光介导技术具有操作简便、远程可控、灵敏度高、时空分辨率高等优点，是生物学研究和临床实践的重要工具。然而，组织内吸收/散射、自身荧光和光辐射下的光损伤问题是荧光成像需要考虑的问题。为此，被认为是生物透明窗口的近红外区域（NIR，700-1700 nm）受到越来越多的关注。近红外光具有可忽略的组织散射/吸收、最小的光损伤和无自体荧光干扰等优点，非常适合于生物应用。近红外染料和稀土掺杂纳米颗粒（RENPs）作为具有代表性的近红外发光探针，由于其独特的光学性质和较低的生物毒性，引起了人们越来越多的兴趣。设计基于NIR染料/RENPs纳米复合材料的发光探针，不仅可以综合两者的优点，还可以通过调节内部能量传递途径来实现额外的功能。本文主要研究了近红外有机染料、稀土纳米颗粒以及其复合材料的发光性能优化和能量传递机制，并将其应用于生物分子的检测和肿瘤组织及血管成像。

一、合成了具有多个结合位点的有机荧光探针，该荧光探针与生物硫醇发生取代、环化、分子内重排等不同反应生成不同的多元环产物来区别半胱氨酸和谷胱甘肽。本文测试了探针检测半胱氨酸和谷胱甘肽的紫外-可见光谱和荧光发射光谱，发现加入生物硫醇后光谱会出现有规律的变化趋势。此外，研究发现，探针在808 nm激光激发下在900 nm出现拖尾荧光，这使其具有在近红外二区成像的潜力。本研究使用功能性磷脂（DSPE-PEG）包覆和四苯基乙烯（TPE）修饰的方法解决了探针在水相中强淬灭的问题，使其在水相中具有良好的生物相容性和稳定性，从而使得探针可以检测活体细胞中的生物硫醇，并且具有在近红外二区成像的潜力。接着，选择c-Met和VEGF作为探针递送靶点，通过复合贝伐单抗（Bevacizumab）和卡马替尼（Capmatinib）两种抗体实现探针对头颈癌肿瘤组织的靶向成像。

二、制备了尺寸为~70 nm的NaYGdF₄:Yb,Er纳米晶，通过改变Gd³⁺, Yb³⁺, Er³⁺的掺杂比例优化了稀土纳米粒子的发光性能，得到了一个发光性能优越的稀土探针。由于有机染料的发射光谱和稀土纳米粒子的吸收光谱有良好的重叠，并且发现该染料在水相中具有良好的稳定性，这种具有较大吸收截面的有机染料可以作为天线吸收更多光子传递给稀土离子，从而增强了稀土纳米粒子的NIR发光。本文通过测试染料/稀土粒子复合材料的紫外吸收光谱、发射光谱、FTIR谱和尺寸证明了染料和稀土粒子的成功结合。在此基础上，进一步探究了染料的浓度对染料敏化稀土纳米粒子荧光强度的影响。选择两亲性聚合物DSPE-PEG和聚丙烯酸（PAA）赋予染料和稀土纳米粒子良好的水溶性和稳定性。测试了优化的水相染料敏化纳米晶的细胞毒性，结果显示不同浓度的探针与细胞共孵育24 h后均保持80%以上的存活率，表明复合材料具有良好的生物相容性。将探针通过小鼠尾静脉注射进行血管成像，30 s后，小鼠后肢血管被清晰地描绘出来。随后在1 min时，小鼠背部血管逐渐显影，并在2 min左右荧光达到最强。这种染料敏化的稀土纳米颗粒能够描绘器官血管和肿瘤血管并富集较长时间，为血管疾病和肿瘤的监测提供了有力工具。

With the advantages of easy operation, remote control, high sensitivity, and high spatial and temporal resolution, light-mediated techniques are important tools for biological research and clinical practice. However, the issues of intra-tissue absorption/scattering, auto-fluorescence and photodamage under light radiation are issues to be considered for fluorescence imaging. For this reason, the near-infrared region (NIR, 700-1700 nm), which is considered to be a biologically transparent window, has received increasing attention. With the advantages of negligible tissue scattering/absorption, minimal photodamage and no autofluorescence interference, NIR light is well suited for biological applications. NIR dyes and rare earth-doped nanoparticles (RENPs) have attracted increasing interest as representative near-infrared luminescent probes due to their unique optical properties and low biotoxicity. Designing luminescent probes based on NIR dye/ RENPs nanocomposites not only can combine the advantages of both, but also can achieve additional functions by modulating the internal energy transfer pathway. In this paper, we focus on the optimization of luminescence properties and energy transfer mechanisms of NIR organic dyes, rare earth nanoparticles and their composites, and apply them to the detection of biomolecules and imaging of tumor tissues and blood vessels.

 

First, an organic fluorescent probe with multiple binding sites was synthesized. The fluorescent probes undergo different reactions with biothiols such as substitution, cyclization and intramolecular rearrangement to generate different polycyclic products to distinguish between cysteine and glutathione. In this paper, we tested the UV-Vis and fluorescence emission spectra of the probe for the detection of cysteine and glutathione and found that the spectra showed a regular trend with the addition of biothiols. In addition, it was found that the probe showed trailing fluorescence at 900 nm under 808 nm laser excitation, which gives it the potential for imaging in the near-infrared second region. In this study, the problem of strong quenching of the probe in the aqueous phase was solved using functional phospholipid (DSPE-PEG) coating and tetraphenylethylene (TPE) modification. The probe has good stability and biocompatibility in the aqueous phase, which allows the probe to detect biothiols in living cells and has the potential for in vivo imaging in the near-infrared second region. Next, c-Met and VEGF were selected as the probe delivery targets, and targeted imaging of head and neck cancer tumor tissue by the probe was achieved by combining two antibodies, Bevacizumab and Capmatinib.

 

Then, NaYGdF₄: Yb,Er nanocrystals with the size of ~70 nm were prepared, and the luminescence properties of the rare-earth nanoparticles were optimized by changing the doping ratios of Gd³⁺, Yb³⁺, and Er³⁺. Due to the good overlap between the emission spectra of the organic dye and the absorption spectra of the rare-earth nanoparticles, this organic dye with a large absorption cross-section can act as an antenna to absorb more photons passed to the rare-earth ions enhancing the NIR luminescence of the rare-earth nanoparticles. In this paper, the successful combination of dye and rare-earth particles is demonstrated by testing the UV absorption spectra, emission spectra, FTIR spectra and dimensions of the dye/rare-earth particle composites. Based on this, the effect of dye concentration on the fluorescence intensity of dye-sensitized nanocrystals was further investigated. The amphiphilic polymers DSPE-PEG and polyacrylic acid (PAA) were chosen to impart good water solubility and stability to the dye and rare-earth nanoparticles. The cytotoxicity of the optimized aqueous phase dye-sensitized nanocrystals was tested, and the results showed that multiple concentration gradients of the probes maintained more than 80% cell viability after 24 h of incubation with cells, indicating that the composites have good biocompatibility. The probe was injected into the venous vessels of the mice and the fluorescence images of the vessels were recorded with an NIR small animal imager. after 30 s, the blood vessels in the hind limbs of mice were clearly depicted. Subsequently, at 1 min, the blood vessels on the back of the mice were gradually visualized, and the fluorescence reached its maximum at around 2 min. This dye-sensitized rare earth nanoparticle is able to depict organ vessels and tumor vessels and enrich them for a long time, providing a powerful tool for monitoring vascular diseases and tumors.

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