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Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid-base coils-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein-protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal to noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, PPI-CLAPon1.1 with higher sensitivity is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-minute calcium-dependent PPI with a SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.

Many medical and chemical applications require target molecules to be delivered in a controlled manner at precise locations. Metal-organic frameworks (MOFs) have high porosity, large surface area, and tunable functionality and are promising carriers for such purposes. Current approaches for incorporating target molecules are based on multistep postfunctionalization. Here, we report a novel approach that combines MOF synthesis and molecule encapsulation in a one-pot process. We demonstrate that large drug and dye molecules can be encapsulated in zeolitic imidazolate framework (ZIF) crystals. The molecules are homogeneously distributed within the crystals, and their loadings can be tuned. We show that ZIF-8 crystals loaded with the anticancer drug doxorubicin (DOX) are efficient drug delivery vehicles in cancer therapy using pH-responsive release. Their efficacy on breast cancer cell lines is higher than that of free DOX. Our one-pot process opens new possibilities to construct multifunctional delivery systems for a wide range of applications.

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude.

Precisely identifying biological targets and accurately extracting their relatively weak signals from complicated physiological environments represent daunting challenges in biological detection and biomedical diagnosis. Fluorescence techniques have become the method of choice and offer minimally invasive and ultrasensitive detections, thus, providing a wealth of information regarding the biological mechanisms in living systems. Despite fluorescence analysis has advanced remarkably, conventional detections still encounter considerable limitations. This stems from the fact that the fluorescence intensity signal (I) is sensitive and liable to numerous external factors including temperature, light source, medium characteristics, and dye concentration. The interferences exasperatingly undermine the precision of measurements, and frequently render the signal undetectable. For example, fluorescence from single-molecule emitters can be measured on glass substrates under optimum conditions, but single-molecule events in complicated physiological environments such as live cells can hardly be detected because of autofluorescence interference and other factors. Furthermore, traditional intensity (I) and wavelength (λ) measurements do not reveal the interactive nature between the donor and the acceptor. Thus, innovative detection strategies to circumvent these aforementioned limitations of the conventional techniques are critically needed. With the use of photoswitching-induced donor-acceptor-fluorescence double modulations, we present a novel strategy that introduces three additional physical parameters: modulation amplitude (A), phase shift (ΔΦ), and lock-in frequency (ω), and demonstrate that such a strategy can circumvent the limitation of the conventional fluorescence detection techniques. Together, these five physical quantities (I, λ, A, ΔΦ, ω) reveal insightful information regarding molecular interactive strength between the probe and the analyte and enable extracting weak-fluorescence spectra from large interfering noises in complex environments.

The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 μmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) M(-1) in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.

[EN] A new molecular sieve, ITQ-38, containing interconnected large and medium pores in its structure has been synthesized. The rational combination of dicationic piperidine-derivative molecules as organic structure directing agents (OSDAs) with germanium and boron atoms in alkaline media has allowed the synthesis of ITQ-38 zeolite. High-resolution transmission electron microscopy (HRTEM) has been used to elucidate the framework topology of ITQ-38, revealing the presence of domains of perfect ITQ-38 crystals as well as very small areas containing nanosized ITQ-38/ITQ-22 intergrowths. The structure of ITQ-38 is highly related to ITQ-22 and the recently described polymorph C of ITQ-39 zeolite. It shares a common building layer with ITQ-22 and contains the same building unit as the polymorph C of ITQ-39. All three structures present similar framework density, 16.1 T atoms/1000 angstrom(3)., Financial support by the Spanish MICINN (MAT2009-14528-C02-01 and MAT2006-14274-C02-01), Consolider Ingenio 2010-Multicat, Generalitat Valenciana by the PROMETEO program, UPV through PAJD-06-11 (n.1952), the Swedish Research Council (VR), the Swedish Governmental Agency for Innovation Systems (VINNOVA) and Goran Gustafsson Foundtion for Natural Sciences and Medical Research, is acknowledged. Manuel Moliner also acknowledges the "Subprograma Ramon y Cajal" for the contract RYC-2011-08972. Wei Wan was supported by a postdoctoral grant from the Carl-Trygger Foundation. The EM facility was supported by Knut and Alice Wallenberg Foundation. The authors thank the beamline BM01B at ESRF in Grenoble for beamtime allocation (exp. CH-2493). Gunnel Karlsson is kindly thanked for TEM sample preparation by ultramicrotomy. Dedicated to Prof. Miguel A. Miranda on his 60th Birthday.

A single probe has limited brightness in time-domain imaging and such limitation frequently renders individual molecules undetectable in the presence of interference or complex cellular structures. However, a single photoswitchable probe produces a signal, which can be separated from interference or noise using photoswitching-enabled Fourier transformation (PFT). As a result, the light-modulated probes can be made super bright in the frequency domain simply by acquiring more cycles in the time domain.

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.