Kuo-Chun Tang

Insulin‐Directed Synthesis of Fluorescent Gold Nanoclusters: Preservation of Insulin Bioactivity and Versatility in Cell Imaging

Fluorescent nanomaterials have received great attention and have been intensively studied, because of their unique optical and photophysical properties, as replacements for conventional organic dyes in optical cell imaging. Although semiconductor quantum dots show promising signals in biomedical imaging, their high inherent cytotoxicity and self-aggregation inside living cells fatally limit pragmatic biomedical applications. Fluorescent nanoclusters (NCs), in contrast, exhibit superior properties such as low toxicity and high biocompatibility. Among the various NCs, much effort has been dedicated to the study of fluorescent Au NCs. Au NCs carry quantum-mechanical properties when their sizes are comparable to or smaller than the Fermi wavelength (ca. 1 nm) of conductive electrons. The fluorescent Au NCs, with their ultrafine size, do not disturb the biological functions of the labeled bioentities; therefore, there is great potential to develop Au NCs as a new luminescent label. For example, Lin et al. successfully used water-soluble fluorescent Au NCs capped with dihydrolipoic acid (AuNC@DHLA) and modified with polyethylene glycol (PEG), bovine serum albumin (BSA), and streptavidin for cell bioimaging. Compared with organic-monolayer-protected Au NCs, the usage of proteins as a green-chemical reducing and stabilizing agent is advantageous because their complex 3D structures can withstand a wide range of pH conditions. Accordingly, Au NC synthesis with BSA and lysozyme has been reported and applied to several devices, such as nanosensors of Hg, CN , and H2O2. [12] Very recently, through the conjugation of BSA–Au NCs to folic acid, targetspecific detection of cancer-cell imaging has been demonstrated. Also, BSA–Au NCs have been applied in MDAMB-45 and HeLa tumor xenograft model imaging. Nevertheless, up to this stage, there has been a lack of reports on bioactive protein-directed fluorescent Au NCs that can still preserve their own biological role. Conversely, using Au nanoparticles encapsulated in certain enzymes, several reports claimed significant changes of enzymatic functionality. The goal of this project is thus to search for a bioactive protein to exploit as a template to direct the growth of fluorescent Au NCs. The resulting protein–Au NC nanocomposites are able to retain bioactivity, so that the associated biological role can be pursued by various imaging techniques. Among a number of proteins of vital importance, insulin is of prime interest. Insulin is a polypeptide hormone comprising only 51 amino acids. Its function primarily lies in the regulation of insulin-responsive tissues and it is also directly/indirectly related to many diseases, including diabetes, Alzheimer s disease, obesity, and aging. Its signaling pathway controls the growth of an organism, and hence exerts a profound influence on metabolism and reproduction. Herein, we report for the first time the synthesis of fluorescent Au NCs by using insulin as a template. The resulting insulin–Au NCs exhibit intense red fluorescence maximized at 670 nm and, more importantly, retain their bioactivity and biocompatibility. Several key experiments have been performed in vitro and/or in vivo to assess their viability and versatility. Detailed synthetic procedures are elaborated in the Supporting Information. In brief, by mixing insulin and HAuCl4 in Na3PO4 buffer by continuously stirring at 4 8C for 12 h, reddish luminescent insulin–Au NCs were readily prepared. The crude product was then purified by centrifugal filtration (4000g) for 30 min with a cutoff of 5 kDa to obtain the insulin–Au NCs for subsequent applications. The absorption and photoluminescence emission spectra of insulin–Au NCs are shown in Figure 1. The emission quantum yield Ff was determined to be 0.07, with observed lifetimes fitted to be 439 ns (4%) and 2041 ns (96%). The inset of Figure 1 displays a high-resolution transmission electron microscopy (HRTEM) image of insulin–Au NCs. From the respective histograms, the as-prepared insulin– Au NCs revealed a spherical shape and good size uniformity (for size distribution, see Figure S1 in the Supporting Information). The diameters of insulin–Au NCs, upon averaging over 100 particles, were calculated to be (0.92 0.03) nm (mainly for Au NCs). The hydrodynamic radii of [*] C.-L. Liu, Y.-H. Hsiao, Dr. C.-W. Lai, C.-W. Shih, Y.-K. Peng, Dr. K.-C. Tang, H.-W. Chang, Prof. P.-T. Chou Department of Chemistry, National Taiwan University 1, Section 4, Roosevelt Road, Taipei 10617 (Taiwan) Fax: (+886)2-369-5208 E-mail: chop@ntu.edu.tw

A New and Facile Method To Prepare Uniform Hollow MnO/Functionalized mSiO2 Core/Shell Nanocomposites

Trifunctional uniform nanoparticles comprising a manganese nanocrystal core and a functionalized mesoporous silica shell (MnO@mSiO(2)(Ir)@PEG, where Ir is an emissive iridium complex and PEG is polyethylene glycol) have been strategically designed and synthesized. The T(1) signal can be optimized by forming hollow core (H-MnO@mSiO(2)(Ir)@PEG) via a novel and facile etching process, for which the mechanism has been discussed in detail. Systematic investigation on correlation for longitudinal relaxation (T(1)) versus core shapes and shell silica porosity of the nanocomposites (MnO, H-MnO, MnO@SiO(2), MnO@mSiO(2), H-MnO@mSiO(2)) has been carried out. The results show that the worm-like nanochannels in the mesoporous silica shell not only increase water permeability to the interior hollow manganese oxide core for T(1) signal but also enhance photodynamic therapy (PDT) efficacy by enabling the free diffusion of oxygen. Notably, the H-MnO@mSiO(2)(Ir)@PEG nanocomposite with promising r(1) relaxivity demonstrates its versatility, in which the magnetic core provides the capability for magnetic resonance imaging, while the simultaneous red phosphorescence and singlet oxygen generation from the Ir complex are capable of providing optical imaging and inducing apoptosis, respectively.

Enhanced plasmonic resonance energy transfer in mesoporous silica-encased gold nanorod for two-photon-activated photodynamic therapy.

The unique optical properties of gold nanorods (GNRs) have recently drawn considerable interest from those working in in vivo biomolecular sensing and bioimaging. Especially appealing in these applications is the plasmon-enhanced photoluminescence of GNRs induced by two-photon excitation at infrared wavelengths, owing to the significant penetration depth of infrared light in tissue. Unfortunately, many studies have also shown that often the intensity of pulsed coherent irradiation of GNRs needed results in irreversible deformation of GNRs, greatly reducing their two-photon luminescence (TPL) emission intensity. In this work we report the design, synthesis, and evaluation of mesoporous silica-encased gold nanorods (MS-GNRs) that incorporate photosensitizers (PSs) for two-photon-activated photodynamic therapy (TPA-PDT). The PSs, doped into the nano-channels of the mesoporous silica shell, can be efficiently excited via intra-particle plasmonic resonance energy transfer from the encased two-photon excited gold nanorod and further generates cytotoxic singlet oxygen for cancer eradication. In addition, due to the mechanical support provided by encapsulating mesoporous silica matrix against thermal deformation, the two-photon luminescence stability of GNRs was significantly improved; after 100 seconds of 800 nm repetitive laser pulse with the 30 times higher than average power for imaging acquisition, MS-GNR luminescence intensity exhibited ~260% better resistance to deformation than that of the uncoated gold nanorods. These results strongly suggest that MS-GNRs with embedded PSs might provide a promising photodynamic therapy for the treatment of deeply situated cancers via plasmonic resonance energy transfer.

The empirical correlation between hydrogen bonding strength and excited-state intramolecular proton transfer in 2-pyridyl pyrazoles.

A series of 2-pyridyl pyrazoles 1a and 1-5 with various functional groups attached to either pyrazole or pyridyl moieties have been strategically designed and synthesized in an aim to probe the hydrogen bonding strength in the ground state versus dynamics of excited-state intramolecular proton transfer (ESIPT) reaction. The title compounds all possess a five-membered-ring (pyrazole)N-H···N(pyridine) intramolecular hydrogen bond, in which both the N-H bond and the electron density distribution of the pyridyl nitrogen lone-pair electrons are rather directional, so that the hydrogen bonding strength is relatively weak, which is sensitive to the perturbation of subtle chemical substitution and consequently reflected from the associated ESIPT dynamics. Various approaches such as (1)H NMR (N-H proton) to probe the hydrogen bonding strength and absorption titration to assess the acidity-basicity property were made for all the title analogues. The results, together with supplementary support provided by a computational approach, affirm that the increase of acidity (basicity) on the hydrogen bonding donor (acceptor) sites leads to an increase of hydrogen-bonding strength among the title 2-pyridyl pyrazoles. Luminescence results and the associated ESIPT dynamics further reveal an empirical correlation in that the increase of the hydrogen bonding strength leads to an increase of the rate of ESIPT for the title 2-pyridyl pyrazoles, demonstrating an interesting relationship among N-H acidity, hydrogen bonding strength, and the associated ESIPT rate.

Excited-State Intramolecular Proton Transfer (ESIPT) Fine Tuned by Quinoline−Pyrazole Isomerism: π-Conjugation Effect on ESIPT

A series of quinoline/isoquinoline-pyrazole isomers (I-III), in which the pyrazole moiety is in a different substitution position, was strategically designed and synthesized, showing a system with five-membered intramolecular hydrogen bonding. Despite the similarity in molecular structure, however, only I undergoes excited-state intramolecular proton transfer, as evidenced by the distinct 560 nm proton-transfer emission and its associated relaxation dynamics. The experimental results support a recent theoretical approach regarding the conjugation effect on a proton (or hydrogen atom) transfer reaction (J. Phys. Chem. A 2009, 113, 4862-4867). The concept simply predicts that more extended pi conjugation, i.e., resonance, for proton-transfer tautomer species could allow for efficient delocalization of excess charge in the reaction center, resulting in a larger thermodynamic driving force for proton transfer.

Probing water micro-solvation in proteins by water catalysed proton-transfer tautomerism

Scientists have made tremendous efforts to gain understanding of the water molecules in proteins via indirect measurements such as molecular dynamic simulation and/or probing the polarity of the local environment. Here we present a tryptophan analogue that exhibits remarkable water catalysed proton-transfer properties. The resulting multiple emissions provide unique fingerprints that can be exploited for direct sensing of a site-specific water environment in a protein without disrupting its native structure. Replacing tryptophan with the newly developed tryptophan analogue we sense different water environments surrounding the five tryptophans in human thromboxane A₂ synthase. This development may lead to future research to probe how water molecules affect the folding, structures and activities of proteins.

Ortho-branched ladder-type oligophenylenes with two-dimensionally π-conjugated electronic properties.

The synthesis, photochemical and electrochemical properties, and electronic structures of a series of star-shaped ladder-type oligophenylenes Sn (n = 7, 10, 13, 16, 19, and 22), including one multibranched case S19mb, are reported and compared with the linear para-phenylene ladders Rn (n = 2-5 and 8) and the stepladder analogues SFn (n = 10, 16, and 22). The n value refers to the number of π-conjugated phenylene rings. Functionalized isotruxenes are the key synthetic building blocks, and S22 is the largest monodispersed ladder-type oligophenylene known to date. The Sn systems possess the structural rigidity of Rn and the ortho-para phenylene connectivity of SFn. Consequently, Sn represents the first class of branched chromophores with fully two-dimensional conjugation in both ground- and excited-state configurations. Evidences include the excellent linear correlations for the optical 0-0 energies or the first oxidation potentials of Sn and Rn against the reciprocal of their n values, delocalized HOMO and LUMO based on density functional theory calculations, and molecule-like fluorescence anisotropy. The resulting model of effective conjugation plane (ECP) for the two-dimensional π-conjugated systems compliments the concept of effective conjugation length (ECL) for one-dimensional oligomeric systems. Other implications of the observed structure-property relationships are also included.

Studies of Excited-State Properties of Multibranched Triarylamine End-Capped Triazines

Electron donor-acceptor types of multibranched triarylamine end-capped triazines have been systematically investigated by steady-state electronic spectroscopy, electrochemistry, femtosecond fluorescence anisotropy and solvent relaxation dynamics. The results, together with computational approach, have gained in-depth insight into their excited-state properties, especially the interactions between branches. Among different branched triarylamines of one, two and three arms, the interbranch interaction between each arm is weak, as evidenced by their nearly identical absorption spectral profile and frontier orbitals analyses. Upon S(0) → S(1) excitation, the electronic delocalization in the three-branched triarylamine end-capped triazine is resolved to be 680 ± 130 fs, followed by a slow (28 ± 3 ps) electronic localization into one branch and consequently a rotational depolarization of 2.0 ± 0.1 ns. Similar delocalization dynamics was resolved for the two-branched triarylamine end-capped triazine (electronic delocalization, 500 ± 90 fs; twisting localization, 21 ± 5 ps; rotational depolarization, 700 ± 30 ps). The comparable electron delocalization and solvent relaxation time scale may set up a new paradigm to investigate their specific correlation in the early time domain.

Structural tuning intra- versus inter-molecular proton transfer reaction in the excited state

A series of 2-pyridyl-pyrazole derivatives 1-4 possessing five-membered ring hydrogen bonding configuration are synthesized, the structural flexibility of which is strategically tuned to be in the order of 1 > 2 > 3 > 4. This system then serves as an ideal chemical model to investigate the correlation between excited-state intramolecular proton transfer (ESIPT) reaction and molecular skeleton motion associated with hydrogen bonds. The resulting luminescence data reveal that the rate of ESIPT decreases upon increasing the structural constraint. At sufficiently low concentration where negligible dimerization is observed, ESIPT takes place in 1 and 2 but is prohibited in 3 and 4, for which high geometry constraint is imposed. The results imply that certain structural bending motions associated with hydrogen bonding angle/distance play a key role in ESIPT. This trend is also well supported by the DFT computational approach, in which the barrier associated with ESIPT is in the order of 1 < 2 < 3 < 4. Upon increasing the concentration in cyclohexane, except for 2, the rest of the title compounds undergo ground-state dimerization, from which the double proton transfer takes place in the excited state, resulting in a relatively blue shifted dimeric tautomer emission (cf. the monomer tautomer emission). The lack of dimerization in 2 is rationalized by substantial energy required to adjust the angle of hydrogen bond via twisting the propylene bridge prior to dimerization.

Novel oxygen sensor based on terfluorene thin-film and its enhanced sensitivity by stimulated emission

Ter(9,9-diarylfluorene) (TDF) neat film featuring oxygen sensing with advantages of fast response, reversibility and high efficiency is reported. The fast O2 fluorescence quenching process (4.0 × 1010 M−1 s−1) is unique and is mainly due to intact amorphous morphology of the TDF film, providing ample porous sites in nature so that O2 can travel therein freely. This, together with a large S1–T1 energy gap of TDF, leads to efficient O2 sensitization, i.e. the O2 induced S1–T1 intersystem crossing. The sensitivity can be further enhanced up to ∼10-fold and ∼20-fold in amplified spontaneous emission (ASE) and lasing action, respectively. The work thus demonstrated a new class of organic materials suited for high-speed, high-sensitivity oxygen sensing.