ABSTRACT: A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.
Project description:Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.
Project description:DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-?m diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58?000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.
Project description:Research on pure organic room-temperature phosphorescent (RTP) materials has made great advances but near-infrared (NIR) RTP emitting materials are still rare. Novel amorphous acrylamide copolymers containing iodine substituted borondipyrromethene (Bodipy) were prepared to obtain strong absorption in the visible region and moderate RTP in the NIR region with much larger Stokes shift than the fluorescence emission of traditional Bodipy dyes. Expensive metals and crystallization were left out to avoid biotoxicity and strict preparation conditions. Monoiodo and diiodo-Bodipy derivatives were both designed to study the substitution effect of iodine atoms. Photophysical properties, phosphorescence quantum yield and lifetime were characterized. Gels with NIR RTP emission were facilely prepared with the incorporation of ureidopyrimidone (UPy) and N,N'-methylenebisacrylamide (MBAA). The mechanical properties of the gels were measured using a rheometer and the results showed that the gels displayed fast self-healing ability due to the strong quadruple hydrogen bonding between UPy moieties.
Project description:The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ?40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.
Project description:A series of complex boronic acids were prepared through multicomponent reactions (MCRs). Both Passerini and Ugi MCRs were carried out in which one component was an arylboronic acid. The resulting highly functionalized boronic acids participated efficiently in the Liebeskind-Srogl cross-coupling reaction with meso-methylthioBODIPY derivatives to yield complex borondipyrromethene (BODIPY) dyes in good yields. The joined spectroscopic and computational study points out the deep impact of the arylated chromophoric position on the photophysical signatures. Thus, unconstrained aryls grafted at the meso position did not sway the spectral band positions but switched on new nonradiative relaxation channels, whereas additional arylation at the opposite ?-pyrrolic position softened such fluorescence quenching and shifted the emission to the red-edge of the visible spectrum. The conducted biological analysis revealed that peripheral blood mononuclear cells incubated with these new compounds showed reduced cytotoxicity and retained their normal activities. Additionally, the dyes remained stable inside the cells after 24 h of incubation. These results demonstrated that these novel fluorescent probes based on BODIPY can be applied for cell imaging and analysis, expanding their applications.
Project description:With the potential for each droplet to act as a unique reaction vessel, droplet microfluidics is a powerful tool for high-throughput discovery. Any attempt at compound screening miniaturization must address the significant scaling inefficiencies associated with library handling and distribution. Eschewing microplate-based compound collections for one-bead-one-compound (OBOC) combinatorial libraries, we have developed h?SABR (Light-Induced and -Graduated High-Throughput Screening After Bead Release), a microfluidic architecture that integrates a suspension hopper for compound library bead introduction, droplet generation, microfabricated waveguides to deliver UV light to the droplet flow for photochemical compound dosing, incubation, and laser-induced fluorescence for assay readout. Avobenzone-doped PDMS (0.6% w/w) patterning confines UV exposure to the desired illumination region, generating intradroplet compound concentrations (>10 ?M) that are reproducible between devices. Beads displaying photochemically cleavable pepstatin A were distributed into droplets and exposed with five different UV intensities to demonstrate dose-response screening in an HIV-1 protease activity assay. This microfluidic architecture introduces a new analytical approach for OBOC library screening, and represents a key component of a next-generation distributed small molecule discovery platform.
Project description:Chloroquine was among the first of several effective drug treatments against malaria until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine as an antimalarial therapeutic, there is potential in efforts to adapt chloroquine for other clinical applications, such as in combination therapies and in diagnostics. In this context, we designed and synthesized a novel asymmetrical squaraine dye coupled with chloroquine (SQR1-CQ). In this study, SQR1-CQ was used to label live Plasmodium falciparum (P. falciparum) parasite cultures of varying sensitivities towards chloroquine. SQR1-CQ positively stained ring, mature trophozoite and schizont stages of both chloroquine?sensitive and chloroquine?resistant P. falciparum strains. In addition, SQR1-CQ exhibited significantly higher fluorescence, when compared to the commercial chloroquine-BODIPY (borondipyrromethene) conjugate CQ-BODIPY. We also achieved successful SQR1-CQ labelling of P. falciparum directly on thin blood smear preparations. Drug efficacy experiments measuring half-maximal inhibitory concentration (IC50) showed lower concentration of effective inhibition against resistant strain K1 by SQR1-CQ compared to conventional chloroquine. Taken together, the versatile and highly fluorescent labelling capability of SQR1-CQ and promising preliminary IC50 findings makes it a great candidate for further development as diagnostic tool with drug efficacy against chloroquine-resistant P. falciparum.
Project description:Suspension arrays based on optical encoded microspheres have attracted great attention for multiplexed detection in gene analysis, protein profiling, early disease diagnosis, treatment monitoring and so on. However, the fluorescence stability of barcodes and detection sensitivity require further improvement to meet the increasing demands of "precision diagnosis". Methods: This work reports a novel suspension array platform based on extremely stable AIEgens (AIE33 and AIE NIR800) microbeads as barcodes and AIEgens (1,1,2,3,4,5-Hexaphenyl-1H-silole, HPS) nanobeads as fluorescent signal reporter coupled with flow cytometry for multiplexed detection. Results: Due to the excellent fluorescent signal amplification effect of the HPS nanobeads, our multiplex assay showed enhanced detection sensitivity, compared to multiplex assay using QDs nanobeads (up to 3-fold improvement) and commercial organic dye of phycoerythrin (up to 5-fold improvement) as the fluorescent signal reporters. Conclusion: Furthermore, validating experiments showed similar detection performance to the clinical gold-standard method of ImmunoCAP for allergen detection in patient serum samples, demonstrating the suspension array platform based on AIEgens microbeads with excellent fluorescence stability and AIEgens nanobeads with strong signal amplification ability is promising for high-sensitivity multiplexed bioassay applications.
Project description:A borondipyrromethene (BODIPY) chromophore is connected to a benzoxazole, benzothiazole, or nitrobenzothiazole heterocycle through an olefinic bridge with trans configuration. Rotation about the two [C-C] bonds flanking the olefinic bridge occurs with fast kinetics in solution, leading to the equilibration of four conformational isomers for each compound. Ensemble spectroscopic measurements in solutions fail to distinguish the coexisting isomers. They reveal instead averaged absorption and emission bands with dependence of the latter on the excitation wavelength. Using high-throughput single-molecule spectroscopy, two main populations of single molecules with distinct spectral centroids are observed for each compound on glass substrates. Computational analyses suggest the two populations of molecules to be conformational isomers with antiperiplanar and periplanar arrangements of the BODIPY chromophores about its [C-C] bond to the olefinic bridge. Thus, statistical analysis of multiple single-molecule emission spectra can discriminate stereoisomers that would otherwise be impossible to distinguish by ensemble measurements alone.
Project description:Characterization of slow chemical reactions is essential for assessing catalytic efficiency in chemistry and biology. Traditionally, chemical reaction rates are obtained from population relaxation kinetics measurements and the Arrhenius equation. Unfortunately, it is difficult to use this approach to characterize reactions wherein concentrations change slowly. Thus, it is interesting to see whether a dynamical view of chemical reactions may be used to obtain the reaction rates of slow processes. In the present work, we perform Brownian dynamics simulations of an asymmetric double-well potential to investigate how enhanced sampling of barrier crossing at transition states improves the characterization of reaction rate constants. We then present the design of a liquid-filled capillary optical fiber-based fluorescence spectrometer, which, like rare events, is also based on Poissonian statistics. We use the instrument to characterize the slow photochemical degradation kinetics of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) in o-dichlorobenzene. We have employed in situ optical microscopy measurements and electrodynamics simulations to characterize the excitation beam profile inside a liquid-filled capillary fiber. We compare the cuvette and capillary fiber sample holders and show that the MEH-PPV fluorescence line shape is independent of the sample holder, as expected. We characterize the photochemical degradation kinetics of MEH-PPV in o-dichlorobenzene solutions placed in the cuvette versus that in the capillary fiber. We observe small and slow changes in the time-dependent fluorescence spectra when the degradation reaction is performed in the cuvette. On the other hand, we are able to characterize reactant-concentration decay and product-concentration buildup from the time-dependent fluorescence spectra recorded during photochemical degradation of MEH-PPV performed inside the capillary optical fiber. Ultrafast optically heterodyne-detected optical Kerr effect spectroscopy and multimode Brownian oscillator analysis provide further insights into the role of bath oscillator modes of friction in the mechanism of MEH-PPV photochemical degradation. Overall, the work presented herein shows that slow photochemical degradation kinetics of MEH-PPV can be successfully and efficiently assessed in the capillary fiber fluorescence spectrometer.