Near-infrared-absorbing gold nanoclusters and their preparation and use in photoacoustic microscopic imaging

Abstract

This invention relates to the fields of nanomaterial applications and biomedical imaging technology, specifically to near-infrared absorbing gold nanoclusters, their preparation, and their application in photoacoustic microscopy. The near-infrared absorbing gold nanoclusters include Au. 60 NCs and Au 42 NCs, Au 60 The absorption peak of NCs is at 1090 nm, Au 42 Both NCs and Au exhibit strong absorption in the near-infrared region of 700–1700 nm, low absorption at 532 nm, and are water-soluble. 60 NCs and Au 42 NCs can be applied to photoacoustic microscopy, Au 60 NCs are excited using a dual-wavelength method at 532 nm / 1064 nm, with Au 42 NCs were excited using a dual-wavelength method at 532 nm / 808 nm; the Au 60 NCs and Au 42 NCs can be used in photoacoustic microscopy for high-resolution three-dimensional visualization and quantitative analysis of kidney microstructure.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial applications and biomedical imaging technology, specifically to near-infrared absorbing gold nanoclusters, their preparation, and their application in photoacoustic microscopy. Background Technology

[0002] Diabetic nephropathy (DKD) is a leading cause of chronic kidney disease (CKD), affecting over 4% of adults worldwide and accounting for more than 30% of end-stage renal disease cases globally, posing a serious threat to human health. Clinical diagnostic strategies urgently need improvement. Currently used diagnostic markers (such as albuminuria and estimated glomerular filtration rate, eGFR) are ineffective in capturing early pathological changes. This deficiency is particularly pronounced in type 2 diabetic nephropathy (T2DKD), which accounts for over 90% of clinical DKD cases, with up to 50% of T2DKD patients showing no albuminuria. Furthermore, the unpredictable fluctuations in eGFR reduce its sensitivity in detecting early kidney damage. This situation creates a key diagnostic dilemma: it is difficult to effectively distinguish between the true absence of pathological changes in renal tissue and superficially normal functional indicators maintained through compensatory hypertrophy and hyperfiltration mechanisms. This problem is particularly prominent in the diabetic nephropathy population, severely hindering early intervention for kidney disease. Emerging research confirms that morphological changes in multiple components of the renal cortex (including nephrons composed of glomeruli and tubules, and peritubular capillaries) are key predictive indicators of DKD progression. However, existing in vivo imaging techniques have significant limitations: computed tomography (CT) and magnetic resonance imaging (MRI) can only focus on single renal components, lacking the contrast resolution for simultaneous visualization and quantification of multiple components. Fluorescence microscopy, due to optical attenuation, is limited to imaging the surface of renal tissue and cannot reflect deep pathological conditions. Photoacoustic microscopy (PAM), as a three-dimensional imaging technique that combines optical contrast and deep penetration, can clearly resolve renal vessels through endogenous hemoglobin signals, but its application in DKD research is hindered by the lack of tubule-specific probes. Traditional photoacoustic probes are too large to penetrate the glomerular filtration barrier, while existing renal clearance probes have weak absorption and poor contrast, making it difficult to achieve accurate identification of renal tubules and multi-component three-dimensional reconstruction. This hinders research into the core pathological mechanism of DKD (the association between vascular and renal tubular dysfunction) (János Peti-Peterdi, Kengo Kidokoro, Anne Riquier-Brison, Novel in vivo techniques to visualize kidney anatomy and function, Kidney International, Volume 88, Issue 1, 2015, Pages 44-51, https: / / doi.org / 10.1038 / ki.2015.65.).

[0003] In summary, existing diagnostic biomarkers and imaging technologies cannot meet the clinical needs for early and accurate diagnosis of DKD. There is an urgent need to develop novel probes and photoacoustic microscopy applications based on these novel probes to achieve high-resolution three-dimensional reconstruction of multiple components of the renal cortex and fill the technological gap. Summary of the Invention

[0004] To address the shortcomings of existing DKD diagnostic biomarkers (insufficient sensitivity) and imaging techniques (inability to simultaneously analyze multiple components of the renal cortex), this invention provides two near-infrared absorbing gold nanoclusters, Au... 60 NCs (absorption peak at 1090 nm) and Au 42 Both NCs (absorption peak at 785 nm) and NCs exhibit extremely strong absorption in the near-infrared region (700–1700 nm), and both use hydrophilic thiol compounds with opposite charges as co-coating ligands: Au 60 NCs utilize 8-mercaptooctanoic acid (MOA) and 2-(diethylamino)ethanethiol (DAT), Au 42 NCs were synthesized using 6-mercaptohexanoic acid (MHA) and DAT, both with atomic-level precision. Au was used in the synthesis. 60 The hydrodynamic diameter of NCs is only 3.5 ± 0.5 nm, Au 42 NCs are 3.0±0.3 nm, all below the renal filtration threshold, possessing highly efficient renal clearance capacity. They can cross the glomerular filtration barrier, achieving clear distinction between renal tubules and peritubular capillaries, and Au 60 NCs compared to Au 42 NCs penetrate deeper and have better resolution. This invention utilizes near-infrared absorption gold nanoclusters (Au) 60 Based on NCs (nuclear chromatograms), and combined with photoacoustic microscopy, a high-resolution three-dimensional visualization and quantitative analysis platform for kidney microstructure was constructed. This platform integrates 1064nm (Au) microscopy technology. 60 This invention utilizes a dual-wavelength channel (NCs signal) and 532nm (endogenous hemoglobin signal) to simultaneously detect renal tubules, glomeruli, and peritubular capillaries, achieving ultra-high-precision three-dimensional reconstruction of multiple components of the renal cortex at a depth of approximately 412μm below the kidney surface. This enables clear visualization and quantitative analysis of renal microstructure. The high-resolution three-dimensional visualization and quantitative analysis platform for renal microstructure can measure multiple indicators such as peritubular capillary density, glomerular volume, glomerular position, renal tubular density, and renal cortical thickness. This invention overcomes the limitations of existing probe and imaging technologies; the design of two nanoclusters provides multi-dimensional support for renal tissue imaging, enabling simultaneous high-resolution imaging of multiple components of the renal cortex.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides two types of near-infrared absorbing gold nanoclusters (Au). 60 NCs, Au 42 NCs), Au60 The absorption peak of NCs is at 1090 nm, Au 42 The absorption peak of NCs is 785 nm. Both have extremely strong absorption in the near-infrared region of 700~1700 nm, exhibit low absorption characteristics at 532 nm, and are water-soluble.

[0007] Furthermore, Au 60 NCs, Au 42 NCs have strong absorption properties in the near-infrared region, providing a basis for photoacoustic microscopic imaging analysis of kidney microstructure.

[0008] Furthermore, Au 60 NCs, Au 42 NCs, as probes, all exhibit low absorption characteristics at 532 nm. This wavelength-dependent absorption difference enables high-sensitivity photoacoustic microscopy to accurately distinguish between two different signal sources: endogenous hemoglobin vessels dominated by 532 nm signals and probes targeting renal tubules labeled with 808 / 1064 nm signals. This effectively verifies the feasibility of the photoacoustic microscopy method.

[0009] This invention also provides a method for preparing the above-mentioned near-infrared absorbing gold nanoclusters, Au 60 The preparation of NCs includes the following steps:

[0010] (1) Thoroughly mix the thiol ligand 8-mercaptooctanoic acid (MOA), 2-(diethylamino)ethanethiol (DAT), chloroauric acid, and solvent;

[0011] (2) Adjust the well-mixed solution to alkaline, add a reducing agent and continue stirring. Monitor the characteristic absorption peak of the solution. When the characteristic absorption peak is stable, use ultrafiltration to remove excess thiol ligands to obtain Au with near-infrared absorption. 60 NCs (1090nm).

[0012] Preferably, in step (1), the molar ratio of chloroauric acid to the mixed mercapto ligand is 1:2 to 1:8.

[0013] Preferably, in step (1), the molar ratio of MOA to DAT is 5:5 to 9:1.

[0014] This invention also provides a method for preparing the above-mentioned near-infrared absorbing gold nanoclusters, Au 42 NCs include the following steps:

[0015] (1) Thoroughly mix the thiol ligand 6-mercaptohexanoic acid (MHA), DAT, chloroauric acid, and solvent;

[0016] (2) Adjust the pH of the well-mixed solution to alkaline, add a reducing agent and continue stirring. Monitor the characteristic absorption peak of the solution. When the characteristic absorption peak is stable, remove excess thiol ligands by ultrafiltration to obtain Au with near-infrared absorption. 42 NCs (785nm).

[0017] Preferably, in step (1), the molar ratio of chloroauric acid to the mixed mercapto ligand is 1:2 to 1:8.

[0018] Preferably, in step (1), the molar ratio of MHA to DAT is 5:5 to 9:1.

[0019] Preferably, in the two preparation methods described above, the solvent is either deionized water or methanol; the reducing agent is sodium borohydride or carbon monoxide, used to reduce chloroauric acid.

[0020] Preferably, in the two preparation methods described above, the pH is adjusted to be 8-12.

[0021] Preferably, in the two preparation methods described above, the ultrafiltration tubes used in the purification process have a rejection capacity of 10,000-30,000 Da.

[0022] This invention also provides the above two types of near-infrared absorbing gold nanoclusters Au. 60 NCs and Au 42 Application of NCs in photoacoustic microscopy.

[0023] Preferably, the Au 60 NCs are excited using a dual-wavelength method of 532nm / 1064nm, and the Au... 42 NCs are excited using a dual wavelength of 532nm / 808nm; near-infrared absorption gold nanoclusters can be used for high-resolution three-dimensional visualization and quantitative analysis of kidney microstructures in non-disease diagnosis, and for Au-based... 60 The dual-wavelength photoacoustic microscopy system of NCs acquires raw images at 532nm and 1064nm and performs three-dimensional reconstruction. The imaging information of the peritubular capillaries of the glomerulus and renal tubules is accurately extracted in the 532nm channel, and the targeted imaging image of the renal tubules is clearly obtained in the 1064nm channel.

[0024] More preferably, the three-dimensional imaging data of the peritubular capillaries, glomeruli, and renal tubules obtained above are integrated and registered. With the help of high-precision multi-component image fusion technology, ultra-high precision three-dimensional (3D) reconstruction of the renal cortex multistructures is successfully achieved within a depth range of about 412 μm from the kidney surface. This not only fully presents the spatial distribution and anatomical relationship between the microstructures of the renal cortex, but also allows for precise quantitative measurement of key structural parameters such as the diameter of peritubular capillaries, the diameter of glomeruli, the diameter of renal tubules, the spatial position of glomeruli, and the thickness of the renal cortex.

[0025] Compared with the prior art, the present invention has the following advantages and technical effects:

[0026] (1) This invention is the first to prepare two types of near-infrared absorbing gold nanoclusters Au. 60 NCs (absorption peak at 1090 nm) and Au 42 Both NCs (absorption peak at 785 nm) and NCs exhibit extremely strong absorption in the near-infrared region (700~1700 nm). Furthermore, the preparation method is simple, the conditions are mild, the yield is high, and it is easy to industrialize.

[0027] (2) The present invention uses the near-infrared absorption gold nanoclusters prepared for photoacoustic microscopy to construct an ultra-high precision renal cortex imaging platform. It is the first to integrate a dual-wavelength photoacoustic microscopy imaging system with 1064nm (probe signal) and 532nm (endogenous hemoglobin signal), which can simultaneously detect multiple structures such as renal tubules and glomeruli, peritubular capillaries, etc., and realize ultra-high precision 3D reconstruction of renal cortex. Attached Figure Description

[0028] Figure 1 The UV absorption spectra of near-infrared gold nanoclusters prepared in Example 1 with the molar ratio of thiol ligand MOA to DAT set to 5:5, 6:4, 7:3, 8:2 and 9:1, respectively.

[0029] Figure 2 The UV absorption spectra of near-infrared gold nanoclusters prepared in Example 5 with the molar ratios of thiol ligand MHA to DAT set to 5:5, 6:4, 7:3, 8:2, and 9:1, respectively.

[0030] Figure 3 The ultraviolet absorption spectra of the two near-infrared absorbing gold nanoclusters in Examples 1 and 5 (molar ratio of MOA to DAT = 6:4, Au) 60 NCs; Au with a molar ratio of 7:3 for thiol ligands MHA and DAT. 42 NCs).

[0031] Figure 4 The fluorescence emission spectra of the two near-infrared absorbing gold nanoclusters in Examples 1 and 5 (Molar ratio of MOA to DAT = 6:4, Au) are shown. 60 NCs; Au with a molar ratio of 7:3 for thiol ligands MHA and DAT. 42 NCs).

[0032] Figure 5 The near-infrared absorbing gold nanoclusters Au in Example 1 60 Electrospray ionization mass spectrum of NCs (molar ratio of MOA to DAT = 6:4 Au)60 NCs).

[0033] Figure 6 The near-infrared absorbing gold nanoclusters Au in Example 5 42 Electrospray ionization mass spectra of NCs (Au with a molar ratio of 7:3 for mercaptoligands MHA and DAT). 42 NCs).

[0034] Figure 7 The near-infrared absorbing gold nanoclusters Au in Examples 1 and 5 60 NCs and Au 42 TEM images and particle size distribution of NCs (Au with a molar ratio of MOA to DAT of 6:4). 60 NCs; Au with a molar ratio of 7:3 for thiol ligands MHA and DAT. 42 NCs).

[0035] Figure 8 The near-infrared absorbing gold nanoclusters Au in Examples 1 and 5 60 NCs and Au 42 Statistical analysis of hydrated particle size of NCs (molar ratio of MOA to DAT = 6:4 for Au). 60 NCs; Au with a molar ratio of 7:3 for thiol ligands MHA and DAT. 42 NCs).

[0036] Figure 9 The near-infrared absorbing gold nanoclusters Au in Examples 1 and 5 60 NCs and Au 42 In vitro photoacoustic and fluorescence imaging performance of NCs (molar ratio of MOA to DAT = 6:4 Au) 60 NCs; Au with a molar ratio of 7:3 for thiol ligands MHA and DAT. 42 NCs).

[0037] Figure 10 The near-infrared absorbing gold nanoclusters Au in Example 9 60 NCs and Au 42 In vivo fluorescence imaging results of NCs in mice.

[0038] Figure 11 The near-infrared absorbing gold nanoclusters Au in Example 9 60 NCs and Au 42 Images of silver-enhanced staining and hematoxylin-eosin (H&E) stained sections of kidney tissue after intravenous injection of NCs.

[0039] Figure 12 This is a schematic diagram of the construction of the dual-wavelength photoacoustic microscope (PAM) in Example 10.

[0040] Figure 13 In Example 10, Au was injected intravenously. 60 NCs (1064nm channel), Au 42 Maximum intensity projection (MAP) analysis of PAM images after NCs (808nm channel).

[0041] Figure 14 This is a three-dimensional reconstruction image of the renal tubules achieved by dual-wavelength channel PAM based on two types of near-infrared absorbing gold nanoclusters (AuNCs) in Example 10.

[0042] Figure 15 The image shown is a three-dimensional reconstruction of the renal vascular network and a 50μm layer-by-layer segmentation image obtained using Imaris software in Example 11.

[0043] Figure 16 The images show magnified views of the dense peritubular capillary network (pink), extracted glomeruli, and a single reconstructed glomeruli reconstructed under the 532nm channel in Example 11; the three-dimensional visualization results of the renal tubules (green) reconstructed under the 1064nm channel; and the three-dimensional visualization results of the renal cortex integration, which fuses the vascular system (including glomeruli and peritubular capillaries) with the renal tubules.

[0044] Figure 17 The three-dimensional visualization results of the reconstructed renal cortex in Example 11, and the volume rendering map obtained by continuous volume rendering of the glomeruli, renal tubules and peritubular capillaries.

[0045] Figure 18 The results of measuring the diameters of blood vessels, renal tubules, and glomeruli using Imaris software are shown in Example 11.

[0046] Figure 19 The gold nanoclusters Au based on near-infrared absorption in Example 11 60 Magnified multi-angle views of PAM 3D reconstructed renal cortex images of NCs.

[0047] Figure 20 These are representative PAS-stained images of glomeruli and renal tubules from T2DKD mice at different pathological stages in Example 12.

[0048] Figure 21 The results of the physiological indicators of the T2DKD group mice in Example 12 include body weight, fasting blood glucose, serum creatinine (CREA), and blood urea nitrogen (BUN).

[0049] Figure 22 The results of three-dimensional reconstruction of mouse renal cortex during the T2DKD process based on PAM in Example 12 are shown.

[0050] Figure 23This is a volumetric rendering of the three-dimensional reconstruction of the renal cortex of T2DKD mice at different disease progression stages in Example 12.

[0051] Figure 24 This serves as a quantitative comparison and consistency verification of the effects of PAS staining and PAM on the progression of T2DKD damage in Example 12.

[0052] Figure 25 The results of the physiological indicators of the mice in the T1DKD group in Example 13 include body weight, fasting blood glucose, creatinine (CREA), and blood urea nitrogen (BUN).

[0053] Figure 26 These are representative PAS-stained images of glomeruli and renal tubules in T1DKD mice at different pathological stages in Example 13.

[0054] Figure 27 The results of three-dimensional reconstruction of mouse renal cortex during the T1DKD process based on PAM in Example 13 are shown.

[0055] Figure 28 This is a volumetric rendering of the three-dimensional reconstruction of the renal cortex of T1DKD mice at different disease progression stages in Example 13.

[0056] Figure 29 This serves as a quantitative comparison and consistency verification of the effects of PAS staining and PAM on the progression of T1DKD damage in Example 13.

[0057] Figure 30 This is a flowchart illustrating the application of near-infrared absorbing gold nanoclusters prepared in this invention to perform three-dimensional reconstruction of multiple components of mouse renal cortex in photoacoustic microscopy. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0059] In the following specific embodiments, the gold trichloride trihydrate (HAuCl4) involved 3H2O was purchased from Shanghai Mairui Chemical Technology Co., Ltd.; 6-mercaptohexanoic acid and 8-mercaptooctanoic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; diethylaminoethyl mercaptan hydrochloride was purchased from Fluorochem Ltd., UK; sodium borohydride (NaBH4) was purchased from Chengdu Kelong Chemical Reagent Co., Ltd. All reagents were used directly without purification.

[0060] Absorption spectra were measured using a UV-2600 UV-Vis spectrophotometer (Shimadzu, Japan); emission spectra of gold nanoclusters were acquired using a Chronos DFD transient spectrometer (ISS, USA); ESI-MS data were obtained using a TIMS-TOF mass spectrometer (Bruker, Inc.); hydrated particle size was measured using a Nano-ZS nanoparticle size analyzer (Malvin, UK); the laser for the 532nm / 1064nm photoacoustic microscope PAM system was purchased from the Nanjing Institute of Advanced Laser Technology, and the 808nm laser in the 532nm / 808nm photoacoustic microscope PAM system was provided by an Ekapla NT-200 optical parametric oscillator; in vivo NIR-II fluorescence imaging was performed using an InGaAs camera (Photon Etc., Canada, ZephIR 1.7) with a 970nm long-pass filter and an 808nm laser as the excitation source.

[0061] Example 1

[0062] This invention prepares near-infrared absorbing gold nanoclusters Au 60 The implementation scheme for NCs (absorption peak at 1090 nm) is as follows:

[0063] At room temperature, 5.2 mL of 1 mM (0.0052 mmol) chloroauric acid (HAuCl4) aqueous solution was placed in a 10 mL EP tube. A mixed solution of thiol ligands, consisting of 8-mercaptooctanoic acid (MOA) and 2-(diethylamino)ethanethiol (DAT), was added. The molar ratio of MOA to DAT was controlled at 5:5, 6:4, 7:3, 8:2, or 9:1, and the molar ratio of chloroauric acid to the mixed thiol ligand MOA+DAT was 1:4. After thorough mixing, sodium hydroxide solution was added to adjust the pH of the system to 10 (the solution immediately changed from turbid to colorless and transparent). Then, a reducing agent was added (200 μL of freshly prepared NaBH4 solution of 114 mM dissolved in 0.2 M sodium hydroxide solution, or carbon monoxide gas was passed through for 5 min). The reaction was stirred at 25 °C, and the characteristic absorption peak of the solution was monitored in real time. The reaction was terminated when the characteristic absorption peak stabilized. The solution after the reaction was completed was transferred to a 15 mL tube. In a 10 kDa ultrafiltration tube, the sample was ultrafiltered and centrifuged at 8000 rpm for 15 min. It was then washed six times with deionized water to remove unreacted substrate. The purified and concentrated near-infrared absorbing gold nanoclusters (Au) were then collected. 60 NCs were stored at 4°C for later use.

[0064] Experimental results show that when the molar ratio of MOA to DAT is 6:4, the characteristic absorption peak intensity of the product at 1090 nm reaches its maximum. Under other ratios, although the characteristic absorption peak at 1090 nm can still be detected, the height of this peak is significantly reduced due to impurities generated in the reaction system. In summary, a molar ratio of 6:4 for MOA to DAT is optimal for synthesizing high-purity, high-characteristic-absorption-intensity near-infrared gold nanoclusters (Au). 60 The optimal ratio of NCs.

[0065] Figure 1 The ultraviolet absorption spectra of near-infrared gold nanoclusters prepared with the molar ratio of MOA to DAT in the thiol ligand set to 5:5, 6:4, 7:3, 8:2 and 9:1, respectively, show that the absorption peak intensity of the synthesized AuNCs is highest at 1090 nm only when the molar ratio of the two is 6:4.

[0066] Example 2

[0067] The steps are the same as in Example 1, except that the molar ratio of MOA to DAT is fixed at 6:4, and the molar ratio of chloroauric acid to the mixed thiol ligand MOA+DAT is adjusted to 1:2, 1:4, or 1:8.

[0068] Experimental verification shows that the ultraviolet absorption spectra of the three groups of near-infrared absorbing gold nanoclusters prepared in Example 2 are all consistent with... Figure 1 The graph shows a molar ratio of 6:4 for MOA to DAT, indicating that the molar ratio of chloroauric acid to mixed thiol ligands (1:2, 1:4, 1:8) has no significant effect on the reaction process and products.

[0069] Example 3

[0070] The steps are the same as in Example 1, except that the ratio of MOA to DAT is fixed at 6:4, and the pH value is adjusted to 8, 10 or 12.

[0071] Experimental verification shows that the ultraviolet absorption spectra of the three groups of near-infrared absorbing gold nanoclusters prepared in Example 3 are all consistent with... Figure 1 The graph shows a molar ratio of 6:4 for MOA to DAT, indicating that pH adjustment values ​​(8, 10, 12) have no significant effect on the reaction process and products.

[0072] Example 4

[0073] The steps are the same as in Example 1, except that the ratio of MOA to DAT is fixed at 6:4, and a 15mL 30kDa ultrafiltration tube is selected.

[0074] Experimental verification shows that the ultraviolet absorption spectrum of the near-infrared absorbing gold nanoclusters prepared in Example 4 is consistent with... Figure 1 The image shows a 6:4 molar ratio of MOA to DAT, indicating that a 15 mL 10 kDa or 30 kDa ultrafiltration tube was used during the purification stage for Au. 60 The purification effect of NCs was not substantially affected.

[0075] Example 5

[0076] This invention prepares near-infrared absorbing gold nanoclusters Au 42 The implementation scheme for NCs (absorption peak at 785nm) is as follows:

[0077] One-pot synthesis of Au with enhanced 785nm absorption 42 For NCs, at room temperature, take 5.2 mL of 1 mM (0.0052 mmol) chloroauric acid (HAuCl4) aqueous solution and place it into 10 mL EP tubes. Add a mixed solution of thiol ligands composed of 6-mercaptohexanoic acid (MHA) and DAT to each tube, adjusting the molar ratio of MHA to DAT to 5:5, 6:4, 7:3, 8:2, or 9:1, and the molar ratio of chloroauric acid to the mixed thiol ligand MHA+DAT to 1:5. After thorough mixing, add sodium hydroxide solution to adjust the pH of the system to 10 (the solution immediately changes from turbid to colorless and transparent). Then add a reducing agent (choose 200 μL of freshly prepared NaBH4 solution of 114 mM dissolved in 0.2 M sodium hydroxide solution, or purge carbon monoxide gas for 5 min). Stir the reaction at 25 °C and monitor the characteristic absorption peak of the solution in real time until the characteristic absorption peak stabilizes and remains unchanged, then terminate the reaction. The remaining operations are the same as for Au. 60 The post-processing steps of NCs synthesis (transferring the reaction solution to a 15 mL 10 kDa ultrafiltration tube, centrifuging at 8000 rpm for 15 min each time, and washing with deionized water 6 times to remove unreacted substrate) ultimately yielded near-infrared absorbing gold nanoclusters Au. 42 NCs should be stored at 4°C for later use.

[0078] Experimental results show that when the molar ratio of MHA to DAT is 7:3, the characteristic absorption peak intensity of the product at 785 nm reaches its highest level. Under other ratios, although the characteristic absorption peak at 785 nm is still detectable, its intensity is significantly weakened due to the formation of impurities in the reaction system. In conclusion, a molar ratio of MHA to DAT of 7:3 is the optimal ratio for synthesizing high-purity, high-intensity near-infrared absorbing gold nanoclusters (Au). 42 The optimal ratio of NCs.

[0079] Figure 2The ultraviolet absorption spectra of near-infrared gold nanoclusters prepared with the molar ratio of MHA to DAT in the thiol ligand set to 5:5, 6:4, 7:3, 8:2 and 9:1, respectively, show that the absorption peak intensity of the synthesized AuNCs is highest at 785 nm only when the molar ratio of the two is 7:3.

[0080] Example 6

[0081] The steps are the same as in Example 5, except that the molar ratio of MHA to DAT is fixed at 7:3, and the molar ratio of chloroauric acid to the mixed thiol ligand MHA+DAT is adjusted to 1:2, 1:5, or 1:8.

[0082] Experimental verification shows that the ultraviolet absorption spectra of the three groups of near-infrared absorbing gold nanoclusters prepared in Example 6 are all consistent with... Figure 2 The graph shows a molar ratio of MHA to DAT of 7:3, which is similar to the graph, indicating that the molar ratio of chloroauric acid to mixed thiol ligands (1:2, 1:5, 1:8) has no significant effect on the reaction process and products.

[0083] Example 7

[0084] The steps are the same as in Example 5, except that the ratio of MHA to DAT is fixed at 7:3, and the pH value is adjusted to 8, 10 or 12.

[0085] Experimental verification shows that the ultraviolet absorption spectra of the three groups of near-infrared absorbing gold nanoclusters prepared in Example 7 are all consistent with... Figure 2 The graph shows a molar ratio of MHA to DAT of 7:3, which is similar to the graph, indicating that the pH adjustment values ​​(8, 10, 12) of the system have no significant effect on the reaction process and products.

[0086] Example 8

[0087] The steps are the same as in Example 5, except that the ratio of MHA to DAT is fixed at 7:3, and a 15mL 30kDa ultrafiltration tube is selected.

[0088] Experimental verification shows that the ultraviolet absorption spectrum of the near-infrared absorbing gold nanoclusters prepared in Example 8 is consistent with... Figure 2 The image shows a 7:3 molar ratio of MHA to DAT, indicating that a 15 mL 10 kDa or 30 kDa ultrafiltration tube was used during the purification stage for Au. 60 The purification effect of NCs was not substantially affected.

[0089] The two gold nanoclusters Au prepared in Examples 1 and 5 with the highest near-infrared absorption peaks were examined. 60 NCs (molar ratio of thiol ligand MOA to DAT = 6:4) (absorption peak at 1090 nm) and Au 42The fluorescence emission spectrum and ultraviolet absorption spectrum of NCs (molar ratio of mercapto ligand MHA to DAT = 7:3) (absorption peak 785 nm) and their morphology were characterized.

[0090] Figure 3 Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 The UV absorption spectra of NCs (with a molar ratio of thiol ligands MHA and DAT of 7:3). Specifically, near-infrared absorbing gold nanoclusters with a strong near-infrared absorption peak at 1090 nm were synthesized in aqueous solution using MOA and DAT as thiol surface ligands and NaBH4 as a reducing agent; near-infrared absorbing gold nanoclusters with a strong near-infrared absorption at 785 nm were synthesized using MHA and DAT as thiol surface ligands.

[0091] Figure 4 Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 Fluorescence emission spectra of NCs (molar ratio of thiol ligand MHA to DAT = 7:3). The results show that both near-infrared absorbing gold nanoclusters exhibit specific fluorescence emission in the near-infrared II region (NIR-II). 60 NCs: 1115nm; Au 42 NCs: 1025nm).

[0092] Figure 5 Au 60 (MOA) 14-20 (DAT) 30-24 Electrospray ionization mass spectra (ESI-MS) of (molar ratio of mercapto ligand MOA to DAT = 6:4), magnified views of the 7-, 6-, and 5- charge peaks, respectively. ESI-MS analysis results show that the mass spectral signals are similar to [Au]... 60 (MOA) x (DAT) y ] 7- 、[Au 60 (MOA) x (DAT) y ] 6- and [Au] 60 (MOA) x (DAT) y ] 5- The three ion species showed high agreement, all exhibiting peaks corresponding to x, y = (14, 30), (15, 29), (16, 28), (17, 27), (18, 26), (19, 15), and (20, 14), thus confirming that the prepared near-infrared absorbing gold nanoclusters were Au.60 NCs.

[0093] Figure 6 Au 42 (MHA) 11-16 (DAT) 21-16 Electrospray ionization mass spectra (ESI-MS) of (molecular ratio of mercapto ligand MHA to DAT = 7:3), magnified views of the 5+ and 4+ charge peaks, respectively. ESI-MS analysis shows that the mass spectral signal is related to [Au] 42 (MHA) x (DAT) y ] 5+ and [Au] 42 (MHA) x (DAT) y ] 4+ The two ion species were highly matched, both showing peaks corresponding to x, y = (11, 21), (12, 20), (13, 19), (14, 18), (15, 17), and (16, 16), thus confirming that the prepared gold nanoclusters were Au. 42 NCs.

[0094] Figure 7 Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 Transmission electron microscopy (TEM) images of NCs (molar ratio of thiol ligand MHA to DAT = 7:3) at the 20 nm scale and particle size statistical analysis. Au 60 The average particle size of NCs was 2.2 ± 0.3 nm, and Au 42 The average particle size of NCs is 1.8 ± 0.2 nm, indicating that both near-infrared absorbing gold nanoclusters have ultra-small core sizes.

[0095] Figure 8 Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 Statistical analysis of hydrated particle size of NCs (molar ratio of thiol ligand MHA to DAT = 7:3). Au 60 The average hydrated particle size of NCs was 3.5 ± 0.5 nm, and Au 42 The average hydrated particle size of the NCs was 3.0 ± 0.3 nm. Both are smaller than the renal filtration threshold (approximately 5.5 nm), indicating their good potential for kidney-related applications.

[0096] Figure 9 For different concentrations of Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au42 Results of in vitro photoacoustic and fluorescence imaging performance determination of NCs (molar ratio of thiol ligand MHA to DAT = 7:3). As Au 60 Preliminary studies on the application of photoacoustic imaging in vivo with NCs included studies on a series of concentrations (0–500 μM) of Au. 60 Fluorescence and photoacoustic signals at 1064 nm were measured in aqueous solutions of NCs. The results showed a linear relationship between signal enhancement and concentration increase, indicating that they can be used for in vivo photoacoustic / fluorescence imaging. Similarly, a series of concentrations (0~500 μM) of Au were also tested. 42 Fluorescence and 808nm photoacoustic signal were measured in aqueous solution of NCs, and the signal enhancement was linearly related to the increase in concentration.

[0097] In the following examples, near-infrared absorbing gold nanoclusters Au were used. 60 NCs and Au 42 NCs is Au 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 NCs (molar ratio of thiol ligand MHA to DAT = 7:3).

[0098] Example 9

[0099] Two types of near-infrared absorbing gold nanoclusters (Au) prepared in this invention 60 NCs (molar ratio of mercapto ligand MOA to DAT = 6:4) and Au 42 The renal targeting ability implementation scheme and renal tissue sub-organ distribution characteristics of NCs (with a molar ratio of thiol ligand MHA to DAT of 7:3) are investigated as follows:

[0100] Six-week-old male C57BL / 6J mice were selected. Hair was removed before imaging. An NIR-II imaging system equipped with a deeply cooled InGaAs camera (ZephIR 1.7, 640×512 pixels) and a 970nm long-pass filter (Edmund Optics) was used, with an 808nm laser as the excitation source. Au... 60 NCs (300 μL, 400 μM) and Au 42 After intravenous injection of NCs (300 μL, 400 μM) into mice, fluorescence signals were collected at 0.5, 1, 6, and 12 hours post-injection. The biodistribution characteristics of the two probes were evaluated by fluorescence imaging, and the fluorescence signal values ​​at different time points were statistically analyzed using PhySpecV2 image processing software. Simultaneously, representative images of the two near-infrared absorbing gold nanoclusters were obtained 1 hour after injection by silver-enhanced staining and H&E staining of kidney tissue to clarify their specific suborganizational distribution within the kidney.

[0101] Figure 10 The near-infrared absorbing gold nanoclusters Au in Example 9 60 NCs and Au 42 In vivo fluorescence imaging of NCs in mice, normal mice injected with Au 60 NCs and Au 42 In vivo fluorescence imaging results after NCs showed that, by evaluating the in vivo biodistribution characteristics of the two probes through fluorescence imaging, both showed significant fluorescence signals in the kidneys 0.5 hours after injection, and synchronous accumulation was detected in the bladder, indicating that the two near-infrared absorbing gold nanoclusters are mainly cleared through the renal pathway.

[0102] Figure 11 In Example 9, Au was injected intravenously. 60 NCs and Au 42 Images of kidney tissue sections stained with silver enhancement and hematoxylin and eosin (H&E) after injection of NCs. The images show that one hour after injection (300 μL, 400 μM), both near-infrared absorbing gold nanoclusters were uniformly distributed along the surface of the renal tubules, with no aggregation detected in the glomeruli (glomeruli are marked with dashed boxes). This selective distribution enables specific imaging of the renal tubules without interfering with the visualization of the glomeruli and the surrounding capillaries.

[0103] Example 10

[0104] The present invention constructs a dual-wavelength photoacoustic microscopy imaging system at 532nm / 808nm and 532nm / 1064nm, and Au 42 NCs and Au 60 The imaging analysis and renal tubular photoacoustic imaging depth quantization scheme for NCs probes is as follows:

[0105] To achieve high-resolution visualization of renal microstructures (blood vessels and renal tubules), a dual-focus photoacoustic depth microscopy system was constructed, incorporating a dual-wavelength combination of 532nm / 808nm and 532nm / 1064nm. The 532nm channel was used to capture the intrinsic signal of hemoglobin within the blood vessels, while the 808nm / 1064nm channel was used to capture the extrinsic signal from the probe. This system was used to visualize intravenously injected Au... 42 NCs and Au 60 Maximum projection imaging was performed on mouse kidneys 1 hour after NCs probe insertion, and Au was also analyzed to determine the level of Au. 42 NCs (808nm) and Au 60 The photoacoustic (PA) imaging depth difference of renal tubules by NCs (1064nm) was first reconstructed in three dimensions by photoacoustic reconstruction of renal tubules located by two probes, and the area to be measured was delineated. Then, the depth was accurately quantified by magnification imaging.

[0106] Figure 12This is a schematic diagram of the setup of the dual-wavelength photoacoustic microscope (PAM) in Example 10. The device is a dual-focus photoacoustic deep microscopy imaging system that includes a dual-wavelength combination of 532nm / 808nm and 532nm / 1064nm. It can emit laser pulses with two different wavelength combinations to achieve precise capture and imaging of the corresponding signals.

[0107] Figure 13 In Example 10, Au was injected intravenously. 60 NCs (1064nm channel), Au 42 The results of maximum intensity projection (MAP) analysis of PAM images after NCs (808nm channel) showed that the Pearson correlation coefficients between the two and the endogenous hemoglobin signal (532nm channel) were 0.01 and -0.01, respectively, with almost no overlap and surrounding each other, indicating that both probes can clearly distinguish between renal tubules and peritubular capillaries.

[0108] Figure 14 The results of three-dimensional reconstruction of renal tubules using dual-wavelength channel PAM based on two near-infrared absorbing gold nanoclusters (AuNCs) in Example 10 are as follows: Au 60 The renal tubular imaging depth of NCs (1064nm channels) is approximately 405μm, and strong signals are still maintained at a depth of 500μm. 42 NCs (808nm channel) have an imaging depth of only about 208μm, and the signal attenuates significantly with depth, therefore Au 60 NCs offer superior imaging depth and stable spatial resolution, making them more suitable for high-precision three-dimensional reconstruction of renal tubules.

[0109] Example 11

[0110] This invention is based on Au 60 The implementation plan for high-precision three-dimensional reconstruction of normal renal cortex using NCs' PAM technology is as follows:

[0111] To achieve Au-based 60 High-precision 3D reconstruction of the renal cortex using PAM in NCs. In the glomerular visualization, the renal vascular network signal acquired by the 532nm channel was reconstructed in 3D using Imaris software. The glomerular signal and the dense peritubular capillary network signal were manually separated by segmenting the images at different depths, and the complete signal of a single glomerulus was extracted.

[0112] Simultaneously, by combining renal tubular data acquired through the 1064nm channel with separate three-dimensional imaging results of peritubular capillaries, glomeruli, and renal tubules, three-dimensional visualization and volume rendering were performed. The diameters of the three components were measured using the software, successfully achieving an integrated and precise presentation of the microscale structure of the renal cortex, providing high-resolution imaging support for research related to renal physiology and pathology.

[0113] Figure 15 The results of three-dimensional reconstruction of the renal vascular network and layer-by-layer segmentation with a layer thickness of 50 μm, as described in Example 11, show that glomeruli with a diameter of 70-90 μm were successfully observed in the deep vascular layer about 120 μm from the kidney surface, and the glomerular signals were separated from the dense peritubular capillary network by manual differentiation.

[0114] Figure 16 The images show the three-dimensional visualization results of the dense peritubular capillary network (pink), extracted glomeruli, and magnified views of a single reconstructed glomeruli reconstructed under the 532nm channel in Example 11, the renal tubules (green) reconstructed under the 1064nm channel, and the fusion of the three three-dimensional visualizations. The images clearly show that the peritubular capillaries are distributed in a ring-like shape, tightly surrounding the renal tubules.

[0115] Figure 17 The three-dimensional visualization results of the reconstructed renal cortex in Example 11 are shown, as well as the volume rendering map obtained by continuous volume rendering of the glomeruli, renal tubules and peritubular capillaries.

[0116] Figure 18 The results of measuring the diameters of blood vessels, renal tubules, and glomeruli using Imaris software in Example 11 show that their diameters were 14.7±2.7μm, 23.6±3.1μm, and 72.8±5.9μm, respectively. These data are highly consistent with existing literature reports and accurately reveal the morphological characteristics of the microscale structure of the kidney.

[0117] Figure 19 The gold nanoclusters Au based on near-infrared absorption in Example 11 60 A magnified multi-angle view of the PAM 3D reconstructed renal cortex image of NCs. The normal renal cortex is 412 μm thick, and most glomeruli are located at a depth of about 150 μm or more on the surface of the normal kidney, embedded in the complex network of renal tubules and peritubular capillaries.

[0118] Example 12

[0119] Identifying different stages of type 2 diabetic nephropathy (T2DKD) using high-precision three-dimensional reconstruction technology of the renal cortex:

[0120] This study established a type 2 kidney disease (T2DKD) model by combining a high-fat diet with intraperitoneal injection of low-dose streptozotocin (STZ). Six-week-old male C57BL / 6J mice were selected and housed in a normal light-dark cycle environment with free access to food and water, fed a high-fat diet for 5 consecutive weeks. Subsequently, the mice were intraperitoneally injected with STZ at a dose of 40 mg / kg for one week. Dynamic monitoring of mouse weight, blood glucose, serum creatinine, and blood urea nitrogen was conducted, and PAS-stained kidney sections confirmed the successful establishment of T2DKD models at different pathological stages. (Based on Au...) 60The dual-wavelength channel PAM technology of NCs can dynamically monitor the overall changes in the renal cortex and realize the multi-indicator diagnosis of early T2DKD. It provides comprehensive pathological indicators covering peritubular capillary density, glomerular volume and position, renal tubular density and renal cortical thickness, providing support for T2DKD staging. The key pathological changes related to T2DKD (such as glomerular hypertrophy, abnormal glomerular position, renal tubular sparseness and renal cortical thinning) appeared as early as 2 weeks after the model was established.

[0121] Figure 20 The results of physiological index assessment for mice in the T2DKD group in Example 12 include body weight, fasting blood glucose, serum creatinine (CREA), and blood urea nitrogen (BUN). Experimental design related to T2DKD model staging and kidney injury assessment: After intraperitoneal injection of STZ, fasting blood glucose levels were continuously monitored in mice. The day when hyperglycemia was first detected (>11.1 mmol / L) was defined as day 0. Mice were divided into T2DKD-2, T2DKD-4, T2DKD-6, T2DKD-8, T2DKD-12, and T2DKD-16 (corresponding to weeks 2, 4, 6, 8, 12, and 16 after hyperglycemia, respectively). The severity of kidney injury was assessed by measuring blood biochemical indicators at each stage.

[0122] Figure 21 These are representative PAS staining images of glomeruli and tubules in T2DKD mice at different stages of disease progression. Compared with normal mice, T2DKD mice exhibit glomerular hypertrophy, and the proportion of tubular damage gradually increases with disease progression. Tubular vacuolation (diamond-shaped marking) and tubular dilation (star-shaped marking) can be observed in the diseased kidneys.

[0123] Figure 22 The results of three-dimensional reconstruction of the mouse renal cortex during the T2DKD process based on PAM in Example 12 demonstrate the early pathological features and quantitative changes at each stage of the disease: In T2DKD-2 stage, areas with missing photoacoustic signals in the renal tubules are clearly observable, and this phenomenon gradually worsens with disease progression; compared to normal kidneys, the kidneys in T2DKD-2 stage exhibit pathological features such as sparse renal tubules and enlarged glomeruli, and these changes continue to deteriorate with disease progression. Statistical quantitative results show that the glomerular volume distribution in T2DKD mice becomes increasingly dispersed with disease progression, with the proportion of abnormal glomeruli increasing from 2.22% in T2DKD-2 stage to 25.42% in T2DKD-16 stage. This change is attributed to the glomerular hyperfiltration state and compensatory hypertrophy of residual nephrons during DKD progression. Meanwhile, the renal tubular density in T2DKD mice was significantly reduced as early as 2 weeks (18.12±0.96% in T2DKD-2 stage), and gradually decreased to 10.42±1.18% in T2DKD-16 stage (20.26±0.93% in normal mice) as the disease progressed. This phenomenon is related to abnormal renal tubular function or atrophy during disease progression, which in turn disrupts the renal filtration pathway of the probe.

[0124] Figure 23 The images show volumetric renderings of three-dimensional renal cortex reconstructions from T2DKD mice at different disease progression stages in Example 12. Significant differences exist between the three-dimensional renal cortex renderings of T2DKD mice and normal mice. Multiple views show that, compared to normal kidneys, at 2 weeks of age in T2DKD, the frontal view shows sparse renal tubules, the lateral view shows thinning of the renal cortex, and the posterior view shows exposed glomeruli.

[0125] Figure 24 For the quantitative comparison and consistency verification of T2DKD damage progression by PAS staining and PAM in Example 12: the percentage of glomerular and tubular damage measured by both gradually increased with the course of T2DKD, and the upward trend at different stages was highly consistent, confirming that PAM can accurately identify different stages of T2DKD progression.

[0126] Example 13

[0127] This invention identifies different stages of type 1 diabetic nephropathy (T1DKD) using high-precision three-dimensional reconstruction technology of the renal cortex:

[0128] This study established a type 1 diabetic nephropathy (T1DKD) model in 6-week-old male C57BL / 6J mice fed under normal light-dark cycles with free access to food and water. The initial injection dose was 75 mg / kg, followed by a second injection of 150 mg / kg five days later. During model establishment, key indicators such as blood glucose and body weight were dynamically monitored. The characteristic manifestation of persistently elevated blood glucose levels confirmed the successful establishment of the T1DKD model. (Based on Au...) 60 The dual-wavelength channel PAM technology of NCs can dynamically monitor changes in the overall morphology and function of the renal cortex, enabling accurate diagnosis of early T1DKD with multiple indicators. It provides comprehensive pathological quantitative indicators covering peritubular capillary density, glomerular volume and spatial location, renal tubular density, and renal cortical thickness, providing reliable support for the staging and progression assessment of T1DKD. Furthermore, glomerular hypertrophy in T1DKD is clearly visible as early as one week after the model is established, providing important pathological monitoring evidence for early intervention research of T1DKD.

[0129] Figure 25The results of physiological index assessment for mice in the T1DKD group in Example 13 include body weight, fasting blood glucose, creatinine (CREA), and blood urea nitrogen (BUN). Experimental design related to T1DKD model staging and kidney injury assessment: After intravenous injection of STZ, fasting blood glucose levels were continuously monitored in mice. The day when hyperglycemia was first detected (>11.1 mmol / L) was defined as day 0. Mice were divided into T1DKD-1, T1DKD-2, T1DKD-4, T1DKD-6, T1DKD-8, and T1DKD12 (corresponding to weeks 1, 2, 4, 6, 8, and 12 after hyperglycemia, respectively) based on the duration of hyperglycemia. The severity of kidney injury was assessed by measuring blood biochemical indicators at each stage.

[0130] Figure 26 These are representative PAS-stained images of glomeruli and tubules in T1DKD mice at different disease progression stages in Example 13. Compared with normal mice, T1DKD mice exhibit glomerular hypertrophy, and the proportion of tubular damage gradually increases with disease progression. Acute tubular necrosis (triangular marker) and tubular dilation (star marker) are visible in the diseased kidney tissue.

[0131] Figure 27 The results of three-dimensional reconstruction of mouse renal cortex during the T1DKD process (T1DKD-1 to T1DKD-12) based on PAM in Example 13 demonstrate the early pathological features of the disease and the quantitative changes in each stage of the disease: based on Au 60 Dual-wavelength PAM technology was used in NCs to extract and statistically quantify data on glomeruli, peritubular capillaries, and renal tubules. At T1DKD-1, sparse peritubular capillaries and enlarged glomeruli were observed, while tubular density, renal cortical thickness, and glomerular position showed no significant differences compared to normal kidneys. Statistical quantification showed that the normal mouse glomerular volume ranged from 0.5 × 10⁻⁶. 5 –2×10 5 μm 3 The proportion of abnormal glomeruli reached 9.59% in T1DKD-1, rising to 62.21% in T1DKD-12. Peritumoral capillary density was significantly reduced in T1DKD-1 (17.25±0.88%), gradually decreasing from 20.09±1.53% in normal mice to 14.37±1.08% in T1DKD-12. Tubular density remained unchanged in T1DKD-1 but began to decrease in T1DKD-2. The proportion of glomerular and tubular damage quantified by PAM gradually increased with the progression of T1DKD.

[0132] Figure 28The images show volumetric renderings of three-dimensional renal cortex reconstructions from T1DKD mice at different disease progression stages in Example 13. The three-dimensional renderings of the renal cortex from T1DKD mice and normal mice show significant differences. Multiple angle observations reveal that, compared to normal kidneys, the kidneys of T1DKD-2 mice exhibit sparse renal tubules in the frontal view, thinner renal cortex in the side view, and exposed glomeruli in the posterior view.

[0133] Figure 29 To demonstrate the quantitative comparison and consistency verification of the progression of T1DKD damage by PAS staining and PAM in Example 13, the quantitative comparison data of glomerular hypertrophy and renal tubular damage in the progression of T1DKD by PAS staining and PAM are shown: the proportion of glomerular and renal tubular damage quantified by both increases with the course of T1DKD, and the upward trend of each stage is highly consistent, indicating that PAM can accurately distinguish different stages of T1DKD progression.

[0134] Figure 30 This is a flowchart illustrating the application of the near-infrared absorbing gold nanoclusters prepared in this invention to perform three-dimensional reconstruction of multiple components of the mouse renal cortex using photoacoustic microscopy, based on NIR absorption renal clearance Au. 60 NCs' PAM platform enables multi-component three-dimensional reconstruction of the renal cortex: the complex renal cortex consists of nephrons and peritubular capillaries, forming a tightly interwoven network, with each nephron containing a glomerulus and a tubule. This PAM platform integrates a dual-wavelength imaging strategy, using two laser fibers connected to an ultrasound detector to transmit the image through the transilluminated area. 60 The 1064nm signal generated by NCs is combined with the 532nm signal generated by endogenous hemoglobin. This design enables simultaneous visualization of renal vessels (including glomeruli and peritubular capillaries) and renal tubules with high spatial resolution, achieving three-dimensional reconstruction of multiple components of the renal cortex.

[0135] Examples 12 and 13 above illustrate that the near-infrared absorbing gold nanoclusters prepared by this invention, when used in photoacoustic microscopy, enable early dynamic and accurate monitoring of T2DKD and T1DKD through high-resolution three-dimensional visualization and quantitative analysis of kidney microstructure. Based on this platform, a multi-index diagnostic system (covering core parameters such as perivascular capillary density and glomerular volume) can be established, accurately capturing early pathological abnormalities and dynamically tracking disease progression. The detection sensitivity is superior to traditional blood tests, and the results are highly consistent with gold standard tissue sections. This demonstrates the accuracy of high-resolution three-dimensional visualization and quantitative analysis, further illustrating the superior performance of the near-infrared absorbing gold nanoclusters prepared by this invention in photoacoustic microscopy.

[0136] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0137] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for preparing near-infrared absorbing gold nanoclusters, characterized in that, The near-infrared absorbing gold nanoclusters are Au 60 NCs; the Au 60 The preparation of NCs includes the following steps: (1) The thiol ligand 8-mercaptooctanoic acid (MOA), 2-(diethylamino)ethanethiol (DAT), chloroauric acid, and solvent are thoroughly stirred and mixed evenly; the molar ratio of chloroauric acid to the mixed thiol ligand MOA+DAT is 1:2 to 1:8; the molar ratio of MOA to DAT is 5:5 to 9:

1. (2) Adjust the pH of the well-mixed solution to alkaline, add a reducing agent and continue stirring. Monitor the characteristic absorption peak of the solution. When the characteristic absorption peak is stable, remove excess thiol ligands by ultrafiltration to obtain Au with near-infrared absorption. 60 NCs; Au 60 The absorption peak of NCs is 1090 nm. They have extremely strong absorption in the near-infrared region of 700~1700 nm, exhibit low absorption characteristics at 532 nm, and are all water-soluble.

2. A method for preparing near-infrared absorbing gold nanoclusters, characterized in that, The near-infrared absorbing gold nanoclusters are Au 42 NCs; the Au 42 The preparation of NCs includes the following steps: (1) The thiol ligand 6-mercaptohexanoic acid (MHA), 2-(diethylamino)ethanethiol (DAT), chloroauric acid, and solvent are thoroughly stirred and homogenized; the molar ratio of chloroauric acid to the mixed thiol ligand MHA+DAT is 1:2 to 1:8; the molar ratio of MHA to DAT is 5:5 to 9:

1. (2) Adjust the pH of the well-mixed solution to alkaline, add a reducing agent and continue stirring. Monitor the characteristic absorption peak of the solution. When the characteristic absorption peak is stable, remove excess thiol ligands by ultrafiltration to obtain Au with near-infrared absorption. 42 NCs; Au 42 The absorption peak of NCs is 1090 nm. They have extremely strong absorption in the near-infrared region of 700~1700 nm, exhibit low absorption characteristics at 532 nm, and are all water-soluble.

3. The method according to any one of claims 1 or 2, characterized in that, The solvent is either deionized water or methanol; the reducing agent is sodium borohydride or carbon monoxide.

4. The method according to any one of claims 1 or 2, characterized in that, The pH is adjusted to 8-12; the ultrafiltration purification uses an ultrafiltration tube with a rejection capacity of 10000-30000 Da.

5. Near-infrared absorbing gold nanoclusters prepared by the method of any one of claims 1 or 2.

6. The application of the near-infrared absorbing gold nanoclusters as described in claim 5 in photoacoustic microscopy.

7. The application according to claim 6, characterized in that, Au 60 NCs are excited using a dual-wavelength method of 532nm / 1064nm, and the Au... 42 NCs are excited using dual wavelengths of 532nm / 808nm.

8. The application according to claim 7, characterized in that, High-resolution 3D visualization and quantitative analysis of kidney microstructure for non-disease diagnosis, based on Au 60 The dual-wavelength photoacoustic microscopy system of NCs acquires raw images at 532nm and 1064nm and performs three-dimensional reconstruction. The imaging information of the peritubular capillaries of the glomerulus and renal tubules is accurately extracted in the 532nm channel, and the targeted imaging image of the renal tubules is clearly obtained in the 1064nm channel.

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