Photochromic Probe Research Articles

Light-controlled detection of aromatic carboxylate anions using doubly fused naphthopyran derivative

A photochromic naphthopyran derivative, 2,2,11,11-tetraphenyl-2,11-dihydrobenzo[f]pyrano[3,2-h]chromene has been synthesized and its structure was confirmed using various spectroscopic techniques like NMR, infrared spectroscopy (FT-IR), high-resolution mass spectroscopy (HR-MS) and single crystal X-ray diffraction (SCXRD). The synthesized probe has shown a sensitive signal toward salicylic acid and 3,5-diaminobenzoic acid in acetonitrile: water (50:50(v:v)) solvent system at pH 7.6 (1 mM Tris buffer). UV–vis and fluorescence spectroscopic analyses revealed complex formation between these carboxylate anions and the synthesized photochromic probe, with blue shifts in absorption bands & enhancement in fluorescence intensity. Furthermore, photo-switching for the naphthopyran derivative in presence of both carboxylic acids was performed using 365 nm UV light, and results obtained indicated that the release and binding of these carboxylic acids can be controlled using 365 nm UV light. As expected, the fluorescence analysis provided a better limit of detection value as compared to the UV–Vis analysis. Fluorescence spectroscopy yielded a detection limit of 16.29 nM for salicylate ions using the naphthopyran-based probe.

A red-green photochromic bacterial protein as a new contrast agent for improved photoacoustic imaging

The GAF3 domain of the cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803, binding phycocyanobilin as a chromophore, shows photochromicity between two stable, green- and red-absorbing states, characterized by relatively high photoconversion yields. Using nanosecond-pulsed excitation by red or green light, respectively, and suitable cw photoconversion beams, we demonstrate that the light-modulatable photoacoustic waveforms arising from GAF3 can be easily distinguished from background signals originating from non-modulatable competitive absorbers and scattering media. It is demonstrated that this effect can be exploited to identify the position of the photochromic molecule by using as a phantom a cylindrical capillary tube filled with either a GAF3 solution or with an E.coli suspension overexpressing GAF3. These properties identify the high potential of GAF3 to be included in the palette of genetically encoded photochromic probes for photoacoustic imaging.

Background-suppressed tumor-targeted photoacoustic imaging using bacterial carriers

Photoacoustic (PA) imaging offers promise for biomedical applications due to its ability to image deep within biological tissues while providing detailed molecular information; however, its detection sensitivity is limited by high background signals that arise from endogenous chromophores. Genetic reporter proteins with photoswitchable properties enable the removal of background signals through the subtraction of PA images for each light-absorbing form. Unfortunately, the application of photoswitchable chromoproteins for tumor-targeted imaging has been hampered by the lack of an effective targeted delivery scheme; that is, photoswitchable probes must be delivered invivo with high targeting efficiency and specificity. To overcome this limitation, we have developed a tumor-targeting delivery system in which tumor-homing bacteria (Escherichia coli) are exploited as carriers to affect the point-specific delivery of genetically encoded photochromic probes to the tumor area. To improve the efficiency of the desired background suppression, we engineered a phytochrome-based reporter protein (mDrBphP-PCMm/F469W) that displays higher photoswitching contrast than those in the current state of the art. Photoacoustic computed tomography was applied to achieve good depth and resolution in the context of invivo (mice) imaging. The present system effectively integrates a genetically encoded phytochrome-based reporter protein, PA imaging, and synthetic biology (GPS), to achieve essentially background-suppressed tumor-targeted PA monitoring in deep-seated tissues. The ability to image tumors at substantial depths may enable target-specific cancer diagnoses to be made with greater sensitivity, fidelity, and specificity.

Switchable stimulated Raman scattering microscopy with photochromic vibrational probes

Photochromic probes with reversible fluorescence have revolutionized the fields of single molecule spectroscopy and super-resolution microscopy, but lack sufficient chemical specificity. In contrast, Raman probes with stimulated Raman scattering (SRS) microscopy provides superb chemical resolution for super-multiplexed imaging, but are relatively inert. Here we report vibrational photochromism by engineering alkyne tagged diarylethene to realize photo-switchable SRS imaging. The narrow Raman peak of the alkyne group shifts reversibly upon photoisomerization of the conjugated diarylethene when irradiated by ultraviolet (UV) or visible light, yielding “on” or “off” SRS images taken at the photoactive Raman frequency. We demonstrated photo-rewritable patterning and encryption on thin films, painting/erasing of cells with labelled alkyne-diarylethene, as well as pulse-chase experiments of mitochondria diffusion in living cells. The design principle provides potentials for super-resolution microscopy, optical memories and switches with vibrational specificity.

Combined Quantum-Classical Simulation of Photoinduced Electronic Density Redistribution from Biopolymer Segments to Photochromic Probes

The mechanism of fluorescence quenching of the human serum albumin by transferring the energy of the photoinduced electronic excitation from the single tryptophan residue in the structure to the nitrospiropyran donor introduced into its environment is studied by the hybrid computer simulation, including the classical molecular dynamics and the semi-empirical photo-physical calculations for generating the statistical spectra of tryptophan emission and spectra of nitrospiropyran absorption. The probability of the electronic excitation redistribution between the donor and the acceptor is estimated, followed by the photochromic conversion of nitrospiropyran to the merocyanine form, which is readily identifiable due to a significant shift of the longwave absorption band and can be treated as a luminescence detector of the ongoing photoprocesses. The mechanisms of the energy transfer between nonequilibrium fragments in typical combinations of their complex are considered in detail. The general scheme and technical specifics of modeling the optical spectra are illustrated using a simple system of the anthracene molecule in argon. A discussion of several other advanced hybrid approaches of the classical methods in combination with the quantum-mechanical calculations, developed at different theoretical levels and applied in the current computational molecular spectroscopy, is presented.

Palladium induced activation of a substituted naphthopyran for smartphone assisted sensitive and selective sensing in aqueous solution

A substituted naphthopyran moiety was synthesized to act as a probe for metal ions. Structural characterization techniques such as IR, NMR, HRMS, and single-crystal X-ray crystallography were used to establish the structure of the light-responsive photochromic probe. The intermolecular interactions were quantitatively investigated using the crystal explorer program. The affinity of the naphthopyran derivative as a probe for metal ion was investigated using different metal ions in a buffered medium to avoid the effect of change in pH during the recognition process. A bathochromic shift in the absorption band from ~354 nm to ~365 nm (broad band extended up to 450 nm) was observed upon addition of 10 equivalents of palladium ions. The smartphone assisted sensing of palladium ion based on digital images provided an excellent value of LOD (0.69 µM), which was better than the LOD value obtained using UV–visible spectroscopy. The process of complex formation between the probe and the palladium ions was also investigated using NMR and computational methods. The computational methods were used to investigate the effect of water on the stability of the complex geometries. Furthermore, time-dependent DFT calculations were carried out to investigate the UV-Visible absorption spectra observed experimentally for the probe and the complex.

Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmission.

We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chemical properties of the tetrafluoroazobenzene (4FAB) scaffold. Ultraviolet light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks associated with standard AB-based switches. We designed our photoprobes to take advantage of the excellent thermodynamic stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiological buffer) and cis–trans photostationary states above 84%. Furthermore, the well-separated n–π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biological studies. Further chemical functionalization of this 4FAB probe with a maleimide functionality allowed clean cross-linking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacology whilst preserving all the favorable photophysical properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore–protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biologically inert, irradiation with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully saturated heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiological studies of this neurotransmitter.

Target-activated and ratiometric photochromic probe for “double-check” detection of toxic thiols in live cells

Photochromic molecules can achieve reversible isomerization upon alternate light irradiations, which offers a great opportunity to improve the precision of analytes detection and imaging in complicated biological environments. Previous reported photochromic probe exhibited only mono-color switching and an initially fluorescence-ON state that may cause high background signal and impose an adverse impact on the desired sensing precision. To overcome this set-back, we developed a novel photochromic probe with an analyte-activation mode for ratiometric sensing of toxic thiols in both real water samples and live cells. The dynamic dual-fluorescence signal is released only after the fast and selective cleavage of the 2,4-dinitrophenyl sulfonate by the targeted thiophenol derivatives. Consequently, a "double-check" with synchronized dual-fluorescence blinking for analyte detection is successfully employed upon alternate light triggers with rapid response (k=7.2x10(-2) s(-1)), high sensitivity (LOD=6.1 nM) as well as selectivity of thiophenol derivatives over other common thiol species (e.g., GSH, Cys and Hcy). The photochromic probe was successfully introduced to the fast and on-site detection of highly toxic thiophenols in real waste water samples. Moreover, by using confocal laser-scanning microscopy (CLSM) and flow cytometric analysis, the potential applications of this ratiometric photochromic probe for trace toxic thiol sensing in live cells are examined.

Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein–protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.

Remote light-controlled intracellular target recognition by photochromic fluorescent glycoprobes

Development of powerful fluorescence imaging probes and techniques sets the basis for the spatiotemporal tracking of cells at different physiological and pathological stages. While current imaging approaches rely on passive probe–analyte interactions, here we develop photochromic fluorescent glycoprobes capable of remote light-controlled intracellular target recognition. Conjugation between a fluorophore and spiropyran produces the photochromic probe, which is subsequently equipped with a glycoligand “antenna” to actively localize a target cell expressing a selective receptor. We demonstrate that the amphiphilic glycoprobes that form micelles in water can selectively enter the target cell to operate photochromic cycling as controlled by alternate UV/Vis irradiations. We further show that remote light conversion of the photochromic probe from one isomeric state to the other activates its reactivity toward a target intracellular analyte, producing locked fluorescence that is no longer photoisomerizable. We envision that this research may spur the use of photochromism for the development of bioimaging probes.

Encapsulating Naphthalene in an Electron-Deficient MOF to Enhance Fluorescence for Organic Amines Sensing.

Host-guest encapsulation of electron-rich naphthalene molecules into a weakly emissive porous metal-organic framework based on π-electron-deficient (π-acidic) naphthalene diimide tectons leads to orange-emissive crystals, which can be used to sense strongly basic organic amines in a fast response, high photostability, and tunable sensitivity. Moreover, such host-guest inclusion crystals are also a good photochromic probe for the detection of weakly basic N-methyl-2-pyrrolidone and N,N-dimethylformamide molecules.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Photoacoustic tomography (PAT) of genetically encoded probes allows imaging of targeted biological processes with high spatial resolution at depths. Here, we combined multi-scale photoacoustic imaging with, for the first time, a reversibly switchable non-fluorescent bacterial phytochrome BphP1. With a heme-derived biliverdin chromophore, BphP1 has the most red-shifted absorption among reported genetically encoded probes, and is reversibly photoconvertible between its red and near-infrared light absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, enabling differential imaging that substantially removed background signals, enhanced detection sensitivity, increased penetration depth, and improved spatial resolution. In doing so, we monitored tumor growth and metastasis with a ~100 µm resolution at depths approaching 10 mm using photoacoustic computed tomography, and imaged individual cancer cells with a sub-optical-diffraction resolution of ~140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at different length scales.

Analysis of the Solubilisation Power of Detergents by Photochromic Probe

The ability of micelles to solubilize organic substances is a property of fundamental importance in many practical applications, such as in detergents. In this work, a new method to determine the solubilisation power of SDS, in terms of the amount of captured organic substance, was used for the first time. A photochromic probe has been dispersed into an organic phase and its incorporation in the micellar system was monitored. The volume of the organic phase entrapped within the micellar hydrophobic core has been measured by the absorbance peak of the dye sensitizer. In our work, three organic compounds were used to determine the amount of organic solvent solubilised by SDS/water system.

The application of a photochromic probe to monitor the self-assembly of thermosensitive block copolymers

Analysis of the thermal decolouration kinetics of a spirooxazine dye (SOX) incorporated into various thermally responsive, amphiphilc block copolymers, poly(N-isopropyl acrylamide)-block-poly(N-acryloyl morpholine), was used to probe their self-assembly behaviour in water. The Arrhenius plots of the thermal decolouration kinetics for each of the block copolymers displayed a deviation from linearity and this occurred within a temperature range that correlated with their self-assembly into micelles, as measured by dynamic light scattering (DLS). The block copolymers containing SOX units within the PNIPAM section displayed the most notable deviations. The results were interpreted in terms of a change in the local viscosity and polarity sensed by the photochromic probe and its effect on the transition state of the open form on progression to the closed form. The incorporation of the free dye, SOX–PROP, within micelles assembled from amphiphilc poly(n-butyl acrylate)-block-poly(N-acryloyl morpholine), was also apparent from the measured decolouration kinetics.

Synthesis and study of the photochromic behavior of 3-[6′-nitro-1,3,3-trimethylspiro(indolino-2,2′-[2H]-chromen-5-yl)]propenoic acid and its ethyl ester

A new photochromic probe containing a spacer with the reactive carboxyl terminal group at the C5 atom of the photochrome molecule was synthesized. The spectral and kinetic study of the behavior of the new photochromic probes in toluene and ethanol was performed.

Synthesis of N-vinyl-2-pyrrolidone-based branched copolymers via crosslinking free-radical copolymerization in the presence of a chain-transfer agent

Branched copolymers are synthesized via the crosslinking free-radical copolymerization of N-vinyl-2-pyrrolidone and dimethacrylates of various st ructures that is conducted in the presence of the chain-transfer agent 1-decanethiol. The kinetics of this process and the composition of the products are studied by IR spectroscopy, and the content of pendant C=C bonds is estimated. Correlations between the content of dimethacrylate monomer units in the copolymer and the rate of discoloration of a photochromic probe (6-nitrospiropyran) incorporated into the product are established.

Comparative Study of Different Probing Techniques for the Analysis of the Free Volume Distribution in Amorphous Glassy Perfluoropolymers

A comparative study of five different experimental and computational methods is presented for the characterization of the overall free volume (FV) and the free volume element (FVE) size and shape distribution in amorphous glassy perfluoropolymers (PFPs). Experimental results from the photochromic probe (PCP) method, positron annihilation lifetime spectroscopy (PALS), and inverse gas chromatography (IGC) were confronted with literature data from 129Xe NMR spectroscopy, and the experimental data were further compared with molecular dynamics (MD) simulations and a combination of MD studies and the well-known Bondi method as well as a modified Bondi method. An evaluation of the advantages and the limits of each method is presented. This is the first reported study on a so vast number of complementary techniques applied on a single glassy polymer, in this case Hyflon AD perfluoropolymer, and is also the first successful application of the photochromic probe technique in such materials. In two different grades ...

Local molecular dynamics of poly(ethylene glycol) dimethacrylate synthesized in the presence of branched poly(methyl methacrylate): a photochromic probe study

The local molecular dynamics and morphology of poly(ethylene glycol) dimethacrylate networks synthesized in the presence of 0–40% branched poly(methyl methacrylate) were studied using a photochromic probe (6-nitrospiropyran). The change in the free volume of the polymer in the vicinity of the photochromic probe was monitored by the rate of dark bleaching of merocyanine (colored form of 6-nitrospiropyran). The local free volume and the mobility of the polymer chain fragments decrease as the conversion of C=C bonds and the content of branched poly(methyl methacrylate) in the polymer increase.

Free Volume Distribution and Local Environment of a Polymer with Photo-reactive Probe Technique

Free volume distribution of poly (methyl methacrylate) was determined with a photo-reactive probe technique. Fairly large part of the free volume in PMMA has been found to be the more reactive than the reaction in solution using azobenzene as the photochromic probe, while there is some free volume fraction where quantum yield of photoreaction is restricted to zero. Information on the bulk region is clearly observed using a novel photochromic probe, which is obtained by connecting azobenzene to PMMA polymer chain. Effect of polymer relaxation on the quantum yield distribution shows contrast between azobenzene and polymer-connected azobenzene, which reflects the information on vacant or bulk region, respectively. Information on the free volume where the space is crowed by the probe molecule is also obtained by using azobenzene dimer as the photo reactive probe, showing the decrease in the photoreactivity and homogenous quantum yield distribution.

Family of Site-Selective Molecular Optical Switches

[reaction: see text] We describe the design, synthesis, and characterization of a family of thiol-reactive optical switches for labeling proteins and other biomolecules. Site-selective introduction of photochromic probes within biomolecules is being used as part of a new approach for optical control of biomolecular interactions and activities within cells. The thiol-reactive photochromic probes described in this report include a spironaphthoxazine and five spirobenzopyrans. The location of the thiol-reactive group on the spirobenzopyran is different for each probe; this feature can be used to control the geometry of the optical switch within a bioconjugate. The photochromes undergo rapid and reversible, optically driven transitions between a colorless spiro (SP) state and a brightly colored merocyanine (MC) state. The MC absorption of a spironaphthoxazine conjugate is red shifted by more than 100 nm compared to the equivalent spirobenzopyran, which may be exploited for the independent control of the MC to SP transition for up to two different spironaphthoxazine and spirobenzopyran conjugates within the same sample.