Youngsam Kim

Enhanced Fluorescence Turn-on Imaging of Hypochlorous Acid in Living Immune and Cancer Cells.

Two closely related phenyl selenyl based boron-dipyrromethene (BODIPY) turn-on fluorescent probes for the detection of hypochlorous acid (HOCl) were synthesized for studies in chemical biology; emission intensity is modulated by a photoinduced electron-transfer (PET) process. Probe 2 intrinsically shows a negligible background signal; however, after reaction with HOCl, chemical oxidation of selenium forecloses the PET process, which evokes a significant increase in fluorescence intensity. The fluorescence intensity of probes 1 and 2 with HOCl involves an ∼18 and ∼50-fold enhancement compared with the respective responses from other reactive oxygen/nitrogen species (ROS/RNS) and low detection limits (30.9 nm for 1 and 4.5 nm for 2). Both probes show a very fast response with HOCl; emission intensity reached a maximum within 1 s. These probes show high selectivity for HOCl, as confirmed by confocal microscopy imaging when testing with RAW264.7 and MCF-7 cells.

A selective fluorescent probe for cysteine and its imaging in live cells

A probe for the detection of cysteine over homocysteine based on 2-(2′-hydroxyphenyl)benzothiazole (HBT) was prepared and used in confocal microscopy experiments. The probe was designed to block excited state intramolecular proton transfer (ESIPT). When bromopropionyl group protection is removed, HBT is recovered via nucleophilic substitution and intramolecular cyclization. The probe was found to have a detection limit of 2.8 μM and exhibits a ∼20-fold increase. The probe showed cell membrane permeability and efficacy in living Hep3B cells.

Extremely selective fluorescence detection of cysteine or superoxide with aliphatic ester hydrolysis

A novel fluorescence probe modality demonstrated with fluorescein affords a highly selective aqueous-based detection of cysteine over other biothiols, e.g. homocysteine, with a limit of detection of 11.3 μM.

Nerve agent simulant diethyl chlorophosphate detection using a cyclization reaction approach with high stokes shift system

A reaction-based fluorescent probe (CoumNMe2) containing a coumarin-4-dimethyaminoaryl scaffold for the detection of nerve agent simulants was developed. The probe showed fluorescence enhancement selectively with diethyl chlorophosphate (DCP) over close competitors diethyl cyanophosphonate (DECP), and diethyl methylphosphonate (DEMP) with very little interference from metal ions. O–P bond formation by reaction of the benzyl alcohol motif on the probe with DCP, favours intramolecular cyclization that leads to the ammonium salt. The cyclization strongly inhibits the photo-induced electron transfer (PET) process, which leads to the enhancement of fluorescence intensity (∼10 fold). Also, DFT/TDDFT calculations were exploited to explain the nature of the fluorescence “turn-on” process. Herein, we report a new fluorescent probe (CoumNMe2) based on the intramolecular cyclization reaction for the detection of nerve agent simulant and has potential for real applications.

Diselenide-based probe for the selective imaging of hypochlorite in living cancer cells

A non-traditional and robust probe skeleton was derivatized for chemosensing applications to investigate a potential novel mode of hypochlorite detection. The BDPP-DSe probe gave a ∼180-fold “turn-on” response to hypochlorite. Confocal fluorescence imaging demonstrated detection of hypochlorite in living cells and cell membrane permeability.

Substituent Effects in BODIPY in Live Cell Imaging

Small-molecule organoselenium-based fluorescent probes possess great capacity in understanding biological processes through the detection of various analytes such as reactive oxygen/nitrogen species (ROS/RNS), biothiols (cysteine, homocysteine and glutathione), lipid droplets, etc. Herein, we present how substituents on the BODIPY system play a significant part in the detection of biologically important analytes for in vitro conditions and live cell imaging studies. The fluorescence of the probe was quenched by 2-chloro and 6-phenyl selenium groups; the probe shows high selectivity with NaOCl among other ROS/RNS, and gives a turn-on response. The maximum fluorescence intensity is attained within ≈1-2 min with a low detection limit (19.6 nm), and shows a ≈110-fold fluorescence enhancement compared to signals generated for other ROS/RNS. Surprisingly, in live cell experiments, the probe specifically located and accumulated in lipid droplets, and showed a fluorescence turn-on response. We believe this turn-on response occurred because of aggregation-induced emission (AIE), which surprisingly occurred only by introducing one lipophilic mesityl group at the meso position of the BODIPY.

Thiomaleimide functionalization for selective biological fluorescence detection of peroxynitrite as tested in HeLa and RAW 264.7 cells

The role of fluorescent molecules in diagnosis, treatment as well as in biomedical research has great current medicinal significance and is the focus of concentrated effort across the scientific research spectrum. Related research continues to reveal new practical sensing systems that bear enhanced features for interfacing of substituted molecules with biological systems. As part of an effort to better understand chalcogenide systems, a new dithiomaleimide BODIPY (BDP-NGM) probe has been designed, synthesized and characterized. The fluorescence of BDP-NGM was quenched by the incorporation of [3,4-bis(phenylthio)] on the maleimide-4-phenyl moiety which is, in turn, placed at the meso-position of the BODIPY system. The probe shows a turn-on fluorescence response upon reaction with ONOO- ; mass spectral evidence reveals peaks in agreement with products involving oxidation of the sulfur groups to sulfone groups. An about 18.0-fold emission intensity enhancement was found. By comparison, the emission signal from another ROS/RNS, superoxide, gave a modest turn on signal (≈5.0-fold). The reaction is complete within 10 min, judging from the monitoring of the turn-on fluorescence process; the detection limit was found to be 0.4 μm. BDP-NGM can be used for the detection of ONOO- under both acidic and basic conditions. Live cell imaging showed that the current probe can be used for the selective detection of ONOO- in living systems.

Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems

The development of novel fluorescent probes for monitoring the concentration of various biomolecules in living systems has great potential for eventual early diagnosis and disease intervention. Selective detection of competitive species in biological systems is a great challenge for the design and development of fluorescent probes. To improve on the design of fluorescent coumarin-based biothiol sensing technologies, we have developed herein an enhanced dual emission doubly activated system (DACP-1 and the closely related DACP-2) for the selective detection of glutathione (GSH) through the use of one optical channel and the detection of cysteine (Cys) by another channel. A phenylselenium group present at the 4-position completely quenches the fluorescence of the probe via photoinduced electron transfer to give a nonfluorescent species. Probes are selective for glutathione (GSH) in the red region and for cysteine/homocysteine (Cys/Hcy) in the green region. When they were treated with GSH, DACP-1 and DACP-2 showed strong fluorescence enhancement in comparison to that for closely related species such as amino acids, including Cys/Hcy. Fluorescence quantum yields (ΦF) increased for the red channel (<0.001 to 0.52 (DACP-1) and 0.48 (DACP-2)) and green channel (Cys) (<0.001 to 0.030 (DACP-1) and 0.026 (DACP-2)), respectively. Competing fluorescent enhancements upon addition of closely related species were negligible. Fast responses, improved water solubility, and good cell membrane permeability were all properly established with the use of DACP-1 and DACP-2. Live human lung cancer cells and fibroblasts imaged by confocal microscopy, as well as live mice tumor model imaging, confirmed selective detection.

Fluorescent Sensing of a Nerve Agent Simulant with Dual Emission over Wide pH Range in Aqueous Solution

A new 1,8-naphthalimide-based fluorescent probe for the detection of diethyl cyanophosphonate, a very common nerve agent simulant, is designed, synthesized, and characterized fully. The probe shows around 50-fold enhancement of fluorescence intensity over other nerve agent simulants. Importantly, the probe is able to work under aqueous conditions in a wide pH range. Two reactive groups, the oxime and the phenol, allow a dual emission with different kinetic reactions. The reaction of diethyl cyanophosphonate with the oxime group occurs in advance; the resulting time response of the fluorescence enhancement is observed within approximately 30 s. After the oxime reaction, then phenol also undergoes a substitution reaction with diethyl cyanophosphonate, resulting in a blue emission. The real application of this new probe is demonstrated through the use of silica plate assays for the detection of diethyl cyanophosphonate in both gas and liquid phases through dual emission channels.

A fluorogenic and red-shifted diphenyl phosphinate-based probe for selective peroxynitrite detection as demonstrated in fixed cells

The development of NIR fluorescent probes for biomedical applications has outstanding potential because of the lower background fluorescence present involving less scattering and deeper tissue penetration, resulting in limited damage to living cells. Recently, dicyanomethylene-4H-pyran systems have been utilized as an NIR fluorophore for the detection of various analytes and in cell imaging studies. These efforts have involved single- as well as two-photon microscopy. As part of an effort to generate novel probes that can allow biologists/neurobiologists better visualization/quantification of the role of reactive oxygen/nitrogen species in the living system, a new dicyanomethylene-4H-pyran-based fluorescent probe DCPO-DP has been designed, synthesized and characterized. The fluorescence of the probe is pre-quenched by hydroxyl protection (diphenyl phosphinate protecting group). A selective “TURN-ON” fluorescence response involving an ∼120-fold emission intensity enhancement was observed upon reaction with ONOO−, compared with emission signals arising from other ROS/RNS. The reaction was completed within 20 min. The detection limit was determined to be 4.62 μM. Fixed cells were used in imaging experiments and helped demonstrate this novel chemosensing strategy for the detection of ONOO− in living systems with phosphinates.