Multi-wavelength excitation near-infrared resonance Raman probe molecule, preparation method thereof, nano-particles and application of multi-wavelength excitation near-infrared resonance Raman probe molecule

Abstract

The invention belongs to the technical field of detection, and particularly relates to a multi-wavelength excitation near-infrared resonance Raman probe molecule and a preparation method thereof, nanoparticles and application, two donor-acceptor-donor type Raman probes BTA1 and BTA2 are designed and synthesized, the probes have planar molecular structures and are highly conjugated in the alkynyl direction, and the probe has a wide application prospect. The donor-acceptor-donor structure effectively promotes intramolecular charge transfer, so that an absorption spectrum is subjected to red shift to a first near-infrared region, and meanwhile, molecular fluorescence is remarkably reduced. In addition, the planar molecular configuration enhances the intermolecular pi-pi accumulation effect, induces a strong aggregation-induced fluorescence quenching effect, and further inhibits fluorescence emission. Benefited by the synergistic effect of pi electron delocalization and aggregation-induced fluorescence quenching of a molecular skeleton, BTA1 and BTA2 have strong Raman signals under excitation of 488 nanometers, 532 nanometers, 633 nanometers and 785 nanometers, and show excellent compatibility with a commercial laser light source.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a multi-wavelength excited near-infrared resonance Raman probe molecule, its preparation method, nanoparticles, and applications. Background Technology

[0002] As a characteristic vibrational scattering spectrum of molecules, Raman spectroscopy provides detailed information at the molecular level and has become a powerful tool in the fields of bioimaging, analysis, and information storage. To fully realize this potential, developing Raman-active molecular probes has become a key research direction. However, the inherently small Raman scattering cross-section of vibrational probes severely limits the application of spontaneous Raman scattering imaging. To overcome this limitation, research has mainly progressed along two parallel paths: one is the development of signal amplification platforms, such as stimulated Raman scattering, coherent anti-Stokes Raman scattering, and surface-enhanced Raman scattering; the other is the design of Raman molecules with strong intrinsic signals. Although nonlinear optical techniques (stimulated Raman scattering / coherent anti-Stokes Raman scattering, etc.) have brought revolutionary improvements in imaging speed and signal intensity, their equipment availability is limited; while surface-enhanced Raman scattering technology is constrained by inherent factors such as sensitivity reproducibility challenges, large nanoparticle size, and biocompatibility issues. Therefore, current research focus is increasingly shifting to the construction of novel Raman probes—probes that can be used independently for high-sensitivity spontaneous Raman imaging and can also be seamlessly integrated with the aforementioned enhancement platforms to achieve unprecedented signal amplification.

[0003] Resonant Raman scattering amplifies the intrinsic signal (10²–10⁻¹⁰) by matching the excitation wavelength with the electronic transitions of the reporter group. 6 Resonance Raman probes (RMR) are a powerful signal enhancement strategy. However, developing resonant Raman probes suitable for visible or near-infrared excitation requires a strong donor-acceptor conjugated system. Such systems often produce a strong fluorescence background under resonant excitation, which can easily drown out the target Raman signal. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a multi-wavelength excited near-infrared resonance Raman probe molecule, its preparation method, nanoparticles, and applications.

[0005] A multi-wavelength excited near-infrared resonance Raman probe molecule is obtained by a Stille cross-coupling reaction between a thiophene tinane derivative and a benzobisthiadiazole derivative; The structural formula of the thiophenetinane derivative is shown in Formula I or Formula II, where Formula I is... Equation II is ; The structural formula of the benzobisthiadiazole derivative is shown in Formula III, where Formula III is... .

[0006] This invention synthesizes two donor-acceptor-donor type Raman probes, BTA1 and BTA2, which possess planar molecular structures and are highly conjugated along the alkynyl group direction. The donor-acceptor-donor structure effectively promotes intramolecular charge transfer, redshifting the absorption spectrum to the near-infrared region, while significantly reducing molecular fluorescence. Furthermore, the planar molecular configuration enhances intermolecular π-π stacking interactions, inducing a strong aggregation-induced fluorescence quenching effect, further suppressing fluorescence emission. Benefiting from the synergistic effect of π-electron delocalization of the molecular backbone and aggregation-induced fluorescence quenching, BTA1 and BTA2 all exhibit strong Raman signals under excitation at 488, 532, 633, and 785 nm, demonstrating excellent compatibility with commercial laser sources. Specifically, the alkynyl vibrational signal in the Raman-silent region can be efficiently excited by visible light, while the double bond vibrational signal can achieve resonant enhancement under 785 nm excitation.

[0007] Preferably, the structural formula of the multi-wavelength excited near-infrared resonance Raman probe molecule is shown in Formula IV or Formula V: Formula IV is ; Equation V is .

[0008] A method for preparing the multi-wavelength excited near-infrared resonance Raman probe molecule includes the following steps: In a nitrogen atmosphere, using tetrahydrofuran as solvent and butyllithium as base, the alkynylthiophene derivative is reacted with tributyltin chloride to undergo a tin alkylation reaction to obtain a thiophene tinane derivative. In a nitrogen atmosphere, using chlorobenzene as a solvent and palladium dichloride and cuprous iodide as catalysts, the thiophene tinane derivative and benzobisthiadiazole derivative undergo a Stille cross-coupling reaction to obtain the multi-wavelength excited near-infrared resonance Raman probe molecule.

[0009] Preferably, the conditions for the alkynyltin alkylation reaction are: from 85°C to -70°C to 15°C to 25°C and stirring for 6 to 15 hours.

[0010] Preferably, the conditions for the Stille cross-coupling reaction to occur are stirring at 60℃~80℃ for 8h~15h.

[0011] Preferably, the molar ratio of the alkynylthiophene derivative to the tributyltin chloride is 2.8~3.3:3.5~3.8.

[0012] Preferably, the molar ratio of the benzobisthiadiazole derivative to the alkynylthiophene derivative is 0.8~1.3:2.8~3.3.

[0013] A nanoparticle prepared using the aforementioned multi-wavelength excited near-infrared resonance Raman probe molecule is obtained by encapsulating the multi-wavelength excited near-infrared resonance Raman probe molecule with an amphiphilic copolymer DSPE-PEG2000 via a nanoprecipitation method.

[0014] The application of the nanoparticles in Raman imaging.

[0015] Preferably, the nanoparticles are used for staining and labeling imaging of apple tree rot pathogens.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs and synthesizes two donor-acceptor-donor type Raman probes, BTA1 and BTA2. The probes possess a planar molecular structure and are highly conjugated along the alkynyl group direction. The donor-acceptor-donor structure effectively promotes intramolecular charge transfer, redshifting the absorption spectrum to the near-infrared region, while significantly reducing molecular fluorescence. Furthermore, the planar molecular configuration enhances intermolecular π-π stacking interactions, inducing a strong aggregation-induced fluorescence quenching effect, further suppressing fluorescence emission. Benefiting from the synergistic effect of π-electron delocalization of the molecular backbone and aggregation-induced fluorescence quenching, BTA1 and BTA2 all exhibit strong Raman signals under excitation at 488, 532, 633, and 785 nm, demonstrating excellent compatibility with commercial laser sources. Specifically, the alkynyl vibrational signal in the Raman-silent region can be efficiently excited by visible light, while the double bond vibrational signal can achieve resonance enhancement under 785 nm excitation. Taking advantage of its excellent Raman signal and high hydrophobicity, this invention encapsulates the probe with DSPE-PEG2000 to prepare water-soluble nanoparticles, and applies them to the Raman imaging study of apple tree rot pathogens. Attached Figure Description

[0017] Figure 1 The structural formulas for BTA1 and BTA2 are given.

[0018] Figure 2 This is a schematic diagram of the synthesis of BTA1 and BTA2.

[0019] Figure 3 The UV-Vis absorption and fluorescence emission spectra of BTA1 and BTA2 in tetrahydrofuran are shown, where A is the UV-Vis absorption spectrum and B is the fluorescence emission spectrum.

[0020] Figure 4The UV-Vis absorption and fluorescence emission spectra of BTA1 and BTA2 in a water / tetrahydrofuran mixed solvent are shown below. In this spectrum, A is the UV-Vis absorption spectrum of BTA1 in the water / tetrahydrofuran mixed solvent, B is the UV-Vis absorption spectrum of BTA2 in the water / tetrahydrofuran mixed solvent, C is the fluorescence emission spectrum of BTA1 in the water / tetrahydrofuran mixed solvent, and D is the fluorescence emission spectrum of BTA2 in the water / tetrahydrofuran mixed solvent.

[0021] Figure 5 The fluorescence emission spectra of BTA1 and BTA2 in tetrahydrofuran vary with concentration, where A represents the fluorescence emission spectrum of BTA1 in tetrahydrofuran as a function of concentration, and B represents the fluorescence emission spectrum of BTA2 in tetrahydrofuran as a function of concentration.

[0022] Figure 6 The optical properties, morphology, and particle size of BTA1 and BTA2 nanoparticles are shown below. A represents a comparison of the UV-Vis absorption spectra of BTA1 nanoparticles in aqueous solution and tetrahydrofuran solution; B represents a comparison of the UV-Vis absorption spectra of BTA2 nanoparticles in aqueous solution and tetrahydrofuran solution; C represents the fluorescence spectra of BTA1 and BTA2 nanoparticles in aqueous solution; D represents the particle size distribution of BTA1 and BTA2 nanoparticles; E represents the morphology of BTA1 nanoparticles; and F represents the morphology of BTA2 nanoparticles.

[0023] Figure 7 The images show the Raman spectra of BTA1 and BTA2 under 532 nm excitation. In the images, A is the Raman spectrum of solid BTA1, B is the Raman spectrum of solid BTA2, C is the Raman spectrum of BTA1 nanoparticles of different concentrations in aqueous solution, and D is the Raman spectrum of BTA2 nanoparticles of different concentrations in aqueous solution.

[0024] Figure 8 The linear relationship between the specific Raman peak signal intensity of BTA1 and BTA2 nanoparticles and the concentration, and the RIE values ​​of BTA1 and BTA2 are given. Where A represents the linear relationship between the specific Raman peak signal intensity of BTA1 nanoparticles and the concentration, B represents the linear relationship between the specific Raman peak signal intensity of BTA2 nanoparticles and the concentration, and C represents the RIE values ​​of BTA1 and BTA2.

[0025] Figure 9 The images show the Raman spectra of BTA1 and BTA2 under excitation at 488 nm and 633 nm, respectively. Specifically, A represents the Raman spectrum of BTA1 and BTA2 solids under excitation at 488 nm, B represents the Raman spectrum of BTA1 and BTA2 nanoparticles under excitation at 488 nm, C represents the Raman spectrum of BTA1 and BTA2 solids under excitation at 633 nm, and D represents the Raman spectrum of BTA1 and BTA2 nanoparticles under excitation at 633 nm.

[0026] Figure 10 The images show the Raman spectra of BTA1 and BTA2 under 785 nm excitation, where A represents the Raman spectra of BTA1 and BTA2 solids under 785 nm excitation, and B represents the Raman spectra of BTA1 and BTA2 nanoparticles under 785 nm excitation.

[0027] Figure 11 Comparison of the original Raman spectra of BTA1 nanoparticles under excitation at 532, 633, and 785 nm.

[0028] Figure 12 Comparison of the original Raman spectra of BTA2 nanoparticles under excitation at 532, 633, and 785 nm.

[0029] Figure 13 Sunlight images of BTA1 and BTA2 solids and their dissolution in tetrahydrofuran solution.

[0030] Figure 14 The diagram shows the single-crystal packing mode and electrostatic potential distribution of BTA2, where A represents the single-crystal packing mode of BTA2, and B represents the electrostatic potential distribution of BTA2 monomers and trimers.

[0031] Figure 15 The diagrams show the intermolecular interactions of BTA2, where A represents the intermolecular interactions of BTA2 aggregates along the c-axis and B represents the intermolecular interactions of BTA2 aggregates along the a-axis.

[0032] Figure 16 Raman imaging of apple tree rot fungus was performed using BTA1 and BTA2. In the images, A shows the image of apple tree rot fungus by BTA1 under 532 nm excitation, B shows the image of apple tree rot fungus by BTA1 under 785 nm excitation, C shows the image of apple tree rot fungus by BTA2 under 532 nm excitation, and D shows the image of apple tree rot fungus by BTA2 under 785 nm excitation.

[0033] Figure 17 The 1H NMR spectrum of BTA1.

[0034] Figure 18 This is the carbon NMR spectrum of BTA1.

[0035] Figure 19 The 1H NMR spectrum of BTA2.

[0036] Figure 20 This is the carbon NMR spectrum of BTA2. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0038] To date, only azobenzene-based resonance Raman probes have been extensively studied. These probes exhibit fluorescence suppression and Raman enhancement properties in the visible or red light bands (such as 532 nm and 633 nm), providing effective tools for cell imaging. Based on the current research status, expanding the resonance Raman probe system, especially developing near-infrared excitation resonance probes, remains a crucial area for breakthroughs. This is because near-infrared excitation can significantly reduce autofluorescence in organisms, weaken matrix Raman background interference, and provide superior tissue penetration depth.

[0039] On the other hand, clever molecular design can optimize Raman signals by modulating their photophysical properties. For example, while the aggregation-induced fluorescence quenching effect caused by tight molecular stacking suppresses fluorescence emission, it simultaneously creates favorable conditions for Raman signal acquisition by weakening or even eliminating the fluorescence background. Therefore, this invention envisions designing molecules with aggregation-induced fluorescence quenching properties as an important approach to developing high signal-to-noise ratio Raman probes. Furthermore, specific vibrational groups are crucial for the sensitivity and specificity of Raman detection. Double and triple bonds are widely used due to their sharp and intense Raman bands. Among them, the alkyne vibrational peak is located in the cell's Raman silencing region, effectively avoiding background interference from endogenous biomolecules, and is a type of biological orthogonal Raman signal.

[0040] Based on the above theoretical guidance, this invention designed and synthesized two donor-acceptor-donor type Raman probes, BTA1 and BTA2. The probes possess a planar molecular structure and are highly conjugated along the alkynyl group direction. The donor-acceptor-donor structure effectively promotes intramolecular charge transfer, redshifting the absorption spectrum to the near-infrared region I, while significantly reducing molecular fluorescence. Furthermore, the planar molecular configuration enhances intermolecular π-π stacking interactions, inducing a strong aggregation-induced fluorescence quenching effect, further suppressing fluorescence emission. Benefiting from the synergistic effect of π-electron delocalization of the molecular backbone and aggregation-induced fluorescence quenching, BTA1 and BTA2 all exhibit strong Raman signals under excitation at 488, 532, 633, and 785 nm, demonstrating excellent compatibility with commercial laser sources. Specifically, the alkynyl vibrational signal in the Raman-silent region can be efficiently excited by visible light, while the double bond vibrational signal can achieve resonance enhancement under 785 nm excitation. Taking advantage of its excellent Raman signal and high hydrophobicity, this invention encapsulates the probe with DSPE-PEG2000 to prepare water-soluble nanoparticles, and applies them to the Raman imaging study of apple tree rot pathogens.

[0041] Example 1 BTA1 Synthesis: Under a nitrogen atmosphere, at 78°C, n-butyllithium (1.44 mL, 3.6 mmol, 2.5 M hexane solution) was slowly added to S1 (1.0 g, 3.0 mmol) in 10 mL of tetrahydrofuran solvent. The reaction mixture was slowly brought to room temperature and stirred for 15 minutes. The temperature was then lowered to 78°C, and tributyltin chloride (1.17 g, 3.6 mmol) was added to the reaction system. The reaction mixture was then brought to room temperature and stirred overnight. The thiophene group underwent a unilateral tin alkylation reaction. The tetrahydrofuran was removed under reduced pressure to obtain the crude product S2.

[0042] Under a nitrogen atmosphere, all the crude S2 product, S3 (0.35 g, 1.0 mmol), palladium dichloride bis(triphenylphosphine) (70 mg, 0.01 mmol), and cuprous iodide (19 mg, 0.01 mmol) were added sequentially to 40 mL of chlorobenzene. The reaction system was stirred at 70 °C for 12 hours, resulting in a Stille cross-coupling reaction. After the reaction was complete, the reaction solution was cooled to room temperature and then poured into methanol. The product precipitated and was filtered. The filter residue was collected and purified by column chromatography. Compound BTA1 was obtained as a dark green solid (0.2 g, yield: 22%).

[0043] The NMR spectrum of compound BTA1 is shown in [reference needed]. Figure 17 and Figure 18 : 1H NMR (400 MHz, o-Dichlorobenzene-d4) δ 9.17 (s, 2H), 7.42 (d, J=3.5Hz, 2H), 7.14 (d, J=5.2Hz, 2H), 3.21 (t, J=7.7Hz, 4H), 3.11 (t, J=7.7Hz, 4H), 2.19-2.09 (m, 4H), 1.99-1.90 (m, 4H), 1.76 (h, J=7.2Hz, 4H), 1.66-1.54 (m, 20H), 1.20-1.15 (m, 12H).13C NMR (101MHz, o-Dichlorobenzene-d4) δ 150.9, 148.4, 148.1, 138.3, 128.6, 127.65, 127.64, 126.8, 124.6, 118.6, 112.7, 93.1, 90.0, 32.07, 32.00, 30.8, 30.6, 30.2, 30.0, 29.6, 29.4, 23.1, 23.0, 14.34, 14.33. BTA2 Synthesis: The synthesis conditions for BTA2 are the same as those for BTA1, except that S1 is replaced with S4 and S2 is replaced with S5.

[0044] BTA2 is a dark green solid (0.21 g, yield: 22%).

[0045] The NMR spectrum of compound BTA2 is shown in the figure. Figure 19 and Figure 20 : 1H NMR (400MHz, o-Dichlorobenzene-d4) δ 9.15 (s, 2H), 7.33 (d, J=5.2Hz, 2H), 7.01 (d, J=5.1Hz, 2H), 3.12 (t, J=7.7Hz, 4H), 2.98-2.92 (m, 4H), 2.0 4 (p, J=7.7Hz, 4H), 1.83-1.75 (m, 4H), 1.61 (q, J=7.0Hz, 4H), 1.53-1.41 (m, 20H), 1.14-1.07 (m, 12H).13C NMR (101MHz, o-Dichlorobenzene-d4) δ 156.4, 156.0, 155.7, 144.1, 133.7, 133.1, 132.8, 132.28, 132.26, 128.0, 121.9, 117.6, 89.5, 85.6, 84.4, 83.1, 36.6, 36.5, 35.3, 35.1, 34.8, 34.5, 34.0, 33.8, 27.6, 27.5, 18.94, 18.89.

[0046] Information on S1 to S5 during the reaction is shown in Table 1.

[0047] The structural formulas for BTA1 and BTA2 are shown below. Figure 1 The synthetic routes for BTA1 and BTA2 are shown below. Figure 2 .

[0048] Table 1: Information for S1~S5 This invention, based on the design concept of enhancing Raman signals using aggregation-induced fluorescence quenching, synthesized two planar donor-acceptor-donor conjugated compounds, BTA1 and BTA2. Their molecular structures include (…). Figure 1 Benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) acts as a strong electron acceptor, while thiophene linked by an alkynyl group acts as an electron donor, forming a highly delocalized π-conjugated molecular skeleton. Furthermore, a long alkyl chain is introduced to effectively enhance the lipophilicity of the molecule.

[0049] After obtaining BTA1 and BTA2, this invention first investigated their photophysical properties. For example... Figure 3As shown in Figure A, both BTA1 and BTA2 exhibit similar broad-band absorption in tetrahydrofuran solution, covering the visible light region (300-500 nm) and the near-infrared region (600-1000 nm). Simultaneously, both BTA1 and BTA2 show weak near-infrared fluorescence emission in the 750-850 nm range, with a peak around 790 nm. Figure 3 B). By measuring the spectral changes of BTA1 and BTA2 in a tetrahydrofuran / water mixed solvent with increasing water content, this invention investigated their aggregated optical behavior. When the water content increased from 0% to 100%, the absorption spectra of both BTA1 and BTA2 showed a significant red shift and broadening. Figure 4 A, B); its fluorescence signal decays rapidly until it is quenched (A, B); Figure 4 C, D). Concentration-dependent fluorescence experiments conducted in tetrahydrofuran further demonstrated that the fluorescence emission intensity systematically decreased with increasing concentrations of BTA1 and BTA2. Figure 5 Notably, aggregation-induced fluorescence quenching was observed even at a low concentration of 5 micromoles. These results collectively indicate that BTA1 and BTA2 readily form molecular aggregates, and the enhanced π-π stacking ultimately leads to aggregation-induced fluorescence quenching.

[0050] The weak fluorescence of BTA1 and BTA2 in their aggregated state is beneficial for acquiring their Raman signals. To stabilize the aggregates and ensure good water dispersibility, this invention utilizes the amphiphilic copolymer DSPE-PEG2000 to encapsulate BTA1 and BTA2 into nanoparticles via a nanoprecipitation method.

[0051] Preparation of BTA1 and BTA2 nanoparticles: BTA1 or BTA2 (10 mg) and DSPE-PEG2000 (30 mg) were dissolved in tetrahydrofuran (10 mL). Under ultrasonic conditions, the mixture was slowly added dropwise to deionized water (90 mL). After ultrasonication for 30 minutes, the mixture was stirred overnight to allow the tetrahydrofuran to evaporate. The mixture was then filtered through a 0.22 µm polyethersulfone membrane to obtain an aqueous solution of BTA1 or BTA2 nanoparticles.

[0052] The obtained nanoparticles were well dispersed in water, exhibiting a uniform spherical morphology with average sizes of approximately 145 nm and 131 nm, respectively. Figure 6 DF). Compared to their dissolved state in tetrahydrofuran, the absorption spectra of BTA1 and BTA2 nanoparticles in water exhibit a red shift and broadening. Figure 6 A, B), fluorescence completely quenched ( Figure 6 C). These spectral changes confirm that the molecules inside the nanoparticles have formed stable aggregates.

[0053] Subsequently, the spontaneous Raman spectra of BTA1 and BTA2 were recorded in this invention. Under 532 nm excitation, BTA1 exhibited several characteristic Raman peaks, with strong and sharp signals at 889, 1253, 1383, and 2160 cm⁻¹. Figure 7 A). BTA2 exhibits sharp peaks at 889, 1253, and 2160 cm⁻¹, while also showing peaks at 1398, 1423, and 1452 cm⁻¹. Figure 7 B). The signal between 889-1452 cm⁻¹ belongs to the vibration of double bonds in the molecular conjugated backbone, while the peak at 2160 cm⁻¹ is located in the Raman silencing region of the cell, corresponding to the stretching vibration of the alkyne group. Both probes prepared as nanoparticles still retained their Raman spectra. Figure 7 C, D). There is a good linear correlation between the Raman signal intensity and the concentration of the two nanoprobes. Figure 8 A, B). The alkyne signals of BTA1 and BTA2 were 115 and 192 times stronger than those of 5-ethynyl-2'-deoxyuridine (EdU), respectively, confirming that they are Raman probes with excellent signals. Figure 8 C).

[0054] Most reported Raman probes typically rely on near-resonance excitation to balance fluorescence suppression and Raman signal enhancement; unlike them, BTA1 and BTA2 exhibit significant excitation flexibility. Both probes can be excited by lasers at 488, 532, 633, and 785 nm and produce excellent Raman signals. Figure 9 and Figure 10 It is noteworthy that 785 nm excitation can produce resonant Raman enhancement. Under 488 and 532 nm excitation, BTA1 and BTA2 exhibit the same Raman spectra. For BTA1, the peak signal at 1383 cm⁻¹ is the strongest, and the signal intensities of the remaining peaks are also significant. In contrast, the signal intensities of all characteristic peaks of BTA2 are comparable. However, when switching to 633 and 785 nm excitation, only Raman signals at 889 and 1253 cm⁻¹ are observed, while signals above 1383 cm⁻¹ are sharply weakened or even disappear. Analysis of the original Raman spectra of BTA1 and BTA2 nanoparticles under non-resonant (532, 633 nm) and resonant (785 nm) excitation shows that the signals obtained under 532 and 633 nm excitation have a clean fluorescence background, and the fluorescence background under 785 nm excitation is also significantly suppressed. Figure 11-12 These observations indicate that fluorescence masking is not the decisive factor in signal attenuation. This invention suggests that variations in the optical system of resonant excitation or Raman instruments (such as gratings and filters, which change with the laser beam) can significantly affect the spectral measurement results.

[0055] Based on their unique photophysical properties, this invention further investigated the solid-state characteristics of BTA1 and BTA2. BTA1 typically exists in the form of amorphous powder, while BTA2 readily forms layered crystals. Figure 13 By using a tetrahydrofuran / methanol solvent diffusion method, this invention yielded BTA2 single crystals suitable for X-ray diffraction. Crystal structure analysis showed that a single BTA2 molecule exhibits a centrosymmetric linear planar conformation, with its donor-acceptor units maintaining structural stability through intramolecular S···N interactions. The alkyl side chains located adjacent to the alkynyl group are orderly arranged along the b-axis without disrupting the planarity of the molecular backbone. Figure 14 A). BTA2 exhibits typical J-aggregation characteristics, with a glide angle of 69.17° along the c-axis and an interplanar spacing of 3.09 Å, and a glide angle of 72.11° along the a-axis and an interplanar spacing of 3.51 Å ( Figure 4 A). In the c-direction, the C2 atom distance between the terminal thiophene sulfide (S1) of one molecule and the middle thiophene ring of the adjacent molecule is only 3.48 Å. Simultaneously, interlayer slip causes the distance between the upper thiophene sulfide atom (S1) and the C1 atom of the lower benzobisthiadiazole to shorten to 3.46 Å. These close proximity distances indicate strong intermolecular π-π interactions, which can be further visualized through reduced density gradient (RDG) analysis. Figure 15 Significant green isosurfaces are observed between thiophene units along the c-axis and between thiophene and benzobisthiadiazole along the a-axis, indicating a very strong π-π stacking interaction between molecules.

[0056] The electrostatic potential distribution diagram shows that the complementary charge distribution within the molecules drives this ideal packing mode. Figure 14 (B) In this arrangement, the benzobisthiadiazole acceptor unit is surrounded by thiophene donors in multiple directions, promoting intermolecular charge transfer interactions and thus enhancing the overall polarizability. Calculations show that the BTA2 monomer itself possesses high polarizability. When the three molecules slip-pack along the a-axis or c-axis, the molecular polarizability is further increased to three times, which significantly contributes to the enhancement of the Raman signal. Although BTA1 does not form a long-range crystalline ordered structure, its similar optical properties to BTA2 indicate that it still exhibits similar local packing modes and strong intermolecular π-π interactions in its amorphous morphology, thereby promoting its excellent Raman scattering signal.

[0057] The superior Raman performance of BTA1 and BTA2 makes them highly promising for the field of bioimaging. They can achieve orthogonal Raman imaging excited by visible light (e.g., 532 nm) and resonant Raman imaging excited by near-infrared light (785 nm). To verify this capability, this invention applies BTA1 and BTA2 nanoparticles to staining and labeling imaging of Valsa mali, the causal agent of apple tree rot. Figure 16 Apple tree rot pathogens inoculated on bacterial culture dishes were incubated for three days with an aqueous solution of 600 μmol BTA1 or BTA2 nanoparticles, respectively. Afterward, the bacteria were thoroughly washed with phosphate buffer to remove residual nanoparticles, and finally fixed onto a glass slide for Raman imaging. Bright multi-channel images of the stained bacteria at different Raman shifts were obtained under excitation at 532 nm and 785 nm, demonstrating the excellent labeling and imaging performance of the probe. Compared to 532 nm excitation, 785 nm excitation provides a superior imaging depth, enabling signal collection from deeper layers of multi-layered fungal aggregates, thus providing richer spectral information and visual presentation.

[0058] This invention successfully developed two multi-wavelength excitation Raman probes, BTA1 and BTA2. Their molecules employ a planar donor-acceptor-donor conjugated framework, which promotes tight J-shaped aggregation and generates a significant aggregation-induced fluorescence quenching effect, effectively suppressing fluorescence interference. Under the synergistic effect of high π-electron delocalization and aggregation-induced fluorescence quenching, both probes can be excited by lasers of various wavelengths (from visible to near-infrared) and produce excellent Raman signals. Visible light can excite alkyne-based bioorthogonal Raman signals, while near-infrared excitation at 785 nm achieves resonance Raman enhancement. This invention nanoscaled BTA1 and BTA2 and applied them to staining and labeling imaging of *Valsa mali*, successfully achieving visible-light-excited bioorthogonal Raman imaging and near-infrared-excited resonance Raman imaging. This research provides an effective strategy for designing Raman probes that simultaneously achieve signal enhancement and fluorescence suppression.

[0059] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0060] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A multi-wavelength excited near-infrared resonance Raman probe molecule, characterized in that, It is obtained by Stille cross-coupling reaction of thiophene tinane derivative and benzobisthiadiazole derivative; The structural formula of the thiophenetinane derivative is shown in Formula I or Formula II, where Formula I is... Equation II is ; The structural formula of the benzobisthiadiazole derivative is shown in Formula III, where Formula III is... .

2. The multi-wavelength excited near-infrared resonance Raman probe molecule according to claim 1, characterized in that, Its structural formula is shown in formula IV or formula V: Formula IV is ; Equation V is .

3. A method for preparing the multi-wavelength excited near-infrared resonance Raman probe molecule according to claim 1, characterized in that, Includes the following steps: In a nitrogen atmosphere, using tetrahydrofuran as solvent and butyllithium as base, the alkynylthiophene derivative is reacted with tributyltin chloride to undergo a tin alkylation reaction to obtain a thiophene tinane derivative. In a nitrogen atmosphere, using chlorobenzene as a solvent and palladium dichloride and cuprous iodide as catalysts, the thiophene tinane derivative and benzobisthiadiazole derivative undergo a Stille cross-coupling reaction to obtain the multi-wavelength excited near-infrared resonance Raman probe molecule.

4. The preparation method according to claim 3, characterized in that, The conditions for the alkynyltin alkylation reaction are: from 85℃ to -70℃ to 15℃ to 25℃ and stirring for 6 to 15 hours.

5. The preparation method according to claim 3, characterized in that, The conditions for Stille cross-coupling reaction to occur are stirring at 60℃~80℃ for 8h~15h.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the alkynylthiophene derivative to the tributyltin chloride is 2.8~3.3:3.5~3.

8.

7. The preparation method according to claim 3, characterized in that, The molar ratio of the benzobisthiadiazole derivative to the alkynylthiophene derivative is 0.8~1.3:2.8~3.

3.

8. A nanoparticle prepared using the multi-wavelength excited near-infrared resonance Raman probe molecule as described in claim 1, characterized in that, The multi-wavelength excited near-infrared resonance Raman probe molecule was obtained by encapsulating the amphiphilic copolymer DSPE-PEG2000 using a nanoprecipitation method.

9. The application of the nanoparticles according to claim 8 in Raman imaging.

10. The application according to claim 9, characterized in that, The nanoparticles are used for staining and labeling imaging of apple tree rot pathogens.

Images