Bioengineered amyloid peptide for rapid screening of inhibitors against main protease of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evoked a worldwide pandemic. As the emergence of variants has hampered the neutralization capacity of currently available vaccines, developing effective antiviral therapeutics against SARS-CoV-2 and its variants becomes a significant challenge. The main protease (Mpro) of SARS-CoV-2 has received increased attention as an attractive pharmaceutical target because of its pivotal role in viral replication and proliferation. Here, we generated a de novo Mpro-inhibitor screening platform to evaluate the efficacies of Mpro inhibitors based on Mpro cleavage site-embedded amyloid peptide (MCAP)-coated gold nanoparticles (MCAP-AuNPs). We fabricated MCAPs comprising an amyloid-forming sequence and Mpro-cleavage sequence, mimicking in vivo viral replication process mediated by Mpro. By measuring the proteolytic activity of Mpro and the inhibitory efficacies of various drugs, we confirmed that the MCAP-AuNP-based platform was suitable for rapid screening potential of Mpro inhibitors. These results demonstrated that our MCAP-AuNP-based platform has great potential for discovering Mpro inhibitors and may accelerate the development of therapeutics against COVID-19.

Direct observation of surface charge and stiffness of human metaphase chromosomes.

Metaphase chromosomes in which both polynucleotides and proteins are condensed with hierarchies are closely related to life phenomena such as cell division, cancer development, and cellular senescence. Nevertheless, their nature is rarely revealed, owing to their structural complexity and technical limitations in analytical methods. In this study, we used surface potential and nanomechanics mapping technology based on atomic force microscopy to measure the surface charge and intrinsic stiffness of metaphase chromosomes. We found that extra materials covering the chromosomes after the extraction process were positively charged. With the covering materials, the chromosomes were positively charged (ca. 44.9 ± 16.48 mV) and showed uniform stiffness (ca. 6.23 ± 1.98 MPa). In contrast, after getting rid of the extra materials through treatment with RNase and protease, the chromosomes were strongly negatively charged (ca. -197.4 ± 77.87 mV) and showed relatively non-uniform and augmented stiffness (ca. 36.87 ± 17.56 MPa). The results suggested undulating but compact coordination of condensed chromosomes. Additionally, excessive treatment with RNase and protease could destroy the chromosomal structure, providing an exceptional opportunity for multiscale stiffness mapping of polynucleotides, nucleosomes, chromatin fibers, and chromosomes in a single image. Our approach offers a new horizon in terms of an analytical technique for studying chromosome-related diseases.

Proteolysis-driven proliferation and rigidification of pepsin-resistant amyloid fibrils.

Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.

Melanoma Detection by AFM Indentation of Histological Specimens.

Melanoma is visible unlike other types of cancer, but it is still challenging to diagnose correctly because of the difficulty in distinguishing between benign nevus and melanoma. We conducted a robust investigation of melanoma, identifying considerable differences in local elastic properties between nevus and melanoma tissues by using atomic force microscopy (AFM) indentation of histological specimens. Specifically, the histograms of the elastic modulus of melanoma displayed multimodal Gaussian distributions, exhibiting heterogeneous mechanical properties, in contrast with the unimodal distributions of elastic modulus in the benign nevus. We identified this notable signature was consistent regardless of blotch incidence by sex, age, anatomical site (e.g., thigh, calf, arm, eyelid, and cheek), or cancer stage (I, IV, and V). In addition, we found that the non-linearity of the force-distance curves for melanoma is increased compared to benign nevus. We believe that AFM indentation of histological specimens may technically complement conventional histopathological analysis for earlier and more precise melanoma detection.

Isothermal amplification-mediated lateral flow biosensors for in vitro diagnosis of gastric cancer-related microRNAs.

MicroRNAs (miRNAs) are important diagnostic and prognostic biomarkers for various tumors. Currently, many diagnostic systems have been developed to detect miRNAs, but simple techniques for detecting miRNAs are still required. Recently, we reported that the expression of miRNA-135b is upregulated in gastric epithelial cells during gastric inflammation and carcinogenesis. Our aim was to develop an in vitro diagnostic platform to analyze the expression of gastric cancer-related biomarkers in the blood. The diagnostic platform comprised an isothermal amplification-based lateral flow biosensor (IA-LFB) that enables easy diagnosis of gastric cancer through visual observation. In this platform, trace amounts of biomarkers are isothermally amplified through rolling circle amplification (RCA), and the amplified product is grafted to the LFB. The performance of the IA-LFB was confirmed using RNAs extracted from in vitro and in vivo models. The platform could detect target miRNAs within 3 h with excellent sensitivity and selectivity. In particular, the IA-LFB could detect the overexpression of gastric cancer-related markers (miRNA-135b and miRNA-21) in RNAs extracted from the blood of patients with various stages (stages 1-4) of gastric cancer compared to that in healthy volunteers. Therefore, IA-LFB is a simple and sensitive in vitro diagnostic system for detecting gastric cancer-related biomarkers and can contribute to the early diagnosis and prognosis monitoring of gastric cancer. Furthermore, this technology can be applied to systems that can detect multiple biomarkers related to various diseases (such as infectious and genetic diseases).

Colorimetric Nanoparticle-Embedded Hydrogels for a Biosensing Platform.

Hydrogels containing colorimetric nanoparticles have been used for ion sensing, glucose detection, and microbial metabolite analyses. In particular, the rapid chemical reaction owing to both the hydrogel form of water retention and the sensitive color change of nanoparticles enables the rapid detection of target substances. Despite this advantage, the poor dispersibility of nanoparticles and the mechanical strength of nanoparticle-hydrogel complexes have limited their application. In this study, we demonstrate a milliliter agarose gel containing homogeneously synthesized polyaniline nanoparticles (PAni-NPs), referred to as PAni-NP-hydrogel complexes (PNHCs). To fabricate the optimal PNHC, we tested various pH solvents based on distilled water and phosphate-buffered saline and studied the colorimetric response of the PNHC with thickness. The colorimetric response of the prepared PNHC to the changes in the pH of the solution demonstrated excellent linearity, suggesting the possibility of using PNHC as a pH sensor. In addition, it was verified that the PNHC could detect minute pH changes caused by the cancer cell metabolites without cytotoxicity. Furthermore, the PNHC can be stably maintained outside water for approximately 12 h without deformation, indicating that it can be used as a disposable patch-type wearable biosensing platform.

Quantification of doping state of redox sensitive nanoparticles for probing the invasiveness of cancer cells using surface enhanced Raman scattering.

Redox activity is known to regulate migration, invasion, metastasis, proliferation, and vascularization of cancer. Because cancer is heterogeneous, the role of redox activity in different cancers and cancer-related processes vary widely. In this study, water soluble, Tween 80-coated polyaniline (TPAni) nanoparticles were synthesized and used as nano-agents for sensing the redox activities of various cancer cells. To identify the relationship between the redox activity and the aggressiveness of cancer cells, two different cancer cell lines, derived from the same tissue but different with regards to aggressiveness, were selected for study. First, the cancer cell lines were incubated with TPAni nanoparticles, and an absorbance ratio obtained from the cell culture media was used as a colorimetric indicator of the redox activities of the cells. Simultaneously, hydrophobically modified filter papers coated with silver nanosnowflakes (SNSF) were used as sensing substrates for surface enhanced Raman scattering (SERS). SERS spectra obtained from varying concentrations of rhodamine 6G were used to confirm the detection limit of the SNSF-based SERS substrate. Cell culture media containing TPAni nanoparticles were treated with the SNSF-containing SERS substrates to examine the redox activities of the various cancer cell lines.The redox activities of cancer cell lines were confirmed by absorbance spectral analysis, and these redox activities were better identified via an SERS analysis method. A SNSF-containing SERS substrate, fabricated from SNSF and filter paper, was used to sense redox activity in cancer cell lines and to further identify correlations between redox activity and cancer cell line aggressiveness, as indicated by the use of EpCAM as a biomarker. Finally, potential of â€‹in vivo â€‹redox activity sensing was also confirmed.

Hand-Held Raman Spectrometer-Based Dual Detection of Creatinine and Cortisol in Human Sweat Using Silver Nanoflakes.

The conventional tissue biopsy method yields isolated snapshots of a narrow region. Therefore, it cannot facilitate comprehensive disease characterization and monitoring. Recently, the detection of tumor-derived components in body fluids─a practice known as liquid biopsy─has attracted increased attention from the biochemical research and clinical application viewpoints. In this vein, surface-enhanced Raman scattering (SERS) has been identified as one of the most powerful liquid-biopsy analysis techniques, owing to its high sensitivity and specificity. Moreover, it affords high-capacity spectral multiplexing for simultaneous target detection and a unique ability to obtain intrinsic biomolecule-fingerprint spectra. This paper presents the fabrication of silver nanosnowflakes (SNSFs) using the polyol method and their subsequent dropping onto a hydrophobic filter paper. The SERS substrate, which comprises the SNSFs and hydrophobic filter paper, facilitates the simultaneous detection of creatinine and cortisol in human sweat using a hand-held Raman spectrometer. The proposed SERS system affords Raman spectrometry to be performed on small sample volumes (2 µL) to identify the normal and at-risk creatinine and cortisol groups.

Conformation Control of Amyloid Filaments by Repeated Thermal Perturbation.

We report that repeated thermal perturbation by thermal cycling (TC) accelerates the formation rate of amyloid filaments at microliter volumes (10-200 µL) and produces a new conformation of zigzag-shaped filaments. The amyloid filaments have been synthesized under different TC conditions, such as temperature variations (ΔT = 0-86 °C) and the number of cycles (C# = 30-90). In particular, the filament formation was promoted by TC with ΔT ≥ 30 °C. This indicates that the change in binding energy of ß-sheets and the breakage of disulfide bonds induced by TC with large ΔT contributed to the increased filament growth. This molecular interaction was investigated by molecular dynamics simulation. We also found that TC leads to the formation of amyloid filaments with peculiar conformation (zigzag-shaped filaments). Moreover, key structural parameters (tortuosity, segment length, and joint angle) of the amyloid filaments could be fine-tuned by selecting certain ΔT conditions. Taken together, we confirmed that the TC not only promotes the formation of amyloid filaments but also affects the conformational changes of the filaments.

Eco-friendly and enhanced colorimetric detection of aluminum ions using pectin-rich apple extract-based gold nanoparticles.

Aluminum ions are very toxic to human health, especially in relation to neurodegenerative diseases. However, conventional methods of detecting such toxic ions suffer from the use of poisonous chemical probes and complex processes. Herein, we report an eco-friendly and enhanced colorimetric method of aluminum ion detection using green-synthesized gold nanoparticles (AuNPs) from apple (Malus domestica) extract. The apple extract-based AuNPs (AX-AuNPs) contain abundant pectin different from citrate-based AuNPs. The pectin-rich AX-AuNPs improved the sensitivity of the colorimetric detection of aluminum ions. The detection limit was about 20 µM both in artificial and drinking water-based real samples. Interestingly, it is turned out that the AX-AuNPs were aggregated naturally after the chemical assay because of solution getting decayed. For the environmental perspective, it was great that the lump of AX-AuNP aggregates could easily be removed from the solutions before solution discard. Overall, our results indicate that AX-AuNPs offer a high-selectivity, enhanced colorimetric detection of aluminum ions in a short time (less than 1 min), based on an eco-friend synthesis and disposal manner of AuNPs.

Nanoelectrical characterization of individual exosomes secreted by Aß42-ingested cells using electrostatic force microscopy.

Quantifying the physical properties of individual exosomes containing amyloid-ß42 (Aß42) is crucial for a better understanding of an underpinning mechanism of Alzheimer's disease expression which is associated with the Aß42 transfer. Because of the lack of proper tools, however, there have been very few studies on how the amount of Aß42 affects the physical properties of exosomes. To answer the question, we investigated the physical properties of exosomes secreted by neuroblastoma by probing individual exosomes using electrostatic force microscopy. Interestingly, we observed that when the higher concentration of Aß42 oligomers was fed to cells, the higher surface charge of the exosomes appeared. This result indicates that the exosomes contain more Aß42 with the increase in Aß42 concentration in cell media, implying that they serve as transport vesicles for Aß42. Our approach could help to better understand how the neuronal exosomes are related to the propagation of neurodegenerative diseases and to seek how to make an early diagnosis of those diseases.

A bio-inspired highly selective enzymatic glucose sensor using a red blood cell membrane.

In the development of enzymatic glucose sensors, accurate glucose sensing has been a challenging task because of the existence of numerous interfering molecules in the blood. Meanwhile, red blood cells (RBCs) selectively uptake glucose via a membrane protein called glucose transporter-1. In this study, we developed the RBC membrane (RBCM)-coated enzymatic glucose sensors that mimic the glucose uptake. The RBCM-coated sensors were examined via scanning electron microscopy, atomic force microscopy, and ATR-FTIR. We optimized the glucose permeability of the RBCM filter by controlling the thickness of the filter. The sensing range of the optimized sensor was 1-15 mM, the detection limit was 0.66 mM, and the sensitivity was 2.978 µA mM-1. Intriguingly, the RBCM-coated sensor was highly accurate and precise, despite the coexistence of glucose and interfering molecules (e.g., mannose, galactose, ascorbic acid, uric acid, and cysteine). For each interfering molecule, the errors of our sensor were 0.8 to 2.3%, which was 4.8-14.2 times more accurate than the uncoated one. A similar result was verified for a human serum sample containing countless interfering molecules. Also, the sensing performance of the sensor was consistent after 4 weeks of storage. The results suggest that applying RBCM may improve the selectivity of various types of glucose sensors including the continuous monitoring system.

The Formation Mechanism of Segmented Ring-Shaped Aß Oligomers and Protofibrils.

A clear understanding of amyloid formation with diverse morphologies is critical to overcoming the fatal disease amyloidosis. Studies have revealed that monomer concentration is a crucial factor for determining amyloid morphologies, such as protofibrils, annular, or spherical oligomers. However, gaining a complete understanding of the mechanism of formation of the various amyloid morphologies has been limited by the lack of experimental devices and insufficient knowledge. In this study, we demonstrate that the monomer concentration is an essential factor in determining the morphology of beta-amyloid (Aß) oligomers or protofibrils. By computational and experimental approaches, we investigated the strategies for structural stabilization of amyloid protein, the morphological changes, and amyloid aggregation. In particular, we found unprecedented conformations, e.g., single bent oligomers and segmented ring-shaped protofibrils, the formation of which was explained by the computational analysis. Our findings provide insight into the structural features of amyloid molecules formed at low concentrations of monomer, which will help determine the clinical targets (in therapy) to effectively inhibit amyloid formation in the early stages of the amyloid growth phase.

Highly sensitive and wide-range nanoplasmonic detection of fibrinogen using erythrocyte membrane-blanketed nanoparticles.

Fibrinogen, which is a glycoprotein that circulates in the blood, plays various important biological roles, e.g., in blood coagulation, fibroblast proliferation, angiogenesis, and wound healing. Abnormal levels of fibrinogen in plasma have been identified as a key biomarker of a variety of disorders from cardiovascular diseases to hemophilia. Therefore, the development of a quantitative assay for fibrinogen in the blood has emerged as an important issue for the prevention and diagnosis of these diseases. Meanwhile, it is well known that erythrocytes can selectively capture fibrinogen because of the fibrinogen receptor expressed on their plasma membrane. Inspired by these biological interactions, herein, we devised an erythrocyte membrane (EM)-blanketed biosensor based on localized surface plasmon resonance (LSPR) for highly sensitive detection of fibrinogen. By placing the EM onto a nanoparticle-on-substrate, we enhanced the LSPR signal, achieving highly sensitive and selective detection of fibrinogen. We demonstrated that fibrinogen detection is possible over a wide concentration range, 0.001-5.000 mg/mL, which can cover normal and pathological blood fibrinogen levels. In addition, it was verified that the biosensor selectively detects fibrinogen in comparison with other human-blood-plasma components. The nanoplasmonic sensor blanketed with the EM opens up new opportunities for the development of a robust fibrinogen-sensing technology.

Permselective glucose sensing with GLUT1-rich cancer cell membranes.

Enzymatic blood glucose detection with selectivity is one of the most important conundrums, because human blood contains many components that can hinder enzyme-substrate reactions. Meanwhile, cancer cells express much higher levels of glucose transporter-1 on their cell membrane to selectively and excessively uptake more α-D-glucose than do normal cells. Inspired by such cellular permselectivity for glucose, herein we significantly improved the selectivity of a glucose sensor by using a breast cancer cell membrane (BCCM). The BCCM was extracted from MDA-MB-231 cells and coated onto an enzyme-deposited electrode via a vesicle fusion method. We investigated BCCM-coated sensors using ATR-FTIR, SEM, AFM, and cyclic voltammetry. The exceptional permselectivity of BCCM-coated sensors was validated using glucose solutions containing various interfering molecules (e.g., D-(-)-fructose, D-(+)-xylose, D-(+)-maltose, L-cysteine, L-ascorbic acid, and uric acid) and human serum (4.35-7.35 mM of glucose), implying their high potential for practical use.

Adhesive Leaf Created by a Corona Discharge.

Here, we report a new concept of both the adhesive manner and material, named "adhesive leaf (AL)," based on the leaf of the plant Heteropanax fragrans. The treatment of the corona discharge on the leaf surface can cause the nano-/microdestruction of the leaf epidermis, resulting in an outward release of sap. The glucose-containing sap provided the AL with a unique ability to stick to various substrates such as steel, polypropylene, and glass. Moreover, we reveal that the AL adhesion strength depends on the AL size, as well as the corona-discharge intensity. Conventional adhesives, such as glue and bond, lose their adhesive property and leave dirty residues upon the removal of the attached material. Unlike the conventional methods, the AL is advantageous as it can be repeatedly attached and detached thoroughly until the sap liquid is exhausted; its adhesive ability is maintained for at least three weeks at room temperature. Our findings shed light on a new concept of a biodegradable adhesive material that is created by a simple surface treatment.

Contact guidance and collective migration in the advancing epithelial monolayer.

At the edge of a confluent cell layer, cell-free empty space is a cue that can drive directed collective cellular migration. Similarly, contact guidance is also a robust mechanical cue that can drive cell migration. However, it is unclear which of the two effects is stronger, and how each mechanism affects collective migration. To address this question, here we explore the trajectories of cells migrating collectively on a substrate containing micropatterned grooves (10-20 µm in periodicity, 2 µm in height) compared with unpatterned control substrates. Compared with unpatterned controls, the micropatterned substrates attenuated path variance by close to 70% and augmented migration coordination by more than 30%. Together, these results show that contact guidance can play an appreciable role in collective cellular migration. Also, our result can provide insights into tissue repair and regeneration with the remodeling of the connective tissue matrix.

Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression.

Matrix metalloproteinase (MMP) is a key marker and target molecule for cancer diagnosis, as MMP is able to cleave peptide chains resulting in degradation of extracellular matrix (ECM), a necessary step for cancer development. In particular, MMP2 has recently been recognized as an important biomarker for lung cancer. Despite the important role of detecting MMP molecules in cancer diagnosis, it is a daunting task to quantitatively understand a correlation between the status of cancer development and the secretion level of MMP in a blood droplet. Here, we demonstrate a nanoscale cancer diagnosis by nanomechanical quantitation of MMP2 molecules under cancer progression with using a blood droplet of lung cancer patients. Specifically, we measured the frequency dynamics of nanomechanical biosensor functionalized with peptide chains mimicking ECM in response to MMP2 secreted from tumors in lung with different metastasis level. It is shown that the frequency shift of the biosensor, which exhibits the detection sensitivity below 1 nM, enables the quantitation of the secretion level of MMP2 molecules during the progression of cancer cells or tumor growth. More importantly, using a blood droplet of lung cancer patients, nanomechanical biosensor is shown to be capable of depicting the correlation between the secretion level of MMP2 molecules and the level of cancer metastasis, which highlights the cantilever-based MMP2 detection for diagnosis of lung cancer. Our finding will broaden the understanding of cancer development activated by MMP and allow for a fast and point-of-care cancer diagnostics.

Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection.

Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities.

Real-Time Analysis of Cellular Response to Small-Molecule Drugs within a Microfluidic Dielectrophoresis Device.

Quantitative detection of the biological properties of living cells is essential for a wide range of purposes, from the understanding of cellular characteristics to the development of novel drugs in nanomedicine. Here, we demonstrate that analysis of cell biological properties within a microfluidic dielectrophoresis device enables quantitative detection of cellular biological properties and simultaneously allows large-scale measurement in a noise-robust and probeless manner. Applying this technique, the static and dynamic biological responses of live B16F10 melanoma cells to the small-molecule drugs such as N-ethylmaleimide (NEM) and [(dihydronindenyl)oxy]alkanoic acid (DIOA) were quantitatively and statistically examined by investigating changes in movement of the cells. Measurement was achieved using subtle variations in dielectrophoresis (DEP) properties of the cells, which were attributed to activation or deactivation of K(+)/Cl(-) cotransporter channels on the cell membrane by the small-molecule drugs, in a microfluidic device. On the basis of quantitative analysis data, we also provide the first report of the shift of the complex permittivity of a cell induced by the small-molecule drugs. In addition, we demonstrate interesting quantifiable parameters including the drug effectiveness coefficient, antagonistic interaction coefficient, kinetic rate, and full width at half-maximum, which corresponded to changes in biological properties of B16F10 cells over time when NEM and DIOA were introduced alone or in combination. Those demonstrated parameters represent very useful tools for evaluating the effect of small-molecule drugs on the biological properties of cells.