-mercaptobenzonitrile in the nanoprobe of this invention is at 2223 cm⁻¹ -1 The tensile vibration band of the nitrile remains constant, with the Raman characteristic peak of H2S (i.e., 1139 cm⁻¹). -1 () or H2S has a Raman characteristic peak with a large variation in lower peak intensity (i.e., 1329 cm⁻¹). -1 The characteristic peaks of 4-mercaptobenzonitrile (i.e., 2223 cm⁻¹) and 4-mercaptobenzonitrile are also present. -1 Based on the ratio of H2S to 4-nitrophenylthiophenol, this invention achieves accurate detection of H2S in samples. Results show that, compared with H2S quantitative detection based on absolute peak intensity technology, the standard curve corrected by the 4-nitrophenylthiophenol internal standard exhibits excellent reproducibility, small error, and high accuracy, thus improving the reliability of the detection results.

[0033] The internal standard-assisted petal-shaped SERS nanoprobe of this invention can be used to detect endogenous and exogenous H2S in cells, which is of great significance for in-depth research on the dynamic mechanisms of hydrogen sulfide production and transport in organisms, as well as for further understanding the physiological and toxicological effects of hydrogen sulfide. Attached Figure Description

[0034] Figure 1 This is a synthetic route diagram of the internal standard-assisted petal-shaped SERS nanoprobe described in this invention.

[0035] Figure 2 This is a TEM image of the internal standard-assisted petal-shaped SERS nanoprobe in Example 1.

[0036] Figure 3 These are the high-angle annular dark-field scanning TEM and elemental analysis spectra of the internal standard-assisted petal-shaped SERS nanoprobe in Example 1.

[0037] Figure 4 The images show the UV absorption spectra of the Au@MPBN@Au NFs solution and the Au@MPBN@Au@4-NTP in Example 1.

[0038] Figure 5The images show the Raman spectra of Au@MPBN@Au NFs and Au@MPBN@Au@4-NTP in Example 1.

[0039] Figure 6 This is a histogram of the selectivity analysis of the internal standard-assisted petal-shaped SERS nanoprobe of this invention.

[0040] In the figure: the gray-filled histogram represents the ratio of the SERS intensity of NaHS and interfering substances to that of the probe after incubation (the vertical axis is I). 1139 / I 2223 The histogram filled with diagonal lines represents a histogram of the ratio of the interfering substance containing NaHS to the Raman intensity after probe incubation (the vertical axis is I). 1139 / I 2223 ).

[0041] Figure 7 This is the Raman spectrum of the internal standard-assisted petal-shaped SERS nanoprobe after incubation with NaHS.

[0042] Figure 8 This is the standard curve of the internal standard-assisted petal-shaped SERS nanoprobe described in this invention (with relative peak intensity as the ordinate and I as the ordinate). 1139 / I 2223 Or I 1329 / I 2223 (The vertical axis is used as the coordinate).

[0043] Figure 9 This is the standard curve of the internal standard-assisted petal-shaped SERS nanoprobe of the present invention (with I). 1139 Or I 1329 The absolute peak intensity is represented by the ordinate.

[0044] Figure 10 This is an image of the internal standard-assisted petal-shaped SERS nanoprobe in H2S in HepG2 cells as described in this invention.

[0045] Figure 11 This is a schematic diagram of the sensing of H2S in HepG2 cells by the internal standard-assisted petal-shaped SERS nanoprobe described in this invention.

[0046] Figure 12 These are Raman images and Raman spectra of different concentrations of H2S in A549 cells obtained by the internal standard-assisted petal-shaped SERS nanoprobe described in this invention. Detailed Implementation

[0047] The present invention will be described in more detail below through specific embodiments to facilitate understanding by those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all conventional reagents, and the instruments and methods employed are also conventional instruments and methods in the art.

[0048] Example 1: The internal standard-assisted petal-shaped probe Au@MPBN@Au@4-NTP described in this invention

[0049] I. Preparation of internal standard-assisted petal-shaped SERS nanoprobes Au@MPBN@Au@4-NTP

[0050] In this embodiment, gold nanoparticles were used as the core and 4-mercaptobenzonitrile (MPBN) was used as the internal standard to synthesize MPBN-modified gold nanoflowers Au@MPBN@Au NFs. Finally, by modifying Au@MPBN@Au NFs with 4-nitrobenzenethiophenol (i.e., 4-NTP), the internal standard-assisted petal-shaped SERS probe of this invention—Au@MPBN@Au@4-NTP—was obtained. The synthetic route is shown below. Figure 1 .

[0051] Combination Figure 1 It is understood that the preparation method of the internal standard-assisted petal-shaped SERS nanoprobe of the present invention includes the following steps:

[0052] The first step was to prepare gold nanonuclei (AuNPs) with a particle size of 30 nm.

[0053] S11, Gold seeds were prepared using the classic sodium citrate reduction method: 50 mL H2O and 2.5 mL HAuCl4·3H2O (0.2% w / v) were heated to boiling, and then 2 mL of trisodium citrate aqueous solution (1% w / v, containing 0.02% citric acid (w / v)) was quickly added. The solution was then kept boiling for 10 min to obtain a gold seed solution with a particle size of approximately 13 nm.

[0054] S12, gold nanonuclei AuNPs were prepared by seed growth method: 2 ml HAuCl4·3H2O (0.2% w / v) was diluted to 10 mL to obtain precursor solution A;

[0055] Dilute the mixture of 0.5 mL ascorbic acid (1% w / v) and 0.25 mL trisodium citrate (1% w / v, containing 0.02% citric acid) to 10 mL to obtain reducing solution B;

[0056] Add 3 mL of gold seed solution and 20 mL of H2O to the flask. While stirring vigorously at room temperature, slowly add precursor solution A and reducing solution B to the flask. The addition time is 60 min.

[0057] After the sample was added, the above mixture was rapidly heated to boiling and held for 30 min to obtain a wine-red AuNPs solution with AuNPs particles of approximately 30 nm in size.

[0058] The second step is to synthesize 4-mercaptobenzonitrile-modified AuNPs (i.e., Au@MPBN NPs).

[0059] Take 18 mL of AuNPs solution and adjust its pH to 9 with NaOH; then add 90 μL of 20 mM ethanol solution of 4-mercaptobenzonitrile (MPBN), and stir at 30℃ for 3 h. 4-Mercaptobenzonitrile is modified on the surface of AuNPs through Au-S covalent bonds. After the reaction is completed, centrifuge to obtain the precipitate (i.e., Au@MPBN NPs), wash twice with deionized water, and redisperse the precipitate in deionized water to obtain Au@MPBN NPs solution for later use.

[0060] The third step is to synthesize MPBN-doped gold nanoflowers (Au@MPBN@Au NFs).

[0061] Add 4 mL of Au@MPBN NPs solution and 8 mL of 50 mM hexadecyltrimethylammonium chloride solution to a flask. Under sonication, add 900 μL of HAuCl4·3H2O (0.2% w / v). After mixing for 2 min, quickly add 334 μL of ascorbic acid (1% w / v). The color of the reaction system changes rapidly from rose red to light blue. Continue the reaction for 10 min to obtain a dark blue Au@MPBN@Au NFs solution. Store in the dark at 4°C.

[0062] Step 4: Synthesize the internal standard-assisted petal-shaped SERS probe—Au@MPBN@Au@4-NTP

[0063] Centrifuge 24 mL of Au@MPBN@Au NFs solution at 8000 rpm for 6 min. After centrifugation, wash with deionized water 2–4 times. After washing, add freshly prepared p-nitrobenzenethiophenol (i.e., 4-NTP, with a final concentration of 3 μM) and stir at room temperature for 3 h. After the reaction is complete, centrifuge at 8000 rpm for 6 min to obtain the precipitate.

[0064] The precipitate was washed with deionized water and then dispersed in HEPES buffer (20 mM, pH = 7.4) to obtain the internal standard-assisted petal-shaped SERS nanoprobe Au@MPBN@Au@4-NTP, with a concentration of 1.0 mg / ml.

[0065] II. Characterization of the internal standard-assisted petal-shaped SERS nanoprobe in this embodiment

[0066] 1. The size and morphology of Au@MPBN@Au@4-NTP were observed using transmission electron microscopy (TEM). The Au@MPBN@Au@4-NTP nanoprobes have a particle size of approximately 70 nm, and the gold nanospheres exhibit a petal-like shell structure on their outer periphery, as shown in the figure below. Figure 2 As shown.

[0067] 2. Au@MPBN@Au@4-NTP was characterized using high-angle annular dark-field scanning TEM and energy-dispersive X-ray spectroscopy, respectively. See details below. Figure 3 .

[0068] Depend on Figure 3 It can be seen that the elemental spectra of Au and S match well with the bright areas in the HAADF-STEM image, indicating the presence of Au and S elements in Au@MPBN@Au@4-NTP.

[0069] 3. The Au@MPBN@Au NFs solution and Au@MPBN@Au@4-NTP in this embodiment were characterized by ultraviolet absorption spectroscopy, and the results are as follows: Figure 4 As shown.

[0070] Both Au@MPBN@Au NFs and Au@MPBN@Au@4-NTP are dark blue, and the color remained unchanged before and after modification. Figure 4 It can be seen that the ultraviolet absorption peak of Au@MPBN@Au NFs is located at 620 nm; the absorption peak of Au@MPBN@Au@4-NTP probe shifts to 621 nm.

[0071] 4. The Au@MPBN@Au NFs and Au@MPBN@Au@4-NTP of the present invention were characterized by Raman spectroscopy (see Raman spectra). Figure 5 ).Depend on Figure 5 It can be seen that in the Raman silencing region of the cell at 2223 cm⁻¹ -1 The nitrile group vibration of the MPBN molecule exists at 1329 cm⁻¹. -1 A strong SERS peak was observed at the Raman shift, which is the stretching vibration peak of the nitro group in the 4-NTP molecule, indicating that the 4-NTP molecule was successfully modified on the surface of the Au@MPBN@Au NFs nanosubstrate.

[0072] Example 2: Selectivity of the nanoprobe Au@MPBN@Au@4-NTP of the present invention

[0073] S1. Prepare a 40 µM NaHS stock solution using deionized water; prepare solutions of interfering substances such as GSH, L-AA (i.e., L-ascorbic acid), Na2SO3, NaNO2, NaN3, NaCO3, NaClO, KSCN, NaSO4, KNO3, NaHSO3, NaH2PO4, and H2O2 using deionized water respectively. Among them, the concentration of GSH, L-AA, and H2O2 is 2 mM for the stock solution, and the concentration of other interfering substances is 400 µM.

[0074] S2, 50 µL of NaHS stock solution and interfering substances were incubated with 150 µL of Au@MPBN@Au@4-NTP for 30 min. The final concentration of Au@MPBN@Au@4-NTP was 0.1 mg / mL, the final concentration of NaHS stock solution was 10 µM, the final concentration of GSH, L-AA and H2O2 was 500 µM, and the final concentration of other interfering substances was 100 µM.

[0075] After incubation, each mixed probe was dropped onto aluminum foil for SERS characterization (Raman parameters as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, acquisition range 500-2500 cm⁻¹). -1 ), the results are shown Figure 6 The gray-filled histogram in the image;

[0076] Add 50 µL of NaHS stock solution to 50 µL of each interfering substance, and then incubate the NaHS-containing interfering substances with 100 µL of Au@MPBN@Au@4-NTP at a constant temperature for 30 min. The final concentration of NaHS stock solution in each mixed probe solution is 10 µM, the final concentration of the probe is 0.1 mg / mL, the final concentration of GSH, L-AA and H2O2 is 500 µM, and the final concentration of other interfering substances is 100 µM.

[0077] After 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 500-2500 cm⁻¹). -1 ), the Raman peak intensity ratio of each interfering substance (I 1139 / I 2223 )See Figure 6 The histogram filled with diagonal lines.

[0078] Depend on Figure 6 It can be seen that after incubating NaHS and Au@MPBN@Au@4-NTP, the Raman peak intensity ratio (I 1139 / I 2223The Raman peak intensity of Au@MPBN@Au@4-NTP was significantly enhanced, while the Raman peak intensity of interfering substances GSH, L-AA, Na2SO3, NaNO2, NaN3, NaCO3, NaClO, KSCN, NaSO4, KNO3, NaHSO3, NaH2PO4, and H2O2 after incubation with Au@MPBN@Au@4-NTP was significantly enhanced. 1139 / I 2223 There was no significant change, indicating that only hydrogen sulfide was at 1139 cm⁻¹. -1 A strong SERS signal response was induced at 1139 cm⁻¹, while other interfering substances were observed at 1139 cm⁻¹. -1 No obvious response was triggered at this location, indicating that the nanoprobe Au@MPBN@Au@4-NTP has high selectivity for hydrogen sulfide detection;

[0079] When NaHS was mixed with interfering substances and then co-incubated with Au@MPBN@Au@4-NTP of the present invention, it was found that I in each interfering substance after the addition of NaHS... 1139 / I 2223 The ratio peak intensity increased significantly, indicating that other biological interfering substances do not affect the detection of H2S.

[0080] In summary, the Au@MPBN@Au@4-NTP of the present invention is essentially unresponsive to interfering substances such as GSH, L-AA, Na2SO3, NaNO2, NaN3, NaCO3, NaClO, KSCN, NaSO4, KNO3, NaHSO3, NaH2PO4, and H2O2, indicating that the nanoprobe of the present invention has excellent selectivity for H2S.

[0081] Example 3: Accuracy Analysis of the Probe Described in this Invention

[0082] To better illustrate the accuracy of the probe modified with 4-mercaptobenzonitrile as an internal standard, the applicant used NaHS solution as an H2S donor to investigate the effect of 4-mercaptobenzonitrile on the detection accuracy of the probe. Specifically, this included the following:

[0083] The first step is to prepare NaHS stock solutions of different concentrations using deionized water. The concentrations of the NaHS stock solutions are 0, 1 μM, 2 μM, 4 μM, 8 μM, 20 μM, 40 μM and 80 μM respectively.

[0084] The second step involved mixing 150 μL of Au@MPBN@Au@4-NTP with 50 μL of NaHS stock solution of different concentrations, with the final concentration of the probe being 0.1 mg / mL and the final concentrations of the NaHS stock solution being 0, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 20 μM respectively.

[0085] The mixture was incubated at 37℃ for 30 min. After incubation, the probe was dropped onto tin foil for Raman characterization. The Raman spectral parameters were as follows: excitation wavelength 633 nm, magnification 50×, power 0.73 mW, exposure time 10 s, and acquisition range 500-2500 cm⁻¹. -1 ;

[0086] The Raman spectrum of Au@MPBN@Au@4-NTP after incubation with NaHS is shown below. Figure 7 .from Figure 7 It can be seen that: after the nanoprobe of the present invention is mixed with H2S, it reaches a density of 1139 cm⁻¹. -1 1387 cm -1 and 1433 cm -1 Three new characteristic sutures appeared at 1329 cm. -1 The decrease in the Raman characteristic peak at the point indicates that the probe has good recognition performance for hydrogen sulfide.

[0087] To ensure the reliability of the test results, each concentration of NaHS solution was tested in triplicate, and a standard curve was plotted using the average value and relative deviation at each concentration, as well as the logarithm of the concentration.

[0088] Because the probe is at 1139 cm -1 The characteristic peak signal at 1329 cm⁻¹ increases significantly with the addition of H₂S. -1 The Raman characteristic peak at 2223 cm⁻¹ shows a significant decrease. -1 The internal standard peak at a certain location remains essentially unchanged in intensity because it is unaffected by external environmental interference. Therefore, this invention uses I as the detection standard. 1139 / I 2223 Or I 1329 / I 2223 The relative peak intensity is used as the ordinate to correct signal fluctuations during probe detection, so as to minimize errors and improve the reliability of detection results.

[0089] Using the logarithm of NaHS concentration as the x-coordinate, I 1139 / I 2223 Plot a standard curve on the ordinate, and the resulting fitting equation is: Y = 0.168X + 0.683, R 2 =0.995， See results Figure 8 ;

[0090] Using the logarithm of NaHS concentration as the x-coordinate and I as the y-coordinate... 1329 / I 2223 Plot a standard curve on the ordinate, and the resulting fitting equation is: Y = -0.402X + 0.537, R 2 =0.973The limit of detection is as low as 0.2 μM. See the results below. Figure 8 .

[0091] The applicant also fitted a standard curve for the molecule without internal standard modification. Specifically: using the logarithm of the NaHS concentration as the x-coordinate, and plotting the curves at 1139 cm⁻¹. -1 and 1329 cm -1 Using the absolute intensity of the characteristic peak as the ordinate, a linear fit is performed, and the resulting fitting equation is: Y = 1268.230X + 5042.117, R 2 =0.979 ; Y = -4011.367X - 405.240, R 2 = 0.925 The results are shown Figure 9 。

[0092] Take I 1329 Taking the standard curve corresponding to the characteristic peak at a certain point as an example: the working curve after correction using the internal standard modified molecule 4-mercaptobenzonitrile. R 2 The value was 0.973; while the working curve without internal standard correction was... R 2 Only 0.925 This demonstrates that the probe modified with MPBN as an internal standard in this invention has excellent reproducibility, small error, and high accuracy in detecting H2S.

[0093] Example 4: Application of Au@MPBN@Au@4-NTP in live-cell H2S imaging

[0094] This embodiment uses SERS sensing and imaging of H2S in HepG2 cells (cells sourced from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) as an example to provide a more detailed explanation of the present invention. The specific content is as follows:

[0095] The first step is the pretreatment of cell samples.

[0096] Control sample: HepG2 cells were digested and seeded in 6 cm confocal dishes. DMEM was used as the culture medium, and Au@MPBN@Au@4-NTP (final concentration 0.05 mg / mL) was added. The cells were incubated at 37°C for 4 h.

[0097] Exogenous H2S imaging samples: HepG2 cells (undigested) that had adhered for 24 h were seeded into DMEM medium, 100 μM NaHS was added, and the cells were incubated at 37°C for 60 min. After incubation, the cells were washed three times with PBS buffer (pH = 7.4). The washed HepG2 cells were then seeded into DMEM medium, Au@MPBN@Au@4-NTP (final concentration 0.05 mg / mL) was added, and the cells were incubated at 37°C for 4 h.

[0098] Endogenous H2S imaging samples: HepG2 cells (undigested) that had adhered for 24 h were seeded into DMEM medium, and 100 μM sodium nitroprusside (SNP, nitric oxide donor, i.e., NO donor, which can upregulate the activity of H2S-related enzymes (cystathionine γ-lyase, etc.) and stimulate the production of endogenous H2S in HepG2 cells) were added. The cells were incubated at 37℃ for 60 min to stimulate the production of endogenous H2S. Then, the cells were washed three times with PBS buffer (pH=7.4). After washing, HepG2 cells were seeded into DMEM medium, and Au@MPBN@Au@4-NTP nanoprobes (final concentration 0.05 mg / mL) were added. The cells were incubated at 37℃ for 4 h.

[0099] The second step involved placing the cell samples obtained in the first step onto a stage and acquiring Raman signals at room temperature (Raman test parameters: excitation wavelength 633 nm, microscope 50×, spot diameter 1.54 μm, step size 0.8 μm, exposure time 1 s). Imaging images of the three samples were obtained, as detailed in the attached image. Figure 10-11 .

[0100] from Figure 10 The control group showed that when HepG2 cells were incubated with Au@MPBN@Au@4-NTP for only 4 h, the cells at 2223 cm⁻¹... -1 The presence of a significant Raman scattering signal in the channel indicates that the nanoprobe Au@MPBN@Au@4-NTP of this invention has good cellular uptake capacity and intracellular stability.

[0101] Depend on Figure 10 Cell imaging images after NaHS treatment and Figure 11 It was found that incubating HepG2 cells with NaHS followed by treatment with Au@MPBN@Au@4-NTP resulted in a cell growth rate of 1139 cm⁻¹. -1 A novel SERS signal was observed. The results indicate that the Au@MPBN@Au@4-NTP nanoprobe of this invention can detect exogenous H2S in cells.

[0102] Depend on Figure 10 Cell imaging images after SNP treatment and Figure 11 It can be seen that stimulating the production of endogenous H2S in HepG2 cells with SNPs, followed by treatment with Au@MPBN@Au@4-NTPs, can also increase the concentration of H2S at 1139 cm⁻¹. -1 A novel SERS signal was observed. The results indicate that the Au@MPBN@Au@4-NTP of this invention can sense endogenous H2S in cells, thereby enabling the detection of endogenous H2S in cells.

[0103] Example 5: Application of Au@MPBN@Au@4-NTP in semi-quantitative detection of intracellular H2S

[0104] This invention further investigates Au@MPBN@Au@4-NTP as a Raman probe for the quantitative detection of H2S in cells using ratio Raman imaging. The following explanation uses A549 cells as an example:

[0105] After adhering and growing for 24 h, A549 cells (cells were obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were seeded into DMEM medium. Different concentrations of NaHS (0, 20 μM, 50 μM, 100 μM, with 0 μM serving as the control group) were added to the A549 cells and incubated for 60 min.

[0106] After incubation, the cells were washed three times with PBS buffer (pH = 7.4); the washed A549 cells were seeded into DMEM medium, Au@MPBN@Au@4-NTP (final concentration 0.05 mg / mL) was added, and the cells were incubated at 37℃ for 4 h.

[0107] The incubated A549 cell samples were subjected to Raman spectroscopy. The Raman spectroscopy parameters were: excitation wavelength 633 nm, microscope 50×, spot diameter 1.54 μm, step size 0.8 μm, and exposure time 1 s. The results are shown in [Figure number missing]. Figure 12 .

[0108] from Figure 12 As shown in A and B, with the increase of NaHS concentration, a value of 1139 cm⁻¹ was observed. -1 The Raman signal in the channel increased significantly, and at 2223 cm⁻¹ -1 The SERS signal at that location is almost unaffected by the external environment. Figure 12 As shown in C, with the increase of NaHS concentration, the ratio signal I in A549 cell Raman imaging... 1139 / I 2223 The SERS intensity ratio (IL) of A549 cells + 20 μM NaHS, A549 cells + 50 μM NaHS, and A549 cells + 100 μM NaHS increased significantly. 1139 / I2223 The values ​​were 2.90, 4.01, and 6.33 times that of the control group, respectively, showing significant differences (p<0.005).

[0109] The results show that the nanoprobe Au@MPBN@Au@4-NTP of the present invention can achieve semi-quantitative detection of intracellular H2S through ratio Raman imaging technology.

Claims

1. Application of an internal standard-assisted petal-shaped SERS nanoprobe in imaging and quantitative analysis of H2S in living cells for non-disease diagnosis purposes, wherein, The internal standard auxiliary petal-shaped SERS nano probe is a gold nano flower probe with a core-molecule-shell structure composed of a gold nano sphere core, an internal standard modification molecule, a petal-shaped gold nano shell and a recognition molecule; The particle size of the gold nano sphere core is 20-40 nm, the thickness of the petal-shaped gold nano shell is 30-50 nm, the internal standard modification molecule is 4-mercaptobenzonitrile, and the recognition molecule is 4-nitrothiophenol; The internal standard auxiliary petal-shaped SERS nano probe comprises the following preparation steps: First, preparing a gold nano sphere core AuNPs with surface plasmon resonance effect S11, preparing gold seeds by using a classical sodium citrate reduction method; S12, preparing gold nano core AuNPs by using a seed growth method, wherein the AuNPs solution is wine red, and the particle size of AuNPs in the AuNPs solution is about 30 nm; Second, modifying 4-mercaptobenzonitrile on the gold nano sphere core AuNPs: adjusting the pH of the AuNPs solution to 9.0, adding a 20 mM 4-mercaptobenzonitrile ethanol solution, stirring at 30°C for 3 h, removing the unbound 4-mercaptobenzonitrile by centrifugation, and redispersing the precipitate with deionized water to obtain a 4-mercaptobenzonitrile modified AuNPs solution; Third, growing a petal-shaped gold nano shell outside the molecular layer of the internal standard modification molecule to obtain gold nano flowers containing internal standard modification molecules, which are used as SERS substrates: mixing the 4-mercaptobenzonitrile modified AuNPs with a cetyltrimethylammonium chloride solution, then sequentially adding HAuCl4·3H2O and ascorbic acid, and obtaining a deep blue SERS substrate-Au@MPBN@Au NFs solution after the reaction is completed; Fourth, centrifuging the Au@MPBN@Au NFs solution, washing the precipitate with deionized water, adding 4-nitrothiophenol after washing, stirring at room temperature, centrifuging after the reaction is completed, and dispersing the precipitate in a HEPES buffer to obtain a 4-NTP modified internal standard auxiliary petal-shaped SERS nano probe-Au@MPBN@Au@4-NTP; The detection of H2S in cells includes the following contents: cells after adherent growth for 24 h are inoculated into DMEM medium, different concentrations of NaHS solution are added for co-incubation, after incubation, cells are washed with PBS buffer; then DMEM medium containing Au@MPBN@Au@4-NTP is added, constant temperature incubation is carried out, after incubation, cells are washed with PBS buffer, Raman detection is carried out, and extracellular exogenous H2S imaging pictures and Raman peaks are obtained; the SERS intensity ratio I 1139 / I 2223 or I 1329 / I 2223 As the abscissa, the logarithmic value of the concentration of the NaHS solution is taken as the abscissa, and a standard curve is drawn; according to the ratio Raman imaging and the standard curve I 1139 / I 2223 H2S in the cell sample is semi-quantitatively analyzed; The H2S imaging in living cells includes endogenous H2S imaging in cells and exogenous H2S imaging in cells, and the exogenous H2S imaging in cells includes the following contents: the cell sample after adherent growth for 24 hours is inoculated into DMEM medium, NaHS solution is added for incubation, and after the incubation, the cell sample is washed with PBS buffer; then, the DMEM medium containing Au@MPBN@Au@4-NTP is added for incubation, and after the incubation, the cell sample is washed with PBS buffer, Raman detection is performed, and the exogenous H2S imaging picture of cells is obtained; the endogenous H2S imaging in cells includes the following contents: the cell sample after adherent growth for 24 hours is inoculated into DMEM medium, sodium nitroprusside solution is added for incubation, and the sodium nitroprusside is used to stimulate the cells to produce endogenous H2S; after the incubation, the cell sample is washed with PBS buffer; then, the DMEM medium containing Au@MPBN@Au@4-NTP is added for incubation, and after the incubation, the cell sample is washed with PBS buffer, Raman detection is performed, and the endogenous H2S imaging picture of cells is obtained.

Images