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.

Caco-2 cell-derived biomimetic electrochemical biosensor for cholera toxin detection.

Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of ∼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.

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.

Biomimetically Engineered Amyloid-Shelled Gold Nanocomplexes for Discovering α-Synuclein Oligomer-Degrading Drugs.

The assembly of α-synuclein (αS) oligomers is recognized as the main pathological driver of synucleinopathies. While the elimination of toxic αS oligomers shows promise for the treatment of Parkinson's disease (PD), the discovery of αS oligomer degradation drugs has been hindered by the lack of proper drug screening tools. Here, we report a drug screening platform for monitoring the efficacy of αS-oligomer-degrading drugs using amyloid-shelled gold nanocomplexes (ASGNs). We fabricate ASGNs in the presence of dopamine, mimicking the in vivo generation process of pathological αS oligomers. To test our platform, the first of its kind for PD drugs, we use αS-degrading proteases and various small molecular substances that have shown efficacy in PD treatment. We demonstrate that the ASGN-based in vitro platform has strong potential to discover effective αS-oligomer-targeting drugs, and thus it may reduce the attrition problem in drug discovery for PD treatment.

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.

Surface Functionalization of Enzyme-Coronated Gold Nanoparticles with an Erythrocyte Membrane for Highly Selective Glucose Assays.

Colorimetric glucose sensors using enzyme-coronated gold nanoparticles have been developed for high-throughput assays to monitor the blood glucose levels of diabetic patients. Although those sensors have shown sensitivity and wide linear detection ranges, they suffer from poor selectivity and stability in detecting blood glucose, which has limited their practical use. To address this limitation, herein, we functionalized glucose-oxidase-coronated gold nanoparticles with an erythrocyte membrane (EM-GOx-GNPs). Because the erythrocyte membrane (EM) selectively facilitates the permeation of glucose via glucose transporter-1 (GLUT1), the functionalization of GOx-GNPs with EM improved the stability, selectivity (3.3- to 15.8-fold higher), and limit of detection (LOD). Both membrane proteins, GLUT1 and aquaporin-1 (AQP1), on EM were shown to be key components for selective glucose detection by treatment with their inhibitors. Moreover, we demonstrated the stability of EM-GOx-GNPs in high-antioxidant-concentration conditions, under long-term storage (∼4 weeks) and a freeze-thaw cycle. Selectivity of the EM-GOx-GNPs against other saccharides was increased, which improved the LOD in phosphate-buffered saline and human serum. Our results indicated that the functionalization of colorimetric glucose sensors with EM is beneficial for improving selectivity and stability, which may make them candidates for use in a practical glucose sensor.

Highly permselective uric acid detection using kidney cell membrane-functionalized enzymatic biosensors.

Abnormal blood uric acid (UA) levels can lead to its crystallization in the joints, consequently resulting in gout. Accurate detection of UA in the blood is imperative for the early diagnosis of gout. However, electrochemical UA biosensors are vulnerable to antioxidants in the blood, limiting accurate UA detection. To address this issue, we focused on the function of uric acid transporter 1 (URAT1), which is selectively permeable to UA. URAT1 is abundant in the kidney cell membrane (KCM). To apply URAT1 to a sensor, we developed a KCM-coated UA biosensor (called the KCM sensor) that could selectively detect UA through URAT1. The KCM coating in the fabricated KCM sensor was verified via scanning electron microscopy, atomic force microscopy, and confocal microscopy. The KCM sensor enabled the detection of UA in the range of 0-1000 µM, with a limit of detection of 8.5 µM, suggesting that it allows the diagnosis of the early stages of gout. On the other hand, the UA permeability of the KCM sensor was significantly reduced in the presence of a URAT1 inhibitor, implying that URAT1 is a key factor for UA detection. The selectivity of the KCM sensor was demonstrated by measuring the amount for UA in the presence of various antioxidants. Finally, the KCM sensor was capable of measuring UA in human serum and was reproducible with 0.5-1.6% deviation. The UA permeability and selectivity of the KCM sensor were maintained even after 3 weeks of storage.

Erythrocyte-camouflaged biosensor for α-hemolysin detection.

Without appropriate treatment, Staphylococcus aureus (S. aureus) infection can cause life-threatening diseases (e.g., meningitis, pneumonia, bacteremia, and sepsis). However, a rapid and accurate point-of-care test for the infection remains challenging. The bacterium secretes α-hemolysin (Hla), which spontaneously binds to the cell membrane of erythrocyte, and eventually lyses the cell via pore formation. Taking advantage of this phenomenon, we apply the erythrocyte membrane (EM) extracted from human whole blood as a novel bioreceptor for detecting Hla, fabricating erythrocyte-camouflaged biosensors (ECB) by coating EM onto electrochemical impedance electrodes. We verify the existence of EM on the ECB by using confocal microscopy and atomic force microscopy. We demonstrate that ECBs sensitively detect Hla spiked in phosphate buffer saline and human serum. Also, the sensor shows higher sensitivity to Hla than major blood proteins, such as human serum albumin, fibrinogen, and gamma globulin. Specifically, the signal intensities for Hla are 8.8-12.7 times higher than those in the same concentration of those blood proteins. The detection limit of the ECB for Hla is 1.9 ng/ml while the dynamic range is 0.0001-1 mg/ml. Finally, we validate the constant sensing performance of ECB with 99.0 ± 5.6% accuracy for 35 days of storage.

Nanoindentation for Monitoring the Time-Variant Mechanical Strength of Drug-Loaded Collagen Hydrogel Regulated by Hydroxyapatite Nanoparticles.

Hydroxyapatite nanoparticle-complexed collagen (HAP/Col) hydrogels have been widely used in biomedical applications as a scaffold for controlled drug release (DR). The time-variant mechanical properties (Young's modulus, E) of HAP/Col hydrogels are highly relevant to the precise and efficient control of DR. However, the correlation between the DR and the E of hydrogels remains unclear because of the lack of a nondestructive and continuous measuring system. To reveal the correlations, herein, we investigate the time-variant behavior of E for HAP/Col hydrogels during 28 days using the atomic force microscopy (AFM) nanoindentation technique. The initial E of hydrogels was controlled from 200 to 9000 Pa by the addition of HAPs. Subsequently, we analyzed the relationship between the DR of the hydrogels and the changes in their mechanical properties (ΔE) during hydrogel degradation. Interestingly, the higher the initial E value of HAP/Col hydrogels is, the higher is the rate of hydrogel degradation over time. However, the DR of hydrogels with higher initial E appeared to be significantly delayed by up to 40% at a maximum. The results indicate that adding an appropriate amount of HAPs into hydrogels plays a crucial role in determining the initial E and their degradation rate, which can contribute to the properties that prolong DR. Our findings may provide insights into designing hydrogels for biomedical applications such as bone regeneration and drug-delivery systems.

Selective colorimetric urine glucose detection by paper sensor functionalized with polyaniline nanoparticles and cell membrane.

For the diabetes diagnosis, noninvasive methods are preferred to invasive methods; urine glucose measurement is an example of a noninvasive method. However, conventional noninvasive methods for urine glucose measurement are not intuitive. Furthermore, such methods exhibit low selectivity because they can detect interfering molecules in addition to glucose. Herein, we fabricate a noninvasive, intuitive, and highly selective paper sensor consisting of polyaniline nanoparticles (PAni-NPs) and red blood cell membranes (RBCMs). The PAni-NPs (adsorbed on the paper) are highly sensitive to hydrogen ions and change color from emeraldine blue to emeraldine green within a few seconds. The RBCM (coated on the PAni-NP-adsorbed paper) having the glucose transporter-1 protein plays the role of a smart filter that transports glucose but rejects other interfering molecules. In particular, the selectivity of the RBCM-coated PAni-NP-based paper sensor was approximately improved ∼85%, compared to the uncoated paper sensors. The paper sensor could detect urine glucose over the range of 0-10 mg/mL (0-56 mM), with a limit of detection of 0.54 mM. The proposed paper sensor will facilitate the development of a highly selective and colorimetric urine glucose monitoring system.

Sequential dual-drug delivery of BMP-2 and alendronate from hydroxyapatite-collagen scaffolds for enhanced bone regeneration.

The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.

Highly Conductive and Flexible Dopamine-Graphene Hybrid Electronic Textile Yarn for Sensitive and Selective NO2 Detection.

Graphene-based electronic textile (e-textile) gas sensors have been developed for detecting hazardous NO2 gas. For the e-textile gas sensor, electrical conductivity is a critical factor because it directly affects its sensitivity. To obtain a highly conductive e-textile, biomolecules have been used for gluing the graphene to the textile surface, though there remain areas to improve, such as poor conductivity and flexibility. Herein, we have developed a dopamine-graphene hybrid electronic textile yarn (DGY) where the dopamine is used as a bio-inspired adhesive to attach graphene to the surface of yarns. The DGY shows improved electrical conductivity (∼40 times) compared to conventional graphene-based e-textile yarns with no glue. Moreover, it exhibited improved sensing performance in terms of short response time (∼2 min), high sensitivity (0.02 µA/ppm), and selectivity toward NO2. The mechanical flexibility and durability of the DGY were examined through a 1000-cycle bending test. For a practical application, the DGY was attempted to detect the NOx emitted from vehicles, including gasoline, diesel, and fuel cell electric vehicles. Our results demonstrated that the DGYs-as a graphene-based e-textile gas sensor for detecting NO2-are simple to fabricate, cheap, disposable, and mechanically stable.