禽流感病毒流式微球量子点探针免疫诊断新方法

采用微波法水相中合成羧基化的绿色量子点,通过羧基与禽流感单克隆抗体氨基的共价结合,制备了检测禽流感病毒的探针,并结合流式微球技术,建立了量子点生物探针流式微球免疫检测禽流感病毒的新方法.以聚苯乙烯微球为蛋白质载体,将多克隆抗体包被到荧光微球上,依次加入待测抗原和量子点生物探针,形成双抗体夹心复合物,用流式细胞仪进行检测.实验结果表明,多抗和单抗的最佳质量浓度分别为92和4 mg/L,检测禽流感病毒比双抗体夹心ELISA灵敏16倍,比FITC标记单抗检测方法灵敏4倍.对阳性尿囊液的检测与ELISA呈现良好的相关性,不与鸡传染性支气管炎病毒、鸡马力克氏病毒、新城疫病毒等发生交叉反应.     

Fluorescence Detection of Single‐Nucleotide Polymorphisms with a Single,Self‐Complementary,Triple‐Stem DNA Probe

Singled out for its singularity : In a single‐step, single‐component, fluorescence‐based method for the detection of single‐nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self‐complementary DNA strand that forms a triple‐stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F=fluorophore, Q=quencher).

     

Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE‐Cy, a cell‐permeable dye with aggregation‐induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE‐Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE‐Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.     

Biometallization‐Based Electrochemical Magnetoimmunosensing Strategy for Avian Influenza A (H7N9) Virus Particle Detection

A highly sensitive electrochemical immunosensor for avian influenza A (H7N9) virus (H7N9 AIV) detection was proposed by using electrochemical magnetoimmunoassay coupled with biometallization and anodic stripping voltammetry. This strategy could accumulate the enzyme‐generated product on the surface of the magneto electrode by means of silver deposition, which amplified the detection signal about 80 times. The use of magnetic beads (MBs) and the magneto electrode could also amplify the detection signal. Furthermore, a bi‐electrode signal transduction system was introduced into this immunosensor, which is also beneficial to the immunoassay. A concentration as low as 0.011 ng mL^?1 of H7N9 AIV could be detected in about 1.5 h with good specificity. This study not only provides a simple and sensitive approach for virus detection but also offers an effective signal enhancement strategy for the development of highly sensitive MB‐based electrochemical immunoassays.     

Development of a Photoactivatable Phosphine Probe for Induction of Intracellular Reductive Stress with Single‐Cell Precision

Photoactivatable phosphines that induce intracellular reductive stress are reported. The design of these probes takes advantage of the conjugate addition of trialkylphosphines to carbocyanine dyes, which can be reverted photochemically to produce the trialkylphosphine and a fluorescent reporter. The photochemical release depends on the efficiency of photoinduced electron transfer from the indolenine arm of the probe to the coumarin acceptor. These probes readily permeate the mammalian plasma membrane and can be photoactivated in live cells. Upon irradiation of the probe, the released trialkylphosphine induces intracellular reductive stress, which ultimately leads to formation of thioflavin‐positive intracellular protein aggregates. These effects could be induced in individual cells within a monolayer, with minimal disturbance of neighboring cells.     

Voltammetric Detection of a Specific DNA Sequence of Avian Influenza Virus H5N1 Using HS‐ssDNA Probe Deposited onto Gold Electrode

The working principle of a genosensor is based on the mechanism of ion‐channel mimetic sensors. The analytical signals generated upon hybridization processes were recorded by a redox active marker [Fe(CN)₆]^3?/4? present in the sample solution using voltammetric techniques. The developed genosensor was suitable for determination of 20‐mer complementary oligonucleotide sequence, and also of the PCR products containing the complementary 20‐mer sequence in various positions, with detection limits in the 10 pM range. The noncomplementary 20‐mer oligonucleotide sequence as well as the PCR product without complementary region generated very weak response. The good discrimination of the position of the complementary part in the PCR products was observed.     

High Expression Level of α2-3-Linked Sialic Acids on Salivary Glycoproteins of Breastfeeding Women May Help to Protect Them from Avian Influenza Virus Infection

Terminal sialic acids (Sia) on soluble glycoprotein of saliva play an important role in the clearance of influenza virus. The aim of this study is to investigate the alteration of sialylation on the salivary proteins of women during the lactation period and its effect on the saliva binding ability to virus. In total, 210 saliva samples from postpartum women with and without breastfeeding were collected, and the expression level of α2-3/6-linked Sia on the whole salivary proteins and specific glycoproteins of IgA and MUC5B from different groups were tested and verified using lectin microarray, blotting analysis and ELISA based method. The H1N1 vaccine and three strains of Avian influenza virus (AIV) were used for the saliva binding assay. Results showed that the variation in salivary expression level of α2-3-linked Sia was much more obvious than the α2-6-linked Sia, which was up-regulated significantly in the breastfeeding groups compared to the non-breastfeeding groups at the same postpartum stage. Furthermore, the binding abilities of salivary glycoproteins to AIV strains and H1N1 vaccine were increased in breastfeeding groups accordingly. This finding adds new evidence for the maternal benefit of breastfeeding and provides new thinking to protect postpartum women from AIV infection.     

A Reversible Fluorescent Probe for Real‐Time Live‐Cell Imaging and Quantification of Endogenous Hydropolysulfides

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their “intact” function in living cells has been limited owing to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe that reversibly and selectively reacts to hydropolysulfides. The probe enables live‐cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real‐time reversible monitoring of oxidative‐stress‐induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution on the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides.     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

A Nitro‐Functionalized Metal–Organic Framework as a Reaction‐Based Fluorescence Turn‐On Probe for Rapid and Selective H2S Detection

The toxic gas H₂S has recently emerged as one of the important signaling molecules in biological systems. Thus understanding the production, distribution, and mode of action of H₂S in biological system is important, but the fleeting and reactive nature of H₂S makes it a daunting task. Herein we report a biocompatible, nitro‐functionalized metal–organic framework as reaction‐based fluorescence turn‐on probe for fast and selective H₂S detection. The selective turn‐on performance of MOF remains unaffected even in presence of competing biomolecules.     

In Vitro Light‐Up Visualization of a Subunit‐Specific Enzyme by an AIE Probe via Restriction of Single Molecular Motion

Enzymes contain several subunits to maintain different biological functions. However, it remains a great challenge for specific discrimination of one subunit over another. Toward this end, the fluorescent probe TPEMA is now presented for highly specific detection of the B subunit of cytosolic creatine (CK) kinase isoenzyme (CK‐B). Owing to its aggregation‐induced emission property, TPEMA shows highly boosted emission toward CK‐B with a fast response time and very low interference from other analytes, including the M subunit of CK (CK‐M). With the aid of a Job plot assay, ITC assay and molecular dynamics simulation, it was directly confirmed that the remarkably enhanced fluorescence of TPEMA in the presence of CK‐B results from the restriction of single molecular motion in the cavity. Selective wash‐free fluorescence imaging of CK‐B in macrophages under different treatments was successfully demonstrated.     

Identification of Tumor Initiating Cells with a Small‐Molecule Fluorescent Probe by Using Vimentin as a Biomarker

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC‐specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial‐to‐mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti‐TIC activity with low toxicity to non‐TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.     

Construction of a Near‐Infrared Fluorescent Turn‐On Probe for Selenol and Its Bioimaging Application in Living Animals

As selenocysteine (Sec) carries out the majority of the functions of the various Se‐containing species in vivo, it is of high importance to develop reliable and rapid assays with biocompatibility to detect Sec. Herein, an NIR fluorescent turn‐on probe for highly selective detection of selenol was designed and synthesized. The probe exhibits large turn‐on signal upon treatment with selenocysteine (R‐SeH), and it was further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals.     

A Naphthalimide‐Based Fluorescence “Turn‐On” Probe for the Detection of Pb2+ in Aqueous Solution and Living Cells

An easily available naphthalimide‐based fluorescent probe NPA for Pb²⁺ detection was successfully developed. NPA exhibited an obvious fluorescence turn‐on response toward Pb²⁺ in aqueous solution and in living cells. Moreover, a series of model compounds were rationally designed and synthesized in order to explore the sensing mechanism and binding mode of NPA with Pb²⁺.     

NMR Structure of a Triangulenium‐Based Long‐Lived Fluorescence Probe Bound to a G‐Quadruplex

An NMR structural study of the interaction between a small‐molecule optical probe (DAOTA‐M2) and a G‐quadruplex from the promoter region of the c‐myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR‐restrained structural calculations show that binding of DAOTA‐M2 occurs mainly through π–π stacking between the polyaromatic core of the ligand and guanine residues of the outer G‐quartets. Interestingly, the binding affinities of DAOTA‐M2 differ by a factor of two for the outer G‐quartets of the unimolecular parallel G‐quadruplex under study. Unrestrained MD calculations indicate that DAOTA‐M2 displays significant dynamic behavior when stacked on a G‐quartet plane. These studies provide molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G‐quadruplexes.     

A 3,7‐Dihydroxyphenoxazine‐based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide

A novel N‐borylbenzyloxycarbonyl‐3,7‐dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H₂O₂ in living cells is described. The probe could achieve high selectivity for detecting H₂O₂ over other biological reactive oxygen species (ROS). In addition, upon addition of H₂O₂, NBCD exhibited color change from colorless to pink, which makes it a “naked‐eye” probe for H₂O₂ detection. NBCD could not only be used to detect enzymatically generated H₂O₂ but also to detect H₂O₂ in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm . Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)‐stimulated H₂O₂ generation inside the cells.