A benzimidazole-based new fluorogenic differential/sequential chemosensor for Cu2+, Zn2+, CN-, P2O74-, DNA, its live-cell imaging and pyrosequencing applications

Differential chemosensors have emerged as next-generation systems due to their simplicity and favourable responsive properties to produce different signals upon selective binding of various analytes simultaneously. Nevertheless, given their inadequate fluorescence response and laborious synthetic procedures, only a few differential chemosensors have been developed so far. In this work, we have employed a single pot synthesis strategy to establish a new benzimidazole-based Schiff base type fluorogenic chemosensor (DFB) which differentially detects Cu2+ (detection limit (LOD) = 24.4 ± 0.5 nM) and Zn2+ (LOD = 2.18 ± 0.1 nM) through fluorescence “off-on” manner over the library of other metal cations in an aqueous medium. The DFB-derived ‘in situ’ complexes DFB-Cu2+ and DFB-Zn2+ showed fluorescence revival “on-off” responses toward cyanide (CN−) and bio-relevant pyrophosphate (P2O7 4--PPi) ions with a significantly low LOD of 9.43 ± 0.2 and 2.9 ± 0.1 nM, respectively, in water. We have demonstrated the phosphate group-specific binding capability of DFB-Zn2+ , by testing it with both ssDNA and dsDNA samples which displayed fluorescence “turn-off” response (LOD ∼10-7 M), similar to the PPi binding in an aqueous medium, indicating that it interacts explicitly with the phosphate backbone of DNA. We have also harnessed the DFB as a sequential fluorescent probe to detect Cu2+, Zn2+, CN− and P2O7 4- ions in human cervical (HeLa) and breast (MCF-7 and MDA-MB-231 (aggressive and invasive)) cancer cell lines. Moreover, we have explored the PPi recognition capability of DFB-Zn2+ in the polymerase-chain-reaction (PCR) products where PPi is one of the primary by-products during amplification of DNA

Hydrazone-Based Small-Molecule Chemosensors

The hydrazone functional group is widely applied in several fields. The versatility and large use of this chemotype are attributed to its easy and straightforward synthesis and unique structural characteristics which is useful for different chemical and biological purposes. Recently hydrazone scaffold has been widely adopted in the design of small-molecule fluorescent and colorimetric chemosensors for detecting metals and anions because of its corresponding non-covalent interactions. This chapter provides an overview of hydrazone-based fluorescent and colorimetric chemosensors for anions and metals of biological interest, with their representative rational designs in the last 15 years. We hope this chapter inspires the development of novel and powerful fluorescent and colorimetric chemosensors for a broad range of applications

Computational Biology and Chemistry

The use of computers and software tools in biochemistry (biology) has led to a deep revolution in basic sciences and medicine. Bioinformatics and systems biology are the direct results of this revolution. With the involvement of computers, software tools, and internet services in scientific disciplines comprising biology and chemistry, new terms, technologies, and methodologies appeared and established. Bioinformatic software tools, versatile databases, and easy internet access resulted in the occurrence of computational biology and chemistry. Today, we have new types of surveys and laboratories including “in silico studies” and “dry labs” in which bioinformaticians conduct their investigations to gain invaluable outcomes. These features have led to 3-dimensioned illustrations of different molecules and complexes to get a better understanding of nature

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

Synthetic molecular probes, chemosensors, and nanosensors used in combination with innovative assay protocols hold great potential for the development of robust, low-cost, and fast-responding sensors that are applicable in biofluids (urine, blood, and saliva). Particularly, the development of sensors for metabolites, neurotransmitters, drugs, and inorganic ions is highly desirable due to a lack of suitable biosensors. In addition, the monitoring and analysis of metabolic and signaling networks in cells and organisms by optical probes and chemosensors is becoming increasingly important in molecular biology and medicine. Thus, new perspectives for personalized diagnostics, theranostics, and biochemical/medical research will be unlocked when standing limitations of artificial binders and receptors are overcome. In this review, we survey synthetic sensing systems that have promising (future) application potential for the detection of small molecules, cations, and anions in aqueous media and biofluids. Special attention was given to sensing systems that provide a readily measurable optical signal through dynamic covalent chemistry, supramolecular host–guest interactions, or nanoparticles featuring plasmonic effects. This review shall also enable the reader to evaluate the current performance of molecular probes, chemosensors, and nanosensors in terms of sensitivity and selectivity with respect to practical requirement, and thereby inspiring new ideas for the development of further advanced systems

Molecular engineering strategies for expanding the capabilities of fluorescent zinc (II) sensors

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.Includes bibliographical references.(cont.) affords the two fluorophores, such that excitation of the coumarin at 445 nm and measurement of the emission at 488 nm affords information of the amount of sensor present, while excitation of the fluorescein at 505 nm and measurement of the emission at 535 nm indicates the amount of sensor in the zinc(II)-bound form. This system has been characterized and applied to the study of exogenous zinc(II) fluxes in HeLa cells. Chapter 4: Unimolecular Two-Fluorophore Ratiometric Zn²⁺ Sensing Systems. Dichlorofluorescein compounds covalently bound to zinc(II)-insensitive reporter fluorophores via a rigid cyclohexyl linker have been prepared and characterized. Based on favorable photophysical properties, a Zinpyr-1 species covalently bound to coumarin 343 has been prepared and shown to afford a ratiometric response to excess zinc(II). Chapter 5: ZPI Synthons for Functionalization of Biological Targets. Installation of a functional group prior to Mannich reaction is impractical in many cases. This chapter describes the preparation of reactive ZP1 synthons for direct functionalization of biological targets containing an amine or azide, and reports applications to the synthesis of ZPI conjugates. Appendix 1: Synthetic Approaches to Other Isomerically Pure Functionalizable Fluorophores. Crystallization approaches have been applied to separate fluorescein 5- and 6-sulfonic acid, and subsequent generation of the sulfonyl chlorides is discussed. A dibromofluoran approach to isomerically pure rhodamine carboxylates is based on a similar separation. Basic hydrolysis of the previously described 2',7'-dichlorofluorescein ...Chapter 1: The Development and Use of Fluorescent Sensors in the Imaging of Physiological Zinc(II): A Review This chapter presents an overview of fluorescent techniques used to image chelatable zinc(II) in vivo. Many intensity-based sensors take advantage of photoinduced electron transfer quenching pathways. Peptide- and protein-based sensors offer excellent selectivity but are poorly suited to intracellular applications. Recently, ratiometric sensors in which the zinc(II) binding event interrupts or alters conjugation within the fluorophore have been described. Chapter 2: Carboxylate-Functionalized Zinpyr-1 Sensors: Synthesis, Characterization, and In Vivo Staining Patterns. A class of Zinpyr-1 sensors containing a carboxylic acid or ester at the 5- or 6-position of the fluorescein has been prepared. These sensors offer decreased background fluorescence and enhanced fluorescence response compared to the parent Zinpyr-1. The acid-functionalized sensors bear a negative charge at physiological pH, rendering them cell-impermeable. The esterified sensors are cell-permeable, but are hydrolyzed in vivo by intracellular esterases, affording a clear delineation of zinc(II)-containing damaged neurons in mechanically-injured or seizure-induced rats, rather than the punctate staining pattern obtained with Zinpyr-1. Chapter 3: Esterase-Dependent Two-Fluorophore Ratiometric Sensing of Zinc(II). This chapter describes a new approach to ratiometric sensing in which a zinc(II)-sensitive fluorescein fluorophore based on Zinpyr-1 is functionalized with a zinc(lI)-insensitive coumarin fluorophore via a flexible ester linker. The flexible linker enables intramolecular quenching of the two fluorophores. Esterase hydrolysis of the linkerby Carolyn C. Woodroofe.Ph.D

Fluorescence Methods for Investigation of Living Cells and Microorganisms

Fluorescence methods play a leading role in the investigation of biological objects. They are the only non-destructive methods for investigating living cells and microorganisms in vivo. Using intrinsic and artificial fluorescence methods provides deep insight into mechanisms underlying physiological and biochemical processes. This book covers a wide range of modern methods involved in experimental biology. It illustrates the use of fluorescence microscopy and spectroscopy, confocal laser scanning microscopy, flow cytometry, delayed fluorescence, pulse-amplitude-modulation fluorometry, and fluorescent dye staining protocols. This book provides an overview of practical and theoretical aspects of fluorescence methods and their successful application in the investigation of static and dynamic processes in living cells and microorganisms

Novel Carbon Dot-Based Fluorescent Nanomaterials for Biosensing and Bioimaging

Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include optimal synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. In this thesis we have presented the synthesis and development of a series of novel carbon dot based nanoprobes with unique photophysical and biological properties for bioimaging and biosensing applications. These properties include water dispersibility, superior photostability and thermal stability, high quantum yield, excellent two-photon excitability, ease in surface functionalization, rapid cellular uptake, good biocompatibility, rapid detection of targeted molecules with a low detection limit, and high-resolution bioimaging capability. The CD-based probes developed in this study were used for two-photon intensity-based and ratiometric exogenous and endogenous ferric ions sensing in living cells, single- and two-photon deep tissue imaging in synthetic scaffold and complex biological tissue, and two-photon ratiometric real-time intracellular pH monitoring in 3D environment. Furthermore, the influence of CDs synthetic and post-synthetic parameters on parameters on photophysical properties and biological behavior of CDs were comprehensively investigated

Towards an understanding of the role of Connexin26 in breathing

Connexin26 (Cx26) hemichannels expressed in glia at the ventral medullary surface (VMS) have been proposed to play a role in respiratory chemoreception, although this is disputed. At the VMS Cx26 hemichannels open in response to CO2 directly, causing ATP release that is capable of increasing respiratory drive. The main aim of this work was to establish a genetic strategy that can be used in vivo to elegantly remove Cx26 CO2- sensitivity from chemosensitive areas of the VMS, and to thereby investigate the role that Cx26 CO2 sensitivity plays in the chemoreception of awake mice. Using Forster resonance energy transfer and dye loading studies a Cx26 mutant (Cx26DN) was found to coassemble with Cx26WT subunits (forming heteromeric connexon hemichannels) and to remove CO2-induced hemichannel opening from cells stably expressing Cx26WT. In mice aged 12-18 weeks, bilateral lentivirus injections were used to express Cx26DN in GFAP+ cells at the VMS, as a means of removing CO2-induced Cx26 hemichannel opening and subsequent ATP release. As determined by whole-body plethysmography, expression of Cx26DN in the retrotrapezoid nucleus (RTN) had no effect on the hypercapnic ventilatory response in mice. Accidental Cx26DN expression in the caudal chemosensitive area resulted in reduced tidal volume 3 weeks post-transduction, however this was not well supported statistically. Such an auspicious result from suboptimal caudal expression warrants this to be repeated in order to validate these results. The work performed in this thesis outlines the first use of a highly novel genetic tool to remove the CO2-sensitivity property of Cx26 from specific cells, without removing the protein from the system. The results shed light on our understanding of central respiratory chemoreception, suggesting that Cx26 plays no role in chemoreception in the RTN but is likely to play a role in caudal areas of the VMS. Such a tool could aid research into the virtually unexplored role that Cx26 CO2 gating has in the body

TRP Channels in Health and Disease

Almost 25 years ago, the first mammalian transient receptor potential (TRP) channel was cloned and published. TRP channels now represent an extended family of 28 members fulfilling multiple roles in the living organism. Identified functions include control of body temperature, transmitter release, mineral homeostasis, chemical sensing, and survival mechanisms in a challenging environment. The TRP channel superfamily covers six families: TRPC with C for “canonical”, TRPA with A for “ankyrin”, TRPM with M for “melastatin”, TRPML with ML for “mucolipidin”, TRPP with P for “polycystin”, and TRPV with V for “vanilloid”. Over the last few years, new findings on TRP channels have confirmed their exceptional function as cellular sensors and effectors. This Special Book features a collection of 8 reviews and 7 original articles published in “Cells” summarizing the current state-of-the-art on TRP channel research, with a main focus on TRP channel activation, their physiological and pathophysiological function, and their roles as pharmacological targets for future therapeutic options