High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Determining the Subnanometer Thickness of the Water-Depletion Layer at the Interface between Water and the Hydrophobic Substrate.

Surface plasmon resonance (SPR) is one of the most popular and powerful techniques for label-free detecting and quantitatively analyzing the interfacial refractive index (RI). So far, most of the SPR measurements are mainly applied to detect the relative change of RI upon biological and chemical events occurring at the interface, while the determinations on the absolute value of RI remains challenging. However, the absolute value of RI has become increasingly urgent in some cases, such as the existence and physical properties of the water depletion layer (WDL). WDL refers to a subnanometer-thick layer with reduced density between water and the hydrophobic substrate. The detailed explanations of how water meets hydrophobic surface have been studied by several kinds of techniques for decades but it remains under debate. In this work, we successfully established a method to measure the absolute RI at a gold-liquid interface by surface plasmon resonance microscopy (SPRM) and 2D Fourier transformation image processing and further applied this method to study the existence and physical nature of WDL. It was found that a 0.6 nm thick WDL existed at the interface of water and the hydrophobic substrate, leading to a reduced refractive index of 1.3295 ± 0.0006 compared with the standard value of 1.3325. Our results further indicated that the WDL consisted of a uniform layer rather than numerous isolated surface nanobubbles that distributed at the interface with high density.

Label-Free Optical Imaging of the Dynamic Stick-Slip and Migration of Single Sub-100-nm Surface Nanobubbles: A Superlocalization Approach.

The past decade has witnessed theoretical and experimental debates on the extraordinary long lifetime and low contact angle of surface nanobubbles. While several kinds of imaging techniques have provided promising evidence on the lifetime and gaseous nature of single surface nanobubble, each of them suffered from its own limitations before a consensus can be reached. In the present work, we employ a recently developed surface plasmon resonance microscopy (SPRM) to nonintrusively visualize single sub-100-nm surface nanobubble without labeling for the first time. The quantitative dependence between optical signal and nanobubble volume allows for resolving the dissolution kinetics, which is a key for understanding the lifetime. A superlocalization method is further introduced to monitor the trajectory of its mass center during dissolution, which uncovers the stick-slip behavior in the early stage and the migration behavior in the late stage. The label-free, nonintrusive, quantitative and sensitive features of SPRM and the potential compatibility with atomic force microscopy shed new light on the long-standing puzzle behind surface nanobubbles.

Tracking the rotation of single CdS nanorods during photocatalysis with surface plasmon resonance microscopy.

While rotational dynamics of anisotropic nanoobjects has often been limited in plasmonic and fluorescent nanomaterials, here we demonstrate the capability of a surface plasmon resonance microscopy (SPRM) to determine the orientation of all kinds of anisotropic nanomaterials. By taking CdS nanorods as an example, it was found that two-dimensional Fourier transform of the asymmetrical wave-like SPRM image resulted in a peak in its angular spectrum in k space. Consistency between the peak angle and the geometrical orientation of the nanorod was validated by both in situ scanning electron microscope characterizations and theoretical calculations. Real-time monitoring of the rotational dynamics of single CdS nanorods further revealed the accelerated rotation under appropriate reaction conditions for photocatalyzed hydrogen generation. The driving force was attributed to the asymmetric production of hydrogen molecules as a result of inhomogeneous distribution of reactive sites within the nanorod. The present work not only builds the experimental and theoretical connections between the orientation of anisotropic nanomaterials and its SPRM images; the general suitability of SPRM also sheds light on broad types of nonfluorescent and nonplasmonic anisotropic nanoobjects from semiconductors to bacteria and viruses.

Total Internal Reflection-Based Extinction Spectroscopy of Single Nanoparticles.

Herein we report a reflection-mode total internal reflection microscopy (TIRM) to measure the extinction spectrum of individual dielectric, plasmonic, or light-absorbing nanoparticles, and to differentiate absorption and scattering components from the total optical output. These capabilities were enabled via illuminating the sample with evanescent wave of which the lightpath length was comparable with the size of single nanoparticles, leading to a dramatically improved reflectance change (ΔI/I0 ) up to tens of percent. It was further found that scattering and absorption of light contributed to bright and dark centroids, respectively, in the optical patterns of single nanoparticles, allowing to distinguish scattering and absorption components from the extinction spectrum by the use of an appropriate image processing method. In addition, wide-field feature of TIRM enabled the studies on tens of nanoparticles simultaneously with gentle illumination.

Point Spread Function of Objective-Based Surface Plasmon Resonance Microscopy.

Objective-based surface plasmon resonance microscopy (SPRM) is a novel optical imaging technique that can map the spatial distribution of a local refractive index based on propagating surface plasmon polaritons (SPPs). Different from some other optical microscopy that shows a dot-like point spread function (PSF), a nanosized object appears as a wave-like pattern containing parabolic tails in SPRM. The geometrical complexity of the wave-like pattern hampered the quantitative interpretation of the PSF of SPRM. Previous studies have shown that two adjacent rings were obtained in the frequency domain by applying a two-dimensional Fourier transform to such patterns. In the present work, a ring-fitting method was developed to extract geometrical features out of the dual rings and to connect these features with several experimental parameters. It was found that the radius of ring equaled to the wavevector of SPPs. Its orientation revealed the propagation direction of SPPs. The coordinate distance of the center of ring gave the parallel component of the wavevector of the incident light, which was regulated by the incident angle. The ring-broadening factor reflected the propagation length of SPPs in a reciprocal relationship. Systematical and quantitative interpretations in the frequency domain not only advanced the basic understanding on the PSF of SPRM but also opened up the possibility to utilize these frequency-domain features for detection and sensing purposes in future.

Visualizing the Zero-Potential Line of Bipolar Electrodes with Arbitrary Geometry.

In a typical bipolar electrochemistry (BPE) configuration, voltage applied between the two driving electrodes induced a potential drop through solution filled in the microchannel, resulting in an interfacial potential difference between solution and BPE varied along the BPE. In the present work, we employed a recently developed plasmonic imaging technique to map the distribution of surface potential of bipolar electrodes with various geometries including round, triangle, hexagon, star, and rhombus shapes under the nonfaradaic charging process, from which the line of zero potential (LZP) was visualized and determined. We further investigated the dependence of LZP on electrode geometry and the distribution of external electric field and explained the experimental results with a charge balance mechanism. The triangular and star-shaped BPEs show quite different LZP features from the other ones with symmetrical geometry. These experimentally obtained potential distributions are all in good agreement with electromagnetic simulations. Finally, the line of zero overpotential (LZO) of the triangular-shaped BPE under faradaic reactions were investigated. The results confirm the shift of LZO when faradaic reactions occurred at the corresponding ends of BPE. The present work demonstrates the first experimental capability to map the potential distribution of BPE with arbitrary geometry under an arbitrary driving field. It is anticipated to help the design and optimization on the geometry of electrodes and microchannels with implications for boosting their applications in chemical sensing and materials synthesis.

Intermittent photocatalytic activity of single CdS nanoparticles.

Semiconductor photocatalysis holds promising keys to address various energy and environmental challenges. Most studies to date are based on ensemble analysis, which may mask critical photocatalytic kinetics in single nanocatalysts. Here we report a study of imaging photocatalytic hydrogen production of single CdS nanoparticles with a plasmonic microscopy in an in operando manner. Surprisingly, we find that the photocatalytic reaction switches on and off stochastically despite the fact that the illumination is kept constant. The on and off states follow truncated and full-scale power-law distributions in broad time scales spanning 3-4 orders of magnitude, respectively, which can be described with a statistical model involving stochastic reactions rates at multiple active sites. This phenomenon is analogous to fluorescence photoblinking, but the underlying mechanism is different. As individual nanocatalyst represents the elementary photocatalytic platform, the discovery of the intermittent nature of the photocatalysis provides insights into the fundamental photochemistry and photophysics of semiconductor nanomaterials, which is anticipated to substantially benefit broad application fields such as clean energy, pollution treatment, and chemical synthesis.

Plasmonic Imaging of the Interfacial Potential Distribution on Bipolar Electrodes.

Bipolar electrochemistry is based on the gradient distribution of free-electron density along an electrically isolated electrode, which causes a positive electrode potential at one end and a negative potential at the other, allowing for wide applications in analytical chemistry and materials science. To take full advantage of its wireless and high-throughput features, various types of optical probes, such as pH indicators and fluorescence and electrochemiluminescence reagents, have often been used to indirectly monitor the interfacial electron transfer through chromogenic or fluorogenic reactions. Herein, we report the first probe-free imaging approach that can directly visualize the distribution of the interfacial potential in bipolar electrodes, providing essential information for the validation and development of the theory and applications of bipolar electrochemistry. This approach is based on the sensitive dependence of surface plasmon resonance imaging on the local electron density in the electrode, which enables the direct mapping of potential with a spatial resolution close to the optical diffraction limit, a temporal resolution of 50 ms, and a sensitivity of 10 mV. In addition, in contrast to previous optical readouts that relied on faradaic reactions, the present work achieved the impedance-based measurements under non-faradaic conditions. It is anticipated that this technique will greatly expand the application of bipolar electrochemistry as a platform for chemical and biosensing.

Visualizing the bidirectional electron transfer in a Schottky junction consisting of single CdS nanoparticles and a planar gold film.

Bidirectional electron transfer crossing the metal/semiconductor interface regulates the light absorption and carrier separation efficiency of plasmonic-semiconductor hybrid nanomaterials. Existing studies have been largely focused on a localized surface plasmon resonance (LSPR) effect contributed by an ensemble of metal nanomaterials. Herein, we constructed a Schottky junction that consisted of single CdS nanoparticles and a planar gold film, and investigated hot electrons excited by the surface plasmon polaritons (SPPs) propagating in the gold film. When illuminating the interface with blue light, photoinduced electrons were found to inject from the CdS nanoparticle to the gold film. In a reverse process, SPPs were generated on shining a red beam into the gold film via a Kretschmann configuration, resulting in the injection of hot electrons into CdS nanoparticles. A recently developed plasmonic microscopy method was employed to monitor the entire process, providing the ability to image a single nanoparticle to visualize the bidirectional electron transfer dynamics in a Schottky junction involving propagating SPPs. The present study advances the understanding of the mechanism of hot electron transfer, which is anticipated to aid in the rational design and optimization of plasmonic-semiconductor hybrid nanomaterials with broad applications in photocatalysis, photovoltaic devices, and photoelectrochemical sensing.

Optical Imaging of Phase Transition and Li-Ion Diffusion Kinetics of Single LiCoO(2) Nanoparticles During Electrochemical Cycling.

Understanding the phase transition and Li-ion diffusion kinetics of Li-ion storage nanomaterials holds promising keys to further improve the cycle life and charge rate of the Li-ion battery. Traditional electrochemical studies were often based on a bulk electrode consisting of billions of electroactive nanoparticles, which washed out the intrinsic heterogeneity among individuals. Here, we employ optical microscopy, termed surface plasmon resonance microscopy (SPRM), to image electrochemical current of single LiCoO2 nanoparticles down to 50 fA during electrochemical cycling, from which the phase transition and Li-ion diffusion kinetics can be quantitatively resolved in a single nanoparticle, in operando and high throughput manner. SPRM maps the refractive index (RI) of single LiCoO2 nanoparticles, which significantly decreases with the gradual extraction of Li-ions, enabling the optical read-out of single nanoparticle electrochemistry. Further scanning electron microscopy characterization of the same batch of nanoparticles led to a bottom-up strategy for studying the structure-activity relationship. As RI is an intrinsic property of any material, the present approach is anticipated to be applicable for versatile kinds of anode and cathode materials, and to facilitate the rational design and optimization toward durable and fast-charging electrode materials.

Determining the Absolute Concentration of Nanoparticles without Calibration Factor by Visualizing the Dynamic Processes of Interfacial Adsorption.

Previous approaches of determining the molar concentration of nanoparticles often relied on the calibration factors extracted from standard samples or required prior knowledge regarding the geometry, optical, or chemical properties. In the present work, we proposed an absolute quantification method that determined the molar concentration of nano-objects without any calibration factor or prior knowledge. It was realized by monitoring the dynamic adsorption processes of individual nanoparticles with a high-speed surface plasmon resonance microscopy. In this case, diffusing nano-objects stochastically collided onto an adsorption interface and stayed there ("hit-n-stay" scenario), resulting in a semi-infinite diffusion system. The dynamic processes were analyzed with a theoretical model consisting of Fick's laws of diffusion and random-walk assumption. The quantification of molar concentration was achieved on the basis of an analytical expression, which involved only physical constants and experimental parameters. By using spherical polystyrene nanoparticles as a model, the present approach provided a molar concentration with excellent accuracy.

An electrochemiluminescent detector based on multi-wall-carbon-nanotube/Nafion/Ru(bpy)3(2+) composite film modified heated-electrode.

A new electrochemiluminescent detection system containing a modified and electrically controlled heating carbon paste electrode was developed. Ru(bpy)3(2+) was immobilized in multi-wall carbon nanotube/Nafion composite film, the film was then coated on the surface of carbon paste electrode and the temperature of the electrode could be controlled electrically. The tripropylamine was used to evaluate this ECL detection system with heating electrode. The higher sensitivity, wider linear range and lower relative standard deviation were obtained compared with the unheated modified electrode.

Fabrication of an electrically heated indium-tin-oxide electrode for electrochemiluminescent detection system.

An electrochemiluminescence (ECL) detection system equipped with an electrically heated indium-tin-oxide (ITO) electrode was constructed. The heated ITO electrode (HITOE) coupled the advantages of a heated electrode with the optical transparency property of ITO glass. Compared with the conventional heated electrode, the HITOE is integrated into the ECL cell, which could save the expensive luminescent reagent. The temperature of the electrode (T(e)) could be accurately controlled by electrically heating. The Ru(bpy)(3)(2+)/TPrA ECL system and the colchicine/Ru(bpy)(3)(2+) ECL system were used to evaluate the developed ECL detection system. The higher sensitivity and lower RSD of ECL detection for tri-n-propylamine (TPrA) and colchicine were gained by moderately heating the electrode. The proposed method has been successfully utilized to the assay of colchicine in a spiked human serum sample. The results indicated that the developed ECL detection system could provide high sensitivity and excellent reproducibility for analytical practice.

Capillary electrophoresis chemiluminescent detection system equipped with a two-step postcolumn flow interface for detection of some enkephalin-related peptides labeled with acridinium ester.

Despite its low equipment cost and simple design, as one of the sensitive detectors for CE, the chemiluminescence (CL) detector was less developed compared to the detectors of MS and LIF. The main reasons were the limitation of CL reagents, the repeatability problems and the relatively low sensitivity compared to LIF. In this paper, a highly sensitive CE-CL detection system was developed for detection of some enkephalin-related peptides labeled with acridinium ester. A new detection interface was designed for CE with CL detection of acridinium ester and its labeled analytes. The interface included two sections: one was used to acidify the capillary outflow so that the corresponding acridinium pseudo-base form can be changed into acridinium ester form by adding excess acid to the system; the other was designed to provide a suitable solution to produce the CL from acridinium ester. The effect factors, such as pH, the concentration of reaction reagents and the flow rates of the reagents, were investigated. The results showed that acridinium ester had similar CL properties in this interface when pH values of CE BGE were changed from 2.0 to 10.8. The interface was used to detect acridinium ester and three acridinium ester-labeled enkephalin-related peptides, the corresponding LODs were found to be in the attomole range. This CL detection system proved to be of high sensitivity, good repeatability, and relatively low cost.