A handheld device for potential point-of-care screening of cancer

A simple handheld device based on the fluorescence analysis of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) stained cells was established for routine screening and potentially for early detection of cancer cells at extremely low cost. Flow cytometry assay further supported the utility of this simple device, where a preliminary study of tissue biopsy showed highly encouraging results.     

A Novel Method for Screening G‐quadruplex Stabilizers to Human Telomeres

We present a simple method based on the Cu²⁺ induced unfolding of G‐quadruplex (G4) of human telomere sequence d[AG₃(T₂AG₃)₃] to screen a number of 3,6‐bis(1‐methyl‐4‐vinylpyridinium)carbazole diiodide (BMVC) analogues for better G4 stabilizers. Using circular dichroism (CD), the screening results suggest that the tri‐cations of 9‐substituted BMVC derivatives are better G4 stabilizers than the bi‐cations of BMVC. In addition, 3,6‐bis(1‐methyl‐4‐vinylpyrazinium)carbazole diiodide (BMVC4) is likely a better core molecule than BMVC for G4 stabilizers.     

A Carbazole Derivative Synthesis for Stabilizing the Quadruplex Structure

A new molecule of 3,6‐Bis(1‐methyl‐4‐vinylpyridium iodine)carbazole ( BMVC ) was synthesized for stabilizing the quadruplex structure of human telomeric sequence of d(T₂AG₃)₄ in vitro. Mixing BMVC with the DNA can raise the melting temperature of the d(T₂AG₃)₄ by ～ 13 °C, implying that BMVC could be a useful telomerase inhibitor. In addition, the fluorescence of the BMVC increased significantly upon interacting with the d(T₂AG₃)₄ which may be useful as a G‐quadruplex specific marker.     

Interaction between human telomere and a carbazole derivative: a molecular dynamics simulation of a quadruplex stabilizer and telomerase inhibitor

The mechanism of inhibition of telomerase by drugs is a key factor in an understanding of guanine-quadruplex complex stabilization during human cancer. This study describes a simulated annealing docking and molecular dynamics simulation to investigate a synthesized potent inhibitor, 3,6-bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC), which stabilizes the quadruplex structure of the human telomeric DNA sequence d[AG3(T(2)AG(3))3] and inhibits telomerase activity. The compound was predicted to selectively interact with the quadruplex structure. During our simulation, the binding affinities were calculated and used to predict the best drug-binding sites as well as enhanced selectivity compared with other compounds. Our studies suggest that the simulation results quite coincide with the experimental results. In addition, molecular modeling shows that a 2:1 binding model involving the external binding of BMVC to both ends of the G-quartet of d[AG(3)(T(2)AG)3))3] is the most stable binding mode and this agrees with the absorbance titration results that show two binding sites. Of particular interest is that one pyridinium ring and carbazole moiety of the BMVC can stack well at the end of G-quartet. This implies that BMVC is a good human quadruplex stabilizer and also a good telomerase inhibitor.     

Delineation of G‐Quadruplex Alkylation Sites Mediated by 3,6‐Bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole‐Aniline Mustard Conjugates

A new G‐quadruplex (G‐4)‐directing alkylating agent BMVC‐C3M was designed and synthesized to integrate 3,6‐bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G‐4 structures (hybrid‐2 type and antiparallel) and an oncogene promoter, c‐MYC (parallel), were constructed to react with BMVC‐C3M, yielding 35 % alkylation yield toward G‐4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI‐MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on G‐4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC‐C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c‐MYC), respectively, as monoalkylated adducts and formed A15–C3M–A21 (H26), G12–C3M–G4 (H24), and G2–C3M–G4/G17 (c‐MYC), respectively, as cross‐linked dialkylated adducts. Collectively, the stability and site‐selective cross‐linking capacity of BMVC‐C3M provides a credible tool for the structural and functional characterization of G‐4 DNAs in biological systems.     

Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging

Fluorescent nanoparticles (FNs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. In this study, anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) (anti-EGFR antibody conjugated FNs) probe was used to detect breast cancer cells. FNs with excellent character such as non-toxicity and photostability were first synthesized with a simple, cost-effective and environmentally friendly modified Stőber synthesis method, and then successfully modified with anti-EGFR antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated FNs has been used to detect breast cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-EGFR antibody conjugated FNs can effectively recognize breast cancer cells and exhibited good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of breast cancer cells and offer a new method in detecting EGFR.     

Synthesis of Folate Conjugated Fluorescent Nanoparticle Probe and Its Application in Cervical Cancer Cell Imaging

Abstract

Cervical cancer is the second most common cancer in women worldwide with 80% of cases arising in the developing world, following cancer of the breast. The mortality associated with cervical cancer can be reduced if this disease is detected in a timely fashion. In this study, a folate conjugated fluorescent nanoparticle (FCFN) probe was used to detect cervical cancer cells. Fluorescent nanoparticles (FNs), with excellent characteristics such as nontoxicity and photostability, were first synthesized with a simple, cost-effective, and environmentally friendly modified St?ber synthesis method and then successfully modified with folate. This kind of fluorescence probe based on FCFNs has been used to detect cervical cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the FCFNs can effectively recognize cervical cancer cells and exhibit good sensitivity and exceptional photostability; they would provide a novel way for the diagnosis and curative effect observation of cervical cancer cells and offer a new method in detecting folate receptors (FR).     

水溶性量子点荧光探针用于胃癌细胞相关抗原CA242的检测

基于量子点荧光探针对胃癌细胞相关抗原CA242进行了检测。首先在水溶液中直接合成性能优良的量子点荧光纳米颗粒,并在其表面成功修饰了羊抗小鼠IgG和聚乙二醇,制得功能化的水溶性量子点荧光探针,并利用探针对胃癌细胞相关抗原CA242进行检测,进一步与传统的基于荧光染料标记的免疫荧光分析方法进行了比较。实验结果表明:该功能化的探针能够有效地识别胃癌细胞相关抗原CA242,并且在光稳定性和灵敏度方面都较传统的基于荧光染料标记的免疫荧光分析方法有明显的改善,从而为CA242的相关检测以及胃癌的诊断与愈后判断提供了新的方法。     

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.     

Boronic acid-containing carbon dots array for sensitive identification of glycoproteins and cancer cells

Discrimination of glycoproteins and cell types is a significant but difficult issue. Herein, we presented a novel fluorescence sensor array for the detection and identification of glycoproteins and cancer cells based on the specific affinity between boronic acid-containing carbon dots (BA-CDs) and cis-diol residues of polysaccharides. The differential binding affinity of three BA-CDs to various glycoproteins resulted in a different fluorescence turn-on signal pattern caused by aggregation-enhanced emission (AEE), along with negligible response from other proteins. Therefore, BA-CDs encompassing sensing elements and signal indicator into one can enable a fast and accurate discrimination of glycoproteins with simple and easy operation. Seven glycoproteins could be well discriminated at a very low concentration of 10 nmol/L. The discriminating capability of glycoproteins is not sacrificed in both human urine and serum. Notably, different glycoprotein compositions of cancer cells provide more recognizable features for identification of cancer cells, comparing to the total protein. Five cell types could be identified in 15 min at a low concentration of 1000 cells/mL. This method is fast, accurate, and easy operation, and has a potential application in cancer diagnosis.     

In situ labeling and imaging of cellular protein via a bi-functional anticancer aptamer and its fluorescent ligand

In this article, we reported a novel approach for in situ labeling and imaging HeLa cancer cells utilizing a bifunctional aptamer (AS1411) and its fluorescent ligand, protoporphyrin IX (PPIX). In the presence of potassium ion, AS1411 folded to G-quadruplex structure, binded fluorescent ligand (PPIX) with fluorescent enhancement, and targeted the nucleolin overexpressed by cancer cells. Consequently, bioimaging of cancer cells specifically were realized by laser scanning confocal microscope. The bioimaging strategy with AS1411–PPIX complex was capable to distinguish HeLa cancer cells from normal cells unambiguously, and fluorescence imaging of cancer cells was also realized in human serum. Moreover, the bioimaging method was very facile, effective and need not any covalent modification. These results illustrated that the useful approach can provide a novel clue for bioimaging based on non-covalent bifunctional aptamer in clinic diagnosis.     

Naphthalimide-rhodamine based fluorescent probe for ratiometric sensing of cellular pH

The pH values of lysosomes in cancer cells is slightly lower than that in normal cells, which can be used to distinguish cancer cells from normal cells. According to this, a naphthalimide-rhodamine based fluorescent probe(hereafter referred to as RBN) with a pK_a of 4.20 was designed and synthesized for ratiometric sensing of cellular pH via fluorescence resonance energy transfer(FRET), which can respond to different pH precisely through ratiometric fluorescence intensity(Ⅰ_(577)/Ⅰ_(540)). RBN can be employed to distinguish cancer cells from normal cells on the basis of different fluorescent response, in particular, RBN showed excellent water solubility and low cell toxicity, all these are quite significant for potential application in cancer diagnose and therapy.     

Differentiation of Normal and Neoplastic Cells by Synchronous Fluorescence: Rat Liver Epithelial and Rat Hepatoma Cell Models

ABSTRACT

In the present study, conventional and synchronous luminescence (SL) were utilized to investigate spectral differences in normal and neoplastic cells. The synchronous fluorescence (SF) method involves scanning simultaneously both emission and excitation wavelengths while keeping a constant wavelength interval between them. This SF procedure simplifies the emission spectrum and provides for greater selectivity and is used to detect subtle differences in the fluorescence emission of the biochemical species of cells from rat tissues. A difference between the fluorescent spectra of the normal rat liver epithelial (RLE) and hepatoma cell lines were detected using synchronous fluorescence. The potential use of SF as a screening tool for cancer diagnosis is discussed.     

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.     

Simultaneous Visualization of Multiple mRNAs and Matrix Metalloproteinases in Living Cells Using a Fluorescence Nanoprobe

Simultaneous monitoring of multiple tumour markers is of great significance for improving the accuracy of early cancer detection. In this study, a fluorescence nanoprobe has been prepared that can simultaneously monitor and visualize multiple mRNAs and matrix metalloproteinases (MMPs) in living cells. Confocal fluorescence imaging results indicate that the nanoprobe could effectively distinguish between cancer cells and normal cells even if one tumour maker of normal cells was overexpressed. Furthermore, it can detect changes in the expression levels of mRNAs and MMPs in living cells. The current approach could provide new tools for early cancer detection and monitoring the changes in expression levels of biomarkers during tumour progression.     

A cancer cell turn-on protein-CuSMn nanoparticle as the sensor of breast cancer cell and CH3O-PEG-phosphatide

A cancer activated protein-inorganic nanoparticle was used as breast cancer cell turn-on fluorescence sensor and NIR activated attenuator.     

Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.     

Self-assembly of amphiphilic peptides to construct activatable nanophotosensitizers for theranostic photodynamic therapy

A variety of nano-engineered photosensitizers have been developed for photodynamic therapy (PDT) of cancer diseases. However, traditional nano-engineering methods usually cannot avoid drug leakage and premature release, and have disadvantages such as low drug load and inaccurate release. The self-assembly strategy based on amphiphilic peptides has been considered to be more attractive nano-engineering method. Here we developed novel acid-activatable self-assembled nanophotosensitizers based on an amphiphilic peptide derivative. The peptide derivative was synthesized from a fluorescein molecule with thermally activated delayed fluorescence (TADF). The self-assembled nanophotosensitizers can specifically enter the tumor cells and disassemble inside lysosomes companied with “turn-on” fluorescence and photodynamic therapy effect. Such smart nanophotosensitizers will open new opportunities for cancer theranostics.     

Hematoporphyrin derivative photosensitization and cellular fluorescent photoproduct formation

A new fluorescence band with an emission maximum at 460 nm was observed in S180 tumour cells and human cancer tissues photosensitized by Y-HPD. This fluorescence was the result of a photochemical reaction involving specific proteins and Y-HPD in the cells.     

Characteristic Visible Fluorescence Emission Spectra of Sera from Cancer Patients

Abstract

The feasibility of using visible fluorimetry as a simple spectroscopic method for cancer diagnosis and/or management was investigated by recording the visible fluorescence emission spectra of sera from cancer patients and healthy individuals. Characteristic visible fluorescence emission spectra were found to occur in sera of some cancer patients, e.g., patients with breast or lung cancer, with spectral features similar to those observed among porphyrin and porphyrin-related compounds. Possible physico-chemical factors responsible for the occurrence of these unique visible fluorescence features among cancer patients were briefly discussed.