Fluorescent optical sensors

Optical sensors are prepared by immobilizing an indicating layer on the distal end of a fiber optic cable. Dyes, enzymes, and antibodies can all be incorporated into the layer using a variety of immobilization techniques. Much of the present work is devoted to developing novel indicating schemes by combining appropriate recognition schemes into polymeric matrices.     

Fluorescent sensors using DNA-functionalized graphene oxide

In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.     

Tunable, colorimetric DNA-based pH sensors mediated by A-motif formation

A colorimetric pH sensor has been developed based on GNP aggregation mediated by A-motif formation under acidic conditions. The pH response of the sensor can be tuned in the range of pH 2-5.5 by changing the length and the sequence of the A-motif forming poly dA tracts.     

Fluorescent sensors based on 2-substituted imidazolidines

A number of 2-substituted imidazolidines were synthesized from N,N′-dibenzylethane-1,2-diamine derivatives and benzaldehyde, naphthalene-1-carbaldehyde, 2-hydroxynaphthalene-1-carbaldehyde, and anthracene-9-carbaldehyde. Examination of the obtained compounds by spectral methods revealed selective sensor properties of 2-(anthracen-9-yl)-1,3-dibenzylimidazolidine and 2-(anthracen-9-yl)-1,3-bis(2-hydroxybenzyl)-imidazolidine for Hg²⁺ and Cu²⁺ ions.     

Fluorescent sensors for organophosphorus nerve agent mimics

We present a small molecule sensor that provides an optical response to the presence of an organophosphorus (OP)-containing nerve agent mimic. The design contains three key features: a primary alcohol, a tertiary amine in close proximity to the alcohol, and a fluorescent group used as the optical readout. In the sensor's rest state, the lone pair of electrons of the basic amine quenches the fluorescence of the nearby fluorophore through photoinduced electron transfer (PET). Exposure to an OP nerve agent mimic triggers phosphorylation of the primary alcohol followed rapidly by an intramolecular substitution reaction as the amine displaces the created phosphate. The quaternized ammonium salt produced by this cyclization reaction no longer possesses a lone pair of electrons, and a fluorescence readout is observed as the nonradiative PET quenching pathway of the fluorophore is shut down.     

Fluorescent proton sensors based on energy transfer

Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical properties of the cassettes, including the pH dependent fluorescence in the xanthene-containing molecules, correlates with the relative energies of the frontier orbitals in these systems. Intracellular sensing of protons is often achieved via sensors that switch off completely at certain pH values, but probes of this type are not easy to locate inside cells in their "off-state". A communication from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642-3) described how the energy transfer cassette 1 could be used for intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the protonation states of the xanthene donors. This work was undertaken to further investigate correlations between structure, photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2-4 were prepared: another one containing pH-sensitive xanthene donors (2) and two "control cassettes" that each have two BODIPY-based donors (3 and 4). Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ～6) and green when the medium is more basic (>～7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer regardless of pH. The cassettes that have BODIPY donors, by contrast, show no significant fluorescence from the donor parts, but the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca. 0.6 and 0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pH-fluorescence profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.     

Fluorescent detection of anions by dibenzophenazine-based sensors

Binding studies of two sulfonamide-functionalized dibenzophenazine-based sensors are reported. Spectroscopic studies showed that both sensors are effective fluorescent turn-on sensors for several anions. Both sensors showed responses to acetate, benzoate, cyanide, and fluoride ions. NMR titrations confirmed the mode of binding of the sensors to be through H-bonding to the sulfonamide groups.     

Enzyme-entrapping membranes for enzyme sensors

A reliable method of physically immobilizing enzymes in cellulose triacetate (TAC) membranes was developed. The method has several advantages compared with analogous ones currently employed; it was possible to prepare enzyme sensors based on immobilized glucose oxidase (GOD) for determination of glucose in standard solutions and control sera, and based on GOD and invertase for determination of sucrose.     

Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging

Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences.     

Fluorescent pH sensors with negligible sensitivity to ionic strength

Optical pH determination has the fundamental disadvantage of measuring a signal that depends on the ionic strength of the sample. The problem originates from the complex relationship between the proton activity and the concentration of the pH-sensitive dye. The effect of ionic strength on the signal depends on the charge of the indicator and its environment, e.g. the immobilisation matrix. We present novel lipophilic fluorescein esters carrying one negative charge. They are embedded in an uncharged, highly proton-permeable hydrogel to give optical pH sensors that show a negligible cross-sensitivity towards ionic strength. The fluorescent dyes differ in their substituents. This variation of substituents results in dissociation constants between 5.5 and 8.5. The indicators were made lipophilic by esterification of the carboxy group with a C(18) alkyl chain. Since their spectral properties are quite similar, two indicators may be used in one sensor. This results in an optical pH sensor with a dynamic range that extends from pH 4.5 to 8.     

Fluorescent sensors for the detection of chemical warfare agents

Along with biological and nuclear threats, chemical warfare agents are some of the most feared weapons of mass destruction. Compared to nuclear weapons they are relatively easy to access and deploy, which makes them in some aspects a greater threat to national and global security. A particularly hazardous class of chemical warfare agents are the nerve agents. Their rapid and severe effects on human health originate in their ability to block the function of acetylcholinesterase, an enzyme that is vital to the central nervous system. This article outlines recent activities regarding the development of molecular sensors that can visualize the presence of nerve agents (and related pesticides) through changes of their fluorescence properties. Three different sensing principles are discussed: enzyme-based sensors, chemically reactive sensors, and supramolecular sensors. Typical examples are presented for each class and different fluorescent sensors for the detection of chemical warfare agents are summarized and compared.     

Fluorescent DNA base replacements: Reporters and sensors for biological systems

We describe the design, synthesis, and properties of nucleoside monomers in which the DNA base is replaced by fluorescent hydrocarbons and heterocycles, and the assembly of these monomers into DNA-like molecules in which the all bases are fluorescent. As monomers, such molecules have useful applications as reporters in the DNA context. The use of fluorescent DNA bases, rather than more traditional fluorophores tethered to the DNA strand, gives a more predictable location and orientation, and yields a more direct response to changes that occur within the helix. In addition to uses as monomers, such compounds can be assembled into polychromophoric oligomers ("oligodeoxyfluorosides" or ODFs). ODFs are water soluble, discrete molecules and are easily arranged into specific sequences by use of a DNA synthesizer. They have displayed a number of properties not readily available in commercial fluorophores, including large Stokes shifts, tunable excitation and emission wavelengths, and sensing responses to physical changes or molecular species in solution. We describe an approach to assembling and screening large sets of oligofluorosides for rapid identification of molecules with desirable properties. Such compounds show promise for applications in biochemistry, biology, environmental and materials applications.     

Suitable potentiometric enzyme sensors for urea and creatinine

Enzyme sensors for urea and creatinine were developed by coupling an ammonia gas-diffusion electrode with triacetate cellulose membranes entrapping urease or creatinine deiminase enzymes. Satisfactory results were obtained by using these sensors both in standard solutions and in authentic biological matrices.