基于迈克尔加成反应的沙门氏菌纳米荧光标记研究

本文通过迈克尔加成反应的荧光标记法,成功构建了纳米荧光探针标记的沙门氏菌(YB1-INPs)。采用三(2-羧乙基)膦将沙门氏菌YB1外膜蛋白表面上的二硫键温和还原为巯基后,与吲哚菁绿(ICG)@纳米探针(INPs)的马来酰亚胺基团(Mal)交联形成YB1-INPs。通过扫描电镜和共聚焦荧光成像对YB1-INPs的形貌、光学性质进行表征。通过活死细菌染色和LB琼脂平板实验对YB1-INPs的活性进行考察。结果表明,INPs成功标记到沙门氏菌YB1表面后对YB1的形态、光学性质、活性及生长均没有产生影响,并且能够用于沙门氏菌YB1体内靶向的荧光分布成像。本研究提供的这种简单、安全、高效的荧光标记方法能够将纳米材料标记到生物载体上,可以应用于生物载体的药物输送和体内监测。     

Multifunctional nanoprobe for cancer cell targeting and simultaneous fluorescence/magnetic resonance imaging

Multifunctional nanoprobes with distinctive magnetic and fluorescent properties are highly useful in accurate and early cancer diagnosis. In this study, nanoparticles of Fe₃O₄ core with fluorescent SiO₂ shell (MFS) are synthesized by a facile improved Stöber method. These nanoparticles owning a significant core-shell structure exhibit good dispersion, stable fluorescence, low cytotoxicity and excellent biocompatibility. TLS11a aptamer (Apt1), a specific membrane protein for human liver cancer cells which could be internalized into cells, is conjugated to the MFS nanoparticles through the formation of amide bond working as a target-specific moiety. The attached TLS11a aptamers on nanoparticles are very stable and can't be hydrolyzed by DNA hydrolytic enzyme in vivo. Both fluorescence and magnetic resonance imaging show significant uptake of aptamer conjugated nanoprobe by HepG2 cells compared to 4T1, SGC-7901 and MCF-7 cells. In addition, with the increasing concentration of the nanoprobe, T₂-weighted MRI images of the as-treated HepG2 cells are significantly negatively enhanced, indicating that a high magnetic field gradient is generated by MFS-Apt1 which has been specifically captured by HepG2 cells. The relaxivity of nanoprobe is calculated to be 11.5 mg⁻¹s⁻¹. The MR imaging of tumor-bearing nude mouse is also confirmed. The proposed multifunctional nanoprobe with the size of sub-100 nm has the potential to provide real-time imaging in early liver cancer cell diagnosis.     

3,4-Dinitrobenzamide Functionalized CdTe/ZnTe Quantum Dots as a Nanoprobe for Imaging Glutathione S-Transferase in Living Cells

A novel fluorescent nanoprobe for glutathione S‐transferase (GST) has been developed by incorporating 3,4‐dinitrobenzamide (a specific substrate of GST) onto CdTe/ZnTe quantum dots. The probe itself displays a low background signal due to the strong quenching effect of the electron‐withdrawing unit of 3,4‐dinitrobenzamide on the quantum dots. However, GST can efficiently catalyze the nucleophilic substitution of reduced glutathione on the p‐nitro group of the nanoprobe, leading to a large fluorescence enhancement. Most notably, this enhancement shows high selectivity and sensitivity towards GST instead of the other biological substances. With this nanoprobe, a simple fluorescence imaging method for intracellular GST has been established, and its applicability has been successfully demonstrated for imaging GST in different living cells, which reveals that A549 cells express GST about 3 times higher than NIH‐3T3 and Hela cells.     

Anti-Her-2 monoclonal antibody conjugated polymer fluorescent nanoparticles probe for ovarian cancer imaging

Fluorescent nanoparticles (FNPs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. A novel kind of polymer fluorescent nanoparticles (PFNPs) was synthesized and its application for ovarian cancer imaging with fluorescence microscopy imaging technology was presented in this study. The PFNPs were synthesized with precipitation polymerization by using methacrylic acid (MAA) as monomer, trimethylolpropane trimethacrylate (Trim) as cross-linker, azobisisobutyronitrile (AIBN) as radical initiator and butyl rhodamine B (BTRB) as fluorescent dye. And the fluorescent dye was embedded into the three-dimensional network of the polymer when the polymer was produced. With this method the PFNPs can be prepared easily. And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody. The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-Her-2 monoclonal antibody conjugated PFNPs can effectively recognize ovarian cancer cells and exhibit good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of ovarian cancer cells.     

A photoinduced electron transfer-based nanoprobe as a marker of acidic organelles in mammalian cells

Photoinduced electron transfer (PET)-based molecular probes have been successfully used for the intracellular imaging of the pH of acidic organelles. In this study, we describe the synthesis and characterization of a novel PET-based pH nanoprobe and its biological application for the signaling of acidic organelles in mammalian cells. A fluorescent ligand sensitive to pH via the PET mechanism that incorporates a thiolated moiety was synthesized and used to stabilize gold nanoparticles (2.4?±?0.6 nm), yielding a PET-based nanoprobe. The PET nanoprobe was unambiguously characterized by transmission electron microscopy, proton nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption, and steady-state/time-resolved fluorescence spectroscopies which confirmed the functionalization of the gold nanoparticles with the PET-based ligand. Following a classic PET behavior, the fluorescence emission of the PET-based nanoprobe was quenched in alkaline conditions and enhanced in an acidic environment. The PET-based nanoprobe was used for the intracellular imaging of acidic environments within Chinese hamster ovary cells by confocal laser scanning microscopy. The internalization of the nanoparticles by the cells was confirmed by confocal fluorescence images and also by recording the fluorescence emission spectra of the intracellular PET-based nanoprobe from within the cells. Co-localization experiments using a marker of acidic organelles, LysoTracker Red DND-99, and a marker of autophagosomes, GFP-LC3, confirm that the PET-based nanoprobe acts as marker of acidic organelles and autophagosomes within mammalian cells.

A ratiometric fluorescent core-shell nanoprobe for sensing and imaging of zinc(II) in living cell and zebrafish

A zinc(II)-responsive ratiometric fluorescent core-shell nanoprobe (referred to as QPNPs) is described. It consist of an optimized combination of an internal reference dye (TBAP) encapsulated in the core, and a Zn(II)-specific indicator dye (PEIQ) in the shell. The nanoprobe was synthesized via single-step graft copolymerization induced by tert-butyl hydroperoxide at 80 °C. QPNPs exhibit a well-defined core-shell nanostructure and well-resolved dual emissions after photoexcitation at 380 nm. After exposure to Zn(II), the QPNPs display a green fluorescence peaking at ~500 nm that increases with the concentration of Zn(II), while the pink fluorescence of the porphine-derived reference dye peaking at ~650 nm remains unchanged. This results in color change from pink to green and thus enables Zn(II) to be detected both spectroscopically and with bare eyes. Zn(II) can be quantified with a 3.1 nM detection limit. The core-shell structured nanoprobe was also applied to real-time imaging of Zn(II) in living HeLa cells and in zebrafish. This work establishes a reliable approach to synthesize ratiometric fluorescent nanoprobes. It enables such nanoprobes to be prepared also by those not skilled in nanomaterial synthesis.

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Graphical abstract A zinc(II)-responsive core-shell nanoprobe (referred to as QPNP) is synthesized via single-step graft copolymerization. Zn(II) can be quantitated with a 3.1 nM detection limit by the QPNPs through ratiometric fluorescence strategy (PEIQ as the Zn(II) indicator and TBAP as the reference dye).

     

A highly selective and sensitive nanoprobe for detection and imaging of the superoxide anion radical in living cells

A novel fluorescent nanoprobe with high sensitivity and selectivity for detection and imaging of the superoxide anion radical O(2)(·-) in living cells was designed and synthesized by a simple self-assembly method based on 2-chloro-1,3-dibenzothiazoline-cyclohexene (DBZTC) and Ag@SiO(2) core-shell nanoparticles.     

二氧化硅-共聚物杂化荧光纳米材料用于动物活体成像

以氧化硅(SiO_2)前驱体与三嵌段共聚物F108合成较小粒径的SiO_2-共聚物杂化纳米体系(SNP),并与高效近红外发射的疏水染料M507自组装,构建了近红外发光纳米探针M507@SNP。同时,研究了M507@SNP的光物理性能和细胞毒性。动物成像实验证明该纳米成像探针可实现活体层次高信噪比的小动物全身成像和前哨淋巴结的指示。     

二氧化硅-共聚物杂化荧光纳米材料用于动物活体成像

以氧化硅（SiO₂）前驱体与三嵌段共聚物F108合成较小粒径的SiO₂-共聚物杂化纳米体系（SNP），并与高效近红外发射的疏水染料M507自组装，构建了近红外发光纳米探针M507@SNP。同时，研究了M507@SNP的光物理性能和细胞毒性。动物成像实验证明该纳米成像探针可实现活体层次高信噪比的小动物全身成像和前哨淋巴结的指示。     

An Activatable Near-Infrared Fluorescent Probe for Precise Detection of the Pulmonary Metastatic Tumors: A Traditional Molecule Having a Stunning Turn

An accurate detection of lung metastasis is of great significance for making better treatment choices and improving cancer prognosis, but remains a big challenge in clinical practice. In this study, we propose a reinventing strategy to develop a pH-activatable near-infrared (NIR) fluorescent nanoprobe, pulmonary metastasis tracer (denoted as PMT), based on assembly of NIR dye IR780 and calcium phosphate (CaP). By delicately tuning the intermolecular interactions during the assembly process and dye doping content, as well as the synthetic condition of probe, the fluorescence of PMT could be finely adjusted via the tumor acidity-triggered disassembly. Notably, the selected PMT9 could sharply convert subtle pH variations into a distinct fluorescence signal to generate high fluorescence ON/OFF contrast, dramatically reducing the background signals. Benefiting from such preferable features, PMT9 is able to precisely identify not only the tumor sites in orthotopic lung cancer models but also the pulmonary metastases in mice with remarkable signal-to-background ratio (SBR). This study provides a unique strategy to turn shortcomings of traditional dye IR780 during in vivo imaging into advantages and further expand the application of fluorescent probe to image lung associated tumor lesions.     

Multiplexed determination of intracellular messenger RNA by using a graphene oxide nanoprobe modified with target-recognizing fluorescent oligonucleotides

A multiplexed graphene oxide (GO) fluorescent nanoprobe is described for quantification and imaging of messenger RNAs (mRNAs) in living cells. The recognizing oligonucleotides (with sequences complementary to those of target mRNAs) were labeled with different fluorescent dyes. If adsorbed on GO, the fluorescence of the recognizing oligonucleotides is quenched. After having penetrated living cells, the oligonucleotides bind to target mRNAs and dissociate from GO. This leads to the recovery of fluorescence. Using different fluorescent dyes, various intracellular mRNAs can be simultaneously imaged and quantified by a high content analysis within a short period of time. Actin mRNA acts as the internal control. This GO-based nanoprobe allows mRNA mimics to be determined within an analytical range from 1 to 400 nM and a detection limit as low as 0.26 nM. Up to 3 intracellular mRNAs (C-myc, TK1, and actin) can be detected simultaneously in a single living cell. Hence, this nanoprobe enables specific distinction of intracellular mRNA expression levels in cancerous and normal cells. It can be potentially applied as a tool for detection of cancer progression and diagnosis.

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Graphical abstract A multiplexed graphene oxide (GO)-based fluorescent nanoprobe is described for quantification and imaging of intracellular messenger RNAs. After penetrating living cells, the recovered fluorescence of the dissociated recognizing oligonucleotides can be analyzed , and this allows for simultaneous detection of up to 3 intracellular messenger RNAs.

     

A novel TICT-based near-infrared fluorescent probe for light-up sensing and imaging of human serum albumin in real samples

Human serum albumin (HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared (NIR) fluorescent probe (7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer (TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.     

嘧啶基双光子荧光染料的设计合成与生物成像

生理条件下光学性质稳定的双光子荧光染料在生物成像领域具有广阔的应用前景。我们使用2,4-二甲基-6-羟基嘧啶与4-(N,N-二甲氨基)苯甲醛进行缩合反应,设计合成了具有双光子荧光性质的化合物2-[(1E)-2-[4-(二甲氨基)苯基]乙烯基]-6-甲基-4(3H)-嘧啶(NHP)。通过质谱(MS)、核磁共振波谱(NMR)、紫外可见吸收光谱和荧光发射光谱等技术手段表征了其结构,研究了其光物理性质,以及外部环境改变对其发射光谱的影响。结果表明,化合物NHP的最佳吸收峰位于400 nm,最佳发射峰位于540 nm左右,且荧光发射不受金属离子、氨基酸和pH等环境因素的影响。生物实验结果表明,化合物NHP细胞毒性较小,且具有很好的活细胞和果蝇脑组织成像效果,是一种较为理想的双光子荧光生物成像染料。     

An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.     

A near-infrared reversible fluorescent probe for peroxynitrite and imaging of redox cycles in living cells

BzSe-Cy is a small-molecule fluorescent probe containing Se, which can respond reversibly to changes in ONOO(-) or reduced ascorbate and exhibit high sensitivity and selectivity for ONOO(-).     

Yellow-green fluorescent carbon dots as fluorescent/colorimetric dual-mode probe for the detection of pH value and its application in fluorescent paintingEI北大核心CSCD

Yellow-green fluorescent carbon dotsY-G-CDswas used as a fluorescent/colorimetric dual-mode nanoprobe for the detection of pH value in solution and fluorescent painting. The fluorescent intensity and absorbance of the nanoprobe showed strong pH dependence. The fluorescent intensity at 520 nm and the ratio of absorbance at 438 and 386 nm showed good linear relationships with the pH valuein the ranges of pH 5.9-7.7 and pH 5.9-8.0respectively. As a fluorescent nanoprobeY-G-CDs had good photo stability at pH 5.96.8 and 8.0and good reversibility between pH 5.9 and pH 7.7. The detection of pH value of solution was not affected by other interfering substances. In additionthe Y-G-CDs with excellent fluorescent performance have been successfully applied in fluorescent painting as a fluorescent ink. Under the irradiation of 365 nm UV lightthe drawing image emitted bright green fluorescence with clear outline. © 2022, Youke Publishing Co.,Ltd. All rights reserved.     

Developing Novel Fabrication and Optimisation Strategies on Aggregation-Induced Emission Nanoprobe/Polyvinyl Alcohol Hydrogels for Bio-Applications

The current study describes a new technology, effective for readily preparing a fluorescent (FL) nanoprobe-based on hyperbranched polymer (HB) and aggregation-induced emission (AIE) fluorogen with high brightness to ultimately develop FL hydrogels. We prepared the AIE nanoprobe using a microfluidic platform to mix hyperbranched polymers (HB, generations 2, 3, and 4) with AIE (TPE-2BA) under shear stress and different rotation speeds (0–5 K RPM) and explored the FL properties of the AIE nanoprobe. Our results reveal that the use of HB generation 4 exhibits 30-times higher FL intensity compared to the AIE alone and is significantly brighter and more stable compared to those that are prepared using HB generations 3 and 2. In contrast to traditional methods, which are expensive and time-consuming and involve polymerization and post-functionalization to develop FL hyperbranched molecules, our proposed method offers a one-step method to prepare an AIE-HB nanoprobe with excellent FL characteristics. We employed the nanoprobe to fabricate fluorescent injectable bioadhesive gel and a hydrogel microchip based on polyvinyl alcohol (PVA). The addition of borax (50 mM) to the PVA + AIE nanoprobe results in the development of an injectable bioadhesive fluorescent gel with the ability to control AIEgen release for 300 min. When borax concentration increases two times (100 mM), the adhesion stress is more than two times bigger (7.1 mN/mm²) compared to that of gel alone (3.4 mN/mm²). Excellent dimensional stability and cell viability of the fluorescent microchip, along with its enhanced mechanical properties, proposes its potential applications in mechanobiology and understanding the impact of microstructure in cell studies.     

Calix[4]pyrroles bearing quinolinium moiety for halide sensing in aqueous solution

Sensing of chloride in aqueous solution with high selectivity is a challenging task and has a great potential for cellular imaging and analytical applications in food chemistry. Supramolecular binding motif calix[4]pyrrole has been conjugated with a known fluorescent probe for chloride – a quinolinium dye - through conformationally flexible and rigid linkers. Effects of the supramolecular host on the properties of the fluorescent dye and vice versa have been investigated by NMR, X-ray crystallographic and spectroscopic methods. New fluorescent probes have demonstrated better binding and quenching properties towards chloride, bromide and iodide in a 1:1 water-methanol mixture as compared to free calix[4]pyrrole and the quinolinium dye.     

Biodegradable nanoprobe based on MnO<Subscript>2</Subscript> nanoflowers and graphene quantum dots for near infrared fluorescence imaging of glutathione in living cells

Near infrared (NIR) emitting semiconductor quantum dots can be excellent fluorescent nanoprobes, but the poor biodegradability and potential toxicity limits their application. The authors describe a fluorescent system composed of graphene quantum dots (GQDs) as NIR emitters, and novel MnO₂ nanoflowers as the fluorescence quenchers. The system is shown to be an activatable and biodegradable fluorescent nanoprobe for the “turn-on” detection of intracellular glutathione (GSH). The MnO₂-GQDs nanoprobe is obtained by adsorbing GQDs onto the surface of MnO₂ nanoflowers through electrostatic interaction. This results in the quenching of the NIR fluorescence of the GQDs. In the presence of GSH, the MnO₂-GQDs nanoprobe is degraded and releases Mn²⁺ and free GQDs, respectively. This gives rise to increased fluorescence. The nanoprobe displays high sensitivity to GSH and with a 2.8 μM detection limit. It integrates the advantages of NIR fluorescence and biodegradability, selectivity, biocompatibility and membrane permeability. All this makes it a promising fluorescent nanoprobe for GSH and for cellular imaging of GSH as shown here for the case of MCF-7 cancer cells.

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Graphical abstract A biodegradable NIR fluorescence nanoprobe (MnO₂-GQDs) for the “turn-on” detection of GSH in living cell was established, with the NIR GQD as the fluorescence reporter and the MnO₂ nanoflower as the fluorescence quencher.

     

Organoboron Schiff bases as cell‐staining fluorescent probes: Synthesis,Chemio‐photophysical characterization,DFT, and X‐ray structures

The fluorescence imaging technologies are becoming the most powerful and noninvasive diagnostic tools in cellular biology and modern medicine where abnormal cell arrangements are associated with diseases. Thus, these techniques require new fluorescent dyes with excellent chemical, physical, and photophysical properties. A series of four new Boron Schiff bases ( 1 – 4 ) has been prepared by condensation between phenylboronic acid with the corresponding ligand. The compounds were characterized by NMR (¹H, ¹³C, and ¹¹B), UV/vis, fluorescence spectroscopy, and high‐resolution mass spectrometry. The crystal structures of three compounds showed tetracoordinated Boron atoms with semiplanar skeleton ligands. Interesting organoboron response to viscosity on their fluorescence (Φ: more than 3‐fold). Additionally, compounds 1 and 2 were found to serve as a fluorescent dye for cell imaging (B16F10, CaCo, and A‐431 cells) since it has the capability to rapidly accumulate within the cells and gave bright green fluorescence, it showed low cytotoxicity activity and high photostability in solution. Additionally, the compounds have also been investigated using DFT.