Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′‐terminal Cy5 dyes

By using a fluorescent exonuclease assay, we reported unusual electrophoretic mobility of 5′‐indocarbo‐cyanine 5 (5′‐Cy5) labelled DNA fragments in denaturing polyacrylamide gels. Incubation time and enzyme concentration were two parameters involved in the formation of 5′‐Cy5‐labelled degradation products, while the structure of the substrate was slightly interfering. Replacement of positively charged 5′‐Cy5‐labelled DNA oligonucleotides (DNA oligos) by electrically neutral 5′‐carboxyfluorescein (5′‐FAM) labelled DNA oligos abolished the anomalous migration pattern of degradation products. MS analysis demonstrated that anomalously migrating products were in fact 5′‐labelled DNA fragments ranging from 1 to 8 nucleotides. Longer 5′‐Cy5‐labelled DNA fragments migrated at the expected position. Altogether, these data highlighted, for the first time, the influence of the mass/charge ratio of 5′‐Cy5‐labelled DNA oligos on their electrophoretic mobility. Although obtained by performing 3′ to 5′ exonuclease assays with the family B DNA polymerase from Pyrococcus abyssi, these observations represent a major concern in DNA technology involving most DNA degrading enzymes.     

Near-infrared small molecular fluorescent dyes for photothermal therapy

This review aims to provide a summary of the progress in fluorescent dyes for photothermal therapy in recent years and it is classified according to the structure of organic molecules including cyanines, phthalocyanines, rhodamine analogues and BODIPYs.     

The staining efficiency of cyanine dyes for single‐stranded DNA is enormously dependent on nucleotide composition

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson‐Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.     

Highly water-soluble,near-infrared emissive BODIPY polymeric dye bearing RGD peptide residues for cancer imaging

Near-infrared emissive BODIPY polymeric dye bearing cancer-homing cyclic arginine–glycine–aspartic acid (RGD) peptide residues (polymer B) was prepared by post-polymerization functionalization of BODIPY polymeric dye bearing bromo groups through tetra(ethylene glycol) tethered spacers (polymer A) with thiol-functionalized RGD cancer-homing peptide through thioether bonds under a mild basic condition. Polymer B possesses excellent water solubility, good photostability, biocompatibility and resistance to nonspecific interactions to normal endothelial cells, and can efficiently detect breast tumor cells through specific cooperative binding of cancer-homing RGD peptides to αvβ3 integrins of cancer cells while its parent polymer A without RGD residues fails to target cancer cells.     

Optical properties of molecular aggregates of oxazine dyes in dispersions of clay minerals

The molecular aggregation of oxazine 1 (Ox1) and oxazine 4 (Ox4) in reduced charge montmorillonite (RCM) colloids was investigated by absorption and fluorescence spectroscopies. The aggregation was significantly influenced by the structure of dye cations. Presence of four hydrophobic ethyl groups attached to the ammonium substituents in Ox1 cation prevented formation of closely packed sandwich-type assemblies (H-aggregates). Significant effect of the layer charge was observed for Ox4/RCMs dispersions. Large amounts of the Ox4 H-aggregates were formed in the systems with RCMs of the highest layer charge and reflected in quenched fluorescence. The presence of J-aggregates was proven by absorption spectra for the systems with Ox4 and low-charge RCMs. The flocculation of the lowest charge RCM colloids led to an extensive reduction of the luminescence. The trends and effects of the dye molecular structure and RCM properties are compared with the results previously published for other types of dyes.     

Oxygen imaging of living cells and tissues using luminescent molecular probes

Oxygen imaging of biological cells and tissues is becoming increasingly important in cell biology and in the pathophysiology of various hypoxia-related diseases. The optical oxygen-sensing method using luminescent probes provides very useful, high spatial resolution information regarding oxygen distribution in living cells and tissues. This review focuses on recent advances in biological oxygen measurements based on the phosphorescence quenching of probe molecules by oxygen, and on hypoxia-sensitive fluorescent probes. Special attention is devoted to metal complex probes, Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes. Current knowledge regarding the mechanism of phosphorescence quenching of metal complexes by oxygen is described in relation to the oxygen sensitivity of the probes, and recent advances in optical oxygen probes and detection techniques for intracellular and tissue oxygen measurements are reviewed, emphasizing the usefulness of chemical modifications for improving probe properties. Tissue oxygen imaging and hypoxic tumor imaging using these metal complex probes demonstrate the vast potential of optical oxygen-sensing methods using luminescent probes.     

Evaluation of a novel EphA2 targeting peptide for triple negative breast cancer based on radionuclide molecular imaging

A new strategy for the early diagnosis of triple-negative breast cancer (TNBC) is urgently needed however specific targets are lacking. EphA2 has been reported to be over-expressed in a variety of tumors, including TNBC, and is closely related to tumor progression. In this study, we designed a novel peptide, SD01, and tested its potential in the diagnosis of TNBC. FITC-SD01 and FITC-YSA were prepared and found to bind to the 4T1 TNBC cell line, the former showing greater affinity. ¹²⁵I-SD01 and ¹²⁵I-YSA were obtained with high radiochemical yield and radiochemical purity, and both showed a high binding affinity to 4T1 cells, with a higher B_max in ¹²⁵I-SD01. Whole-body phosphoautoradiography showed clearer imaging of tumors in the group of ¹²⁵I-SD01 than in ¹²⁵I-YSA. Biodistribution demonstrated higher tumor accumulation in the ¹²⁵I-SD01 group. In group of ¹²⁵I-SD01, the tumor to the opposite muscle tissue (T/NT) ratio was 5.998 ± 0.37, in contrast to 4.69 ± 0.18 in ¹²⁵I-YSA group. Our results indicated that ¹²⁵I-SD01 could selectively and specifically target 4T1 cells in vitro and in vivo, and showed higher binding affinity and better imaging compared to ¹²⁵I-YSA. Therefore, SD01 was deemed to be a novel peptide with more favorable properties in terms of targeting EphA2.     

Investigation of the hybrid molecular probe for intracellular studies

Monitoring gene expression in vivo is essential to the advancement of biological studies, medical diagnostics, and drug discovery. Adding to major efforts in developing molecular probes for mRNA monitoring, we have recently developed an alternative tool, the hybrid molecular probe (HMP). To optimize the probe, a series of experiments were performed to study the properties of HMP hybridization kinetics and stability. The results demonstrated the potential of the HMP as a prospective tool for use in both hybridization studies and in vitro and in vivo analyses. The HMP has shown no tendency to produce false positive signals, which is a major concern for living cell studies. Moreover, HMP has shown the ability to detect the mRNA expression of different genes inside single cells from both basal and stimulated genes. As an effective alternative to conventional molecular probes, the proven sensitivity, simplicity, and stability of HMPs show promise for their use in monitoring mRNA expression in living cells. Figure Hybrid molecular probe (HMP). HMPs consist of two single strands of DNA (green) and a polyethylene glycol (PEG, purple) linker that is used to tether these two sequences together. When a target (orange strand) containing the complementary sequences to both probes at adjacent positions is added, each strand binds to its corresponding target sequence, thus bringing the two fluorophores into close proximity, which allows energy transfer to occur     

BODIPY based ‘click on’ fluorogenic dyes: application in live cell imaging

The design, synthesis, and photophysical properties of new BODIPY-based fluorogenic ‘click on’ dyes are reported. CuAAC reaction of non-fluorescent BODIPY azide with a series of non-fluorescent alkyne molecules resulted in fluorescent triazoles which displayed up to 532-fold enhancement of fluorescence in the red region. Imaging studies confirmed the general trend of cell permeability and a cholesterol linked derivative exhibited selective localization into intracellular membranes.     

A facile synthesis of α-amino-DOTA as a versatile molecular imaging probe

An amino group has been introduced into one ligand of DOTA that can couple to peptidyl carboxylates by coupling α-brominated glycine to DO3A-^tBu (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid, tert-butylester)). α-Amino-DOTA was coupled to the carboxylate backbone terminus of a peptide to demonstrate the utility for derivatization.     

Properties of molecular switch triad compounds for photoinduced intramolecular energy transfer

This paper reports the synthesis and transient absorption decay kinetics of several triad compounds, in which a sulfur aryl section is used as the spacer for intramolecular energy transfer (IET). After flash photolysis the producing sulfur radicals will provide an ‘energy trap’ to stop the IET process, after stopping flash photolysis the sulfur radicals reversibly recombine and the IET process recovers, and then a rapid photoinduced IET switchable function is realized.     

发光ZnSalen配合物在分子荧光成像中的应用进展

荧光分子探针的设计、合成以及应用是分子荧光成像领域重要的化学问题.本文从Znsalen配合物的基本性质出发,概述了Znsalen配合物结构与功能的关系,特别是其发光性质与分子结构及分子聚集状态的相关性及应用.针对Znsalen配合物的发光性质,展示了其应用于分子荧光成像和活细胞中分子事件监测的研究进展.这些最新研究表明,Znsalen配合物探针的细胞毒性低(利于活细胞成像)、发光效率高(适用于单、双光子成像)、发光可调(通过配体的修饰和分子聚集状态的调节),有望作为一类重要的发光金属荧光探针实现在分子荧光成像中的应用.     

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Bursting microbubbles: How nanobubble contrast agents can enable the future of medical ultrasound molecular imaging and image-guided therapy

The field of medical ultrasound has undergone a significant evolution since the development of microbubbles as contrast agents. However, because of their size, microbubbles remain in the vasculature and therefore have limited clinical applications. Building a better—and smaller—bubble can expand the applications of contrast-enhanced ultrasound by allowing bubbles to extravasate from blood vessels—creating new opportunities. In this review, we summarize recent research on the formulation and use of nanobubbles (NBs) as imaging agents and as therapeutic vehicles. We discuss the ongoing debates in the field and reluctance to accepting NBs as an acoustically active construct and a potentially impactful clinical tool that can help shape the future of medical ultrasound. We hope that the overview of key experimental and theoretical findings in the NB field presented in this article provides a fundamental framework that will help clarify NB–ultrasound interactions and inspire engagement in the field.     

可用于活细胞线粒体随机光学重构超分辨成像的分子内可逆环化五甲川菁染料探针

基于单分子定位的随机光学重构超分辨成像作为一种先进的光学成像方法,可用于尺寸小于光学衍射极限的生物结构的超清晰成像,为在单分子层面研究疾病的发病机制及寻找精准的治疗策略提供有力研究工具,在生物医学领域有着广泛的应用前景.随机光学重构超分辨成像技术依赖于标记探针的光物理性质,探针需要在大量缓冲试剂及含巯基试剂存在下才能产生稳定光致闪烁进行超分辨成像,获得理想的超分辨成像结果,但是大量缓冲试剂与巯基试剂对活细胞伤害较大,使得其在活细胞的超分辨成像应用上存在困难,而限制了其在生物医学成像领域的进一步应用,因此,需要开发可用于活细胞的单分子定位超分辨成像的新型光学探针.本工作提出了一种新的可用于单分子定位超分辨成像的五甲川菁染料探针,不需要外加成像缓冲液及巯基试剂就可以产生光致闪烁变化.基于此,开发了一种分子内自发开、关环反应的新型五甲川菁染料探针,具有活细胞膜通透性.探针不需要使用缓冲液体系及对细胞有害的含巯基试剂,在低功率单束激光直接照射下产生光致闪烁,探针对活细胞没有产生明显毒性,适合活细胞的超分辨成像.进入活细胞后探针选择性定位于细胞线粒体上,在激光照射下产生光致闪烁,电子倍增电荷耦合...     

A fluorescent chemosensor for Cu2+ ions and its application in cell imaging

A new on-off fluorescent probe 1 for Cu²⁺ based on Schiff base compound was designed and synthesized by one-step reaction. The single probe 1 exhibited strong green fluorescence emission. A fluorescence quenching effect and faint color change were observed as soon as the Cu²⁺ was added to the probe system in H₂O/EtOH (v/v = 8:2, HEPES buffer, 0.05 M, pH = 7.4) solution. Other common metal cations did not cause the changes in the fluorescence and color of the probe 1. The optical properties were studied by the fluorescence emission and UV–Vis spectra. Meanwhile, the geometry optimizations of probe 1 and the [1-Cu²⁺] coordination complexes were also carried out by DFT using the Gaussian 09 program, in which the B3LYP function was used. Based on experimental measurement and theoretical analysis, we can know that the combination ratio of the probe and Cu²⁺ is 2:1 and the limit of detection (LOD) is as low as 5.3 × 10^?9 M Besides, the probe 1 was also used to analyze the Cu²⁺ in living cells.     

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.     

A selective and sensitive off–on probe for palladium and its application for living cell imaging

A new rhodamine B derivative T1 has been rationally synthesized and displayed selective Pd(Ⅱ)-amplified absorbance and fluorescence emission above 540 nm in methanol–water. Upon the addition of Pd(Ⅱ), the spirolactam ring was unfolded and a 1:1 metal-ligand complex formed, which can be used for ‘‘naked-eyes" detection. In addition, fluorescence imaging experiments of Pd~(2+) in HepG2 living cells showed its valuable application in biological systems.     

Synthesis of a cationic poly(p-phenylenevinylene) derivative for lysosome-specific and long-term imaging

A cationic poly(p-phenylenevinylene) derivative (PPV) is designed and synthesized to bear quaternized N-methyl-imidazole groups, which is successfully utilized in lysosome-specific and long-term imaging.