Pre‐targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles

The pre‐targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions—the condensation reaction of aromatic nitriles and aminothiols and the inverse‐electron demand Diels–Alder reaction between tetrazine and trans‐cyclooctene (TCO)—to develop a novel strategy for pre‐targeted imaging of the activity of proteases. The substrate probe ( TCO‐C‐SNAT4 ) can be selectively activated by an enzyme target (e.g. caspase‐3/7), which triggers macrocyclization and subsequent in situ self‐assembly into nanoaggregates retained at the target site. The tetrazine‐imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO‐C‐SNAT4 can be repeatedly injected to generate and accumulate more TCO‐nanoaggregates for click labeling.     

Molecular‐Sieving Membrane by Partitioning the Channels in Ultrafiltration Membrane by In Situ Polymerization

Commercial ultrafiltration membranes have proliferated globally for water treatment. However, their pore sizes are too large to sieve gases. Conjugated microporous polymers (CMPs) feature well‐developed microporosity yet are difficult to be fabricated into membranes. Herein, we report a strategy to prepare molecular‐sieving membranes by partitioning the mesoscopic channels in water ultrafiltration membrane (PSU) into ultra‐micropores by space‐confined polymerization of multi‐functionalized rigid building units. Nine CMP@PSU membranes were obtained, and their separation performance for H₂/CO₂, H₂/N₂, and H₂/CH₄ pairs surpass the Robeson upper bound and rival against the best of those reported membranes. Furthermore, highly crosslinked skeletons inside the channels result in the structural robustness and transfer into the excellent aging resistance of the CMP@PSU. This strategy may shed light on the design and fabrication of high‐performance polymeric gas separation membranes.     

Modulating Cell‐Surface Receptor Signaling and Ion Channel Functions by In Situ Glycan Editing

Glycans anchored on cell‐surface receptors are active modulators of receptor signaling. A strategy is presented that enforces transient changes to cell‐surface glycosylation patterns to tune receptor signaling. This approach, termed in situ glycan editing, exploits recombinant glycosyltransferases to incorporate monosaccharides with linkage specificity onto receptors in situ. α2,3‐linked sialic acid or α1,3‐linked fucose added in situ suppresses signaling through epidermal growth factor receptor and fibroblast growth factor receptor. We also applied the same strategy to regulate the electrical signaling of a potassium ion channel–human ether‐à‐go‐go‐related gene channel. Compared to gene editing, no long‐term perturbations are introduced to the treated cells. In situ glycan editing therefore offers a promising approach for studying the dynamic role of specific glycans in membrane receptor signaling and ion channel functions.     

In Situ Synthesis of Self‐Assembled Gold Nanoparticles on Glass or Silicon Substrates through Reactive Inkjet Printing

A facile and low cost method for the synthesis of self‐assembled nanoparticles (NPs) with minimal size variation and chemical waste by using reactive inkjet printing was developed. Gold NPs with diameters as small as (8±2) nm can be made at low temperature (120 °C). The size of the resulting NPs can be readily controlled through the concentration of the gold precursor and oleylamine ink. The pure gold composition of the synthesized NPs was confirmed by energy‐dispersive X‐ray spectroscopy (EDXS) analysis. High‐resolution SEM (HRSEM) and TEM (HRTEM), and X‐ray diffraction revealed their size and face‐centered cubic (fcc) crystal structure, respectively. Owing to the high density of the NP film, UV/Vis spectroscopy showed a red shift in the intrinsic plasmonic resonance peak. We envision the extension of this approach to the synthesis of other nanomaterials and the production of tailored functional nanomaterials and devices.     

Real‐time Observation of Deep Lithiation of Tungsten Oxide Nanowires by In Situ Electron Microscopy

An in‐depth mechanistic understanding of the electrochemical lithiation process of tungsten oxide (WO₃) is both of fundamental interest and relevant for potential applications. One of the most important features of WO₃ lithiation is the formation of the chemically flexible, nonstoichiometric Li_xWO₃, known as tungsten bronze. Herein, we achieved the real‐time observation of the deep electrochemical lithiation process of single‐crystal WO₃ nanowires by constructing in situ transmission electron microscopy (TEM) electrochemical cells. As revealed by nanoscale imaging, diffraction, and spectroscopy, it is shown that the rapid and deep lithiation of WO₃ nanowires leads to the formation of highly disordered and near‐amorphous Li_xWO₃ phases, but with no detectable traces of elemental W and segregated Li₂O phase formation. These results highlight the remarkable chemical and structural flexibility of the Li_xWO₃ phases in accommodating the rapid and deep lithiation reaction.     

A Far‐Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging

Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐red chemogenetic reporter of protein–protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.     

Fast Gas–Solid Reaction Kinetics of Nanoparticles Unveiled by Millisecond In Situ Electron Diffraction at Ambient Pressure

Acquiring the kinetics of gas–nanoparticle fast reactions under ambient pressure is a challenge owing to the lack of appropriate in situ techniques. Now an approach has been developed that integrates time‐resolved in situ electron diffraction and an atmospheric gas cell system in transmission electron microscopy, allowing quantitative structural information to be obtained under ambient pressure with millisecond time resolution. The ultrafast oxidation kinetics of Ni nanoparticles in oxygen was vividly obtained. In contrast to the well‐accepted Wagner and Mott–Cabrera models (diffusion‐dominated), the oxidation of Ni nanoparticles is linear at the initial stage (<0.5 s), and follows the Avrami–Erofeev model (n=1.12) at the following stage, which indicates the oxidation of Ni nanoparticles is a nucleation and growth dominated process. This study gives new insights into Ni oxidation and paves the way to study the fast reaction kinetics of nanoparticles using ultrafast in situ techniques.     

Unveiling the Advances of Nanostructure Design for Alloy‐Type Potassium‐Ion Battery Anodes via In Situ TEM

Nanostructure design and in situ transmission electron microscopy (TEM) are combined to demonstrate Sb‐based nanofibers composed of bunched yolk–shell building units as a significantly improved anode for potassium‐ion batteries (PIBs). Particularly, a metal–organic frameworks (MOFs)‐engaged electrospinning strategy coupled to a confined ion‐exchange followed by a subsequent thermal reduction is proposed to fabricate yolk–shell Sb@C nanoboxes embedded in carbon nanofibers (Sb@CNFs). In situ TEM analysis reveals that the inner Sb nanoparticles undergo a significant volume expansion/contraction during the alloying/dealloying processes, while the void space can effectively relieve the overall volume change, and the plastic carbon shell maintains the structural integrity of electrode material. This work provides an important reference for the application of advanced characterization techniques to guide the optimization of electrode material design.     

Low‐Fouling Fluoropolymers for Bioconjugation and In Vivo Tracking

The conjugation of hydrophilic low‐fouling polymers to therapeutic molecules and particles is an effective approach to improving their aqueous stability, solubility, and pharmacokinetics. Recent concerns over the immunogenicity of poly(ethylene glycol) has highlighted the importance of identifying alternative low fouling polymers. Now, a new class of synthetic water‐soluble homo‐fluoropolymers are reported with a sulfoxide side‐chain structure. The incorporation of fluorine enables direct imaging of the homopolymer by ¹⁹F MRI, negating the need for additional synthetic steps to attach an imaging moiety. These self‐reporting fluoropolymers show outstanding imaging sensitivity and remarkable hydrophilicity, and as such are a new class of low‐fouling polymer for bioconjugation and in vivo tracking.     

Toehold‐initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA‐mediated regulatory circuits and their disease associations. A toehold‐initiated strand‐displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold‐initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.     

Conjugated Polymer Nanoparticles with Appended Photo‐Responsive Units for Controlled Drug Delivery,Release, and Imaging

Carriers that can afford tunable physical and structural changes are envisioned to address critical issues in controlled drug delivery applications. Herein, photo‐responsive conjugated polymer nanoparticles (CPNs) functionalized with donor–acceptor Stenhouse adduct (DASA) and folic acid units for controlled drug delivery and imaging are reported. Upon visible‐light (λ=550 nm) irradiation, CPNs simultaneously undergo structure, color, and polarity changes that release encapsulated drugs into the cells. The backbone of CPNs favors FRET to DASA units boosting their fluorescence. Notably, drug‐loaded CPNs exhibit excellent biocompatibility in the dark, indicating perfect control of the light trigger over drug release. Delivery of both hydrophilic and hydrophobic drugs with good loading efficiency was demonstrated. This strategy enables remotely controlled drug delivery with visible‐light irradiation, which sets an example for designing delivery vehicles for non‐invasive therapeutics.