A chiral molecule detection platform based on hydrogel SERS chip and application thereof in detection of levomilnacipran

Abstract

The application belongs to the technical field of analysis and detection, and discloses a chiral detection platform based on a hydrogel SERS chip and application thereof.The core of the application is that the inherent chiral structure of a hydrogel matrix in a specific hydrogel SERS chip is found and utilized for the first time, so that specific enantiomers of chiral molecules can be directly and highly selectively recognized and detected without any exogenous chiral modifier or complex separation process.When the chip is used for detection with levomeprazole as a model analyte, only levomeprazole can be selectively adsorbed and produce a strong characteristic SERS signal, and the response of the dextrogyratory enantiomer is weak under the optimal conditions of an acid medium with pH 4.The sensor constructed by the method exhibits high sensitivity to levomeprazole, and the detection limit reaches 1.0x10 ‑7 mol / L, excellent selectivity and good anti-biological matrix interference capability, thereby providing a novel, simple and efficient solution for rapid analysis of chiral drugs.

Description

Technical Field

[0001] This invention belongs to the field of chiral analysis sensing technology, specifically relating to a chiral molecule detection platform based on a hydrogel SERS chip and its application in the detection of levo-omeprazole. Background Technology

[0002] Chiral enantiomers are crucial in the life sciences and pharmaceutical fields, and their differentiated biological activities make the development of rapid and accurate chiral analysis methods an urgent need. Traditional methods, such as chiral chromatography, are reliable, but they have limitations such as expensive equipment, complex operation, and long processing time.

[0003] Surface-enhanced Raman scattering (SERS) technology is considered a promising alternative due to its high sensitivity. However, ordinary SERS substrates cannot distinguish between enantiomers and usually require the introduction of exogenous chiral selectors (such as cyclodextrin, antibodies, etc.) for functionalization. This process is often cumbersome and may affect the stability and reproducibility of the substrate.

[0004] In the prior art, a method for preparing a high-performance hydrogel SERS chip has been disclosed (see Chen M, et al. Adv. Opt. Mater. 2023;11:2202762). This chip is fabricated by embedding aggregated silver nanoparticles (a-AgNPs) into polyvinyl alcohol (PVA) hydrogel, and has been used for the detection of various analytes due to its excellent SERS activity and mechanical properties. However, the prior art only discloses the application of this chip as a general SERS substrate, without mentioning or suggesting its application in the field of chiral recognition. In particular, there has been no previous report or understanding regarding whether the PVA hydrogel in this chip possesses, and how its possible chiral structure can be utilized to achieve enantioselective adsorption and detection. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing chiral SERS detection methods, which require complex chiral modifications, by providing a chiral molecule detection platform based on a hydrogel SERS chip and its application in the detection of levomeprazole. This platform requires no exogenous chiral modification, is simple to operate, and can achieve direct detection with high selectivity for chiral recognition. To achieve the above objective, this invention adopts the following technical solution:

[0006] The technical solution of this invention is based on an unexpected discovery: although hydrogel SERS chips themselves are already disclosed, this invention is the first to discover and confirm that the PVA hydrogel matrix in the chip possesses an inherent chiral microstructure, namely nanohelices. This intrinsic property enables it to act as an intrinsic, stable chiral selector, specifically adsorbing levorotatory enantiomer molecules while adsorbing almost no other enantiomers. When levorotatory enantiomer molecules bind to the inherent SERS active components (a-AgNPs) in the chip, an integrated detection platform combining "chiral recognition" and "signal amplification" is naturally formed. The innovation of this platform lies not in the physical structure of the chip, but in the development of its novel functions and applications, namely, utilizing its intrinsic chirality to achieve enantiomer selective detection. The innovative application of this platform in the selective detection of levorotatory enantiomers, especially levorotatory omeprazole (S-OME), has produced unexpectedly excellent detection performance.

[0007] A chiral molecule detection platform based on a hydrogel SERS chip is disclosed, wherein the platform uses an aggregated metal nanoparticle hydrogel SERS chip as the detection element; wherein the hydrogel matrix in the aggregated metal nanoparticle hydrogel SERS chip has an inherent chiral microstructure, and can selectively adsorb specific enantiomers of the target chiral molecule without exogenous chiral modification; wherein the aggregated metal nanoparticles are used to provide SERS enhancement signal.

[0008] Furthermore, the hydrogel matrix is ​​a hydrogel formed by physical cross-linking of polyvinyl alcohol, and its chiral microstructure exhibits a nano-helical morphology.

[0009] Furthermore, the specific enantiomer is a left-handed enantiomer.

[0010] Furthermore, the aggregated metal nanoparticles are aggregated silver nanoparticles with a localized surface plasmon resonance absorption peak at 785 nm to match the corresponding excitation laser.

[0011] The above-mentioned SERS platform is used in the preparation of sensors or detection kits for the selective detection of levorotatory enantiomers.

[0012] The above-mentioned SERS platform is used in the detection of levomethapazole (S-OME).

[0013] Furthermore, the above-mentioned SERS platform is used to detect levo-omeprazole (S-OME), including the following steps: (1) treating the sample to be tested in an acidic medium with a pH of 3.5 to 4.5; (2) incubating the treated sample with the hydrogel SERS chip for 10 to 30 minutes; (3) performing Raman spectroscopy on the chip to obtain the SERS spectrum; (4) achieving qualitative and / or quantitative analysis of levo-omeprazole by analyzing the characteristic Raman peaks of levo-omeprazole in the SERS spectrum.

[0014] Furthermore, the characteristic Raman peak includes one located at 377 cm⁻¹. -1 and / or 1271 cm -1 The peak; the quantitative analysis is based on the linear relationship between the characteristic peak intensity and the logarithm of the levomethapazole concentration, with a linear detection range of 1.0 × 10⁻⁶. -7 mol / L to 1.0×10 -5 mol / L.

[0015] Furthermore, the acidic medium includes hydrochloric acid, sulfuric acid, or nitric acid.

[0016] Furthermore, the sample to be tested is a biological sample, which undergoes protein precipitation pretreatment before detection.

[0017] The beneficial effects of this invention (highlighting its innovativeness and non-obviousness):

[0018] 1. Innovative Principle: This invention reveals and utilizes the intrinsic chiral properties of known hydrogel SERS chips to achieve chiral recognition for the first time, eliminating all complex, unstable, and costly exogenous chiral modification steps. This is a significant breakthrough in the design concept of chiral sensing.

[0019] 2. Excellent Performance: Because chiral recognition originates from the material's inherent stable structure, the platform exhibits excellent and stable enantiomeric selectivity. For the detection of S-OME, the interference signal from its dextrorotatory isomer (R-OME) is negligible. Simultaneously, it boasts high detection sensitivity (LOD up to 10). -7 M), with a wide linear range.

[0020] 3. Simple method: The entire detection process does not require separation or derivatization. SERS readings can be directly performed after the sample is incubated with the chip, which greatly simplifies the operation process of chiral analysis and reduces the technical threshold and time cost.

[0021] 4. Strong anti-interference ability: The platform can still maintain reliable detection performance in complex biological matrices (such as serum) and has a good spike recovery rate, which proves its great potential for practical application. Attached Figure Description

[0022] Figure 1 Example 2: Verification of the chiral detection platform's ability to distinguish between left-handed and right-handed molecules. (a) SERS spectra of a blank hydrogel SERS chip, and D-tryptophan and L-tryptophan on the hydrogel SERS chip; (b) UV-vis spectra of a blank hydrogel chip and a hydrogel chip after being immersed in 0.1 mol / L D-tryptophan and L-tryptophan solutions for 20 minutes. Figure 2 Scanning electron microscope (SEM) images of the PVA hydrogel used in the examples. (a) 5 µm; (b) 500 nm. Figure 3 Figure 1 shows the optimization results of the quantitative detection method for levomethapazole. Effect of solvent on the SERS spectrum of S-OME (a) and 377 cm⁻¹. -1 SERS intensity variation of characteristic peaks at (b); Effect of soaking time on SERS spectrum of S-OME (c) and 377 cm⁻¹ -1 SERS intensity variation at the characteristic peak (d).

[0023] Figure 4 Standard curve for the quantitative detection of levomeprazole. SERS spectra of S-OME at different concentrations recorded using a hydrogel SERS chip (a), 377 cm⁻¹. -1 Linear relationship between peak intensity and S-OME logarithmic concentration (b), 1271 cm⁻¹ -1 The linear relationship between peak intensity and S-OME logarithmic concentration (c).

[0024] Figure 5 Graph of levonorgestrel selectivity assay. Potential interfering substances (1×10⁻⁶) in serum samples. -4 Surface-enhanced Raman spectra (a) of 1 × 10⁻⁶ mol / L. -5 SERS spectra of mol / L S-OME and R-OME and their levorotatory isomers on a hydrogel SERS chip (b).

[0025] Figure 6 SERS spectra of blank serum samples at different S-OME spiking concentrations. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] The hydrogel SERS chip used in this invention can be prepared using existing techniques (e.g., by Ca). 2+AgNPs are induced to aggregate in a PVA solution, and then physically cross-linked hydrogels are formed through freeze-thaw cycles. This invention does not claim protection for the preparation method itself, but rather for innovative applications based on this known product.

[0028] Example 1: Fabrication of a chiral selective hydrogel SERS chip

[0029] AgNPs were prepared using a typical seed growth method with slight modifications: First, 4.2 mL of deionized water, 4 mL of 1 wt% sodium citrate, 200 μL of 20 mM sodium chloride, and 1 mL of 1 wt% AgNO3 solution were added sequentially to a premixed flask at room temperature, and incubated for 5 min to form AgCl nanoparticle seeds; then, 320 μL of 0.10 M ascorbic acid solution was added to 190 mL of boiling water, and the above seed solution was added 1 min later. The reaction solution changed from colorless to yellow within 2 min, and then heating was stopped while stirring continued, and the solution was allowed to cool naturally to room temperature; finally, the solution was centrifuged at 12000 rpm for 10 min to obtain 6 mL of silver nanoparticle (AgNPs) sol with a concentration of 765 μg / mL.

[0030] 300 μL of the above AgNPs sol, 48 μL of 1 mol / L Ca(NO3)2, and 652 μL of deionized water were added to 2 mL of PVA solution (15 wt%) with stirring. After stirring for 30 min, the light yellow solution turned green, indicating the formation of aggregated silver nanoparticles (a-AgNPs) sol with the desired localized surface plasmon resonance (LSPR) characteristics. The resulting aggregated silver nanoparticles (a-AgNPs) sol was then transferred to 96-well plates, with the sol height controlled to be half to two-thirds of the well depth. After six freeze-thaw cycles at -24 °C and room temperature, the sol in the 96-well plate transformed into 96 hydrogel SERS chips.

[0031] Example 2: Verification of the chiral detection platform's ability to distinguish between left-handed and right-handed molecules

[0032] Prepare the same concentration (1×10) separately -5 L-tryptophan (levorotatory) and D-tryptophan solutions (mol / L, dilute hydrochloric acid at pH 4) were prepared. 200 μL of each solution was added to two separate wells of a hydrogel SERS chip and incubated at room temperature for 20 minutes. After removing the chip, cleaning and drying it, SERS detection was performed (laser wavelength 785 nm). The results are as follows: Figure 1As shown in Figure a, the L-tryptophan solution produced a series of strong characteristic peaks in its SERS spectrum; while the SERS spectrum of the D-tryptophan solution was almost indistinguishable from the background spectrum of the blank hydrogel chip. This comparative experiment directly and strongly demonstrates that the platform possesses excellent enantioselectivity, and that this selectivity originates from the material itself, rather than from added reagents.

[0033] Two hydrogels without SERS-active material AgNPs were immersed in D-tryptophan and L-tryptophan solutions, respectively. Figure 1 As shown in b, the hydrogel immersed in L-tryptophan solution exhibits a typical tryptophan absorption band, while another hydrogel shows almost no tryptophan absorption peak. This result confirms the chiral adsorption properties of the hydrogel, indicating that it should have a chiral structure.

[0034] Example 3: Verification of the intrinsic chiral structure of the hydrogel

[0035] The PVA hydrogel (pure gel without AgNPs) prepared according to existing techniques was observed using scanning electron microscopy (SEM). The results are as follows: Figure 2 As shown, a large number of regular nano-helical structures can be clearly observed inside the hydrogel, which directly confirms from a morphological perspective that the hydrogel has an inherent chiral microstructure, providing a material basis for the chiral recognition function of the present invention.

[0036] Example 4: Establishment of a quantitative detection method for levomeprazole

[0037] The detection conditions were optimized: the effects of different solvents (water pH=4, water pH=7, water pH=10, acetone, methanol, ethanol, acetonitrile, isopropanol) on the SERS signal were investigated, and a weakly acidic aqueous solution with pH 4 was determined to be the optimal solvent. Figure 3 As shown in a and 3b. The incubation time was also optimized (0, 5, 10, 15, 20, 25, 30 min), and it was found that the signal reached its strongest point and tended to stabilize after 20 minutes of soaking, as shown in... Figure 3 As shown in c and 3d. Under optimal conditions, the constructed sensor pair achieves an S-OME of 1.0 × 10⁻⁶. -7 Up to 1.0×10 -5 It exhibits a good semi-logarithmic linear relationship within the concentration range of mol / L, with a value of 377 cm⁻¹. -1 and 1271 cm -1 Taking the two characteristic peaks as examples, the quantitative equations are Y = 16827·lg(C) + 119804 and Y = 18231·lg(C) + 130679, respectively, and the lower limit of quantitation for both is 1.0×10⁻⁶. -7 mol / L. Both characteristic peaks can be used to establish a standard working curve (e.g., mol / L). Figure 4 ).

[0038] Example 5: Selectivity study of quantitative detection method for levomeprazole

[0039] Selectivity assessments showed that this SERS sensor exhibited excellent chiral selectivity, effectively distinguishing S-OME from its R-type enantiomer. Furthermore, common serum interfering substances (such as uric acid, glutathione, ascorbic acid, glucose, valine, lysine, phenylalanine, methionine, threonine, leucine, isoleucine, and histidine) did not interfere. While it responded to structurally similar drugs levonorgestrel and levonorgestrel, their characteristic peaks did not overlap with the main quantitative peak of S-OME, thus not affecting detection. Figure 5 As shown. The selective detection results of the quantitative detection method for levomeprazole indicate that the chiral molecule detection platform based on hydrogel SERS chip established in this invention is suitable for reliable detection of S-OME and can be extended to the analysis of similar chiral drugs.

[0040] Example 6: Detection of levomeprazole in actual serum samples

[0041] After establishing a chiral detection platform based on a hydrogel SERS chip, it was applied to the detection of levomeprazole in actual serum samples. First, the serum samples were pretreated: 200 μL of serum was spiked with 20 μL of S-OME standard solutions of different concentrations, followed by the addition of 180 μL of methanol. The mixture was vortexed and sonicated for 2 min to denature and precipitate the protein. Then, it was centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was dried under nitrogen at room temperature. The residue was reconstituted with 200 μL of 0.1 mM dilute hydrochloric acid. The pretreated sample solution was added to a 96-well plate containing a hydrogel SERS chip, incubated for 20 min, and then the chip was removed, rinsed with deionized water, and dried under nitrogen before SERS detection. Experimental results showed that no characteristic S-OME signal was observed in the blank serum sample, indicating the absence of S-OME in the serum; while clear characteristic S-OME peaks were detected in all spiked samples. Figure 6 Although the serum matrix has some inhibitory effect on some weaker peaks, the main characteristic peaks (such as 377 cm⁻¹) are still present. -1 The strength of the sample remains largely unaffected. (Based on 377 cm) -1 The peak intensity at the point and the quantitative equation Y = 18231·lg(C) + 130679 were calculated, and the three spiked concentrations (5×10) were used. -7 2×10 -6 1×10 -5The recoveries of the mol / L serum were 84.1%, 80.5% and 93.3%, respectively, with relative standard deviations (RSD) between 5.91% and 7.31%, indicating that the method still has good accuracy and reliability in complex serum matrices.

[0042] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A chiral molecule detection platform based on a hydrogel SERS chip, characterized in that: The platform uses an aggregated metal nanoparticle hydrogel SERS chip as the detection element; wherein, the hydrogel matrix in the aggregated metal nanoparticle hydrogel SERS chip has an inherent chiral microstructure, which can selectively adsorb specific enantiomers of the target chiral molecule without exogenous chiral modification; wherein the aggregated metal nanoparticles are used to provide SERS enhancement signal.

2. The detection platform according to claim 1, characterized in that, The hydrogel matrix is ​​a hydrogel formed by physical cross-linking of polyvinyl alcohol, and its chiral microstructure exhibits a nano-helical morphology.

3. The detection platform according to claim 1, characterized in that, The specific enantiomer is a left-handed enantiomer.

4. The detection platform according to claim 1, characterized in that, The aggregated metal nanoparticles are aggregated silver nanoparticles with a localized surface plasmon resonance absorption peak at 785 nm to match the corresponding excitation laser.

5. The use of the SERS platform as described in any one of claims 1 to 4 in the preparation of a sensor or detection kit for the selective detection of levorotatory enantiomers.

6. The use of a SERS platform as described in any one of claims 1 to 4 in the detection of levomeprazole (S-omep).

7. The application according to claim 6, characterized in that: Includes the following steps: (1) The sample to be tested is placed in an acidic medium with a pH of 3.5 to 4.5; (2) The treated sample is incubated with the hydrogel SERS chip for 10 to 30 minutes; (3) Raman spectroscopy is performed on the chip to obtain the SERS spectrum; (4) Qualitative and / or quantitative analysis of levo-omeprazole is achieved by analyzing the characteristic Raman peaks of levo-omeprazole in the SERS spectrum.

8. The application according to claim 7, characterized in that: The characteristic Raman peak includes one located at 377 cm. -1 and / or 1271 cm -1 The peak; the quantitative analysis is based on the linear relationship between the characteristic peak intensity and the logarithm of the levomethapazole concentration, with a linear detection range of 1.0 × 10⁻⁶. -7 mol / L to 1.0×10 -5 mol / L.

9. The application according to claim 7, characterized in that: The acidic medium includes hydrochloric acid, sulfuric acid, or nitric acid.

10. The application according to claim 7, characterized in that: The sample to be tested is a biological sample, which has undergone protein precipitation pretreatment before detection.

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