Glucose Oxidase Activity Colorimetric Assay Using Redox-Sensitive Electrochromic Nanoparticle-Functionalized Paper Sensors.

Glucose oxidase (GOx) activity assays are vital for various applications, including glucose metabolism estimation and fungal testing. However, conventional methods involve time-consuming and complex procedures. In this study, we present a colorimetric platform for in situ GOx activity measurement utilizing redox-sensitive electrochromic nanoparticles based on polyaniline (PAni). The glucose-adsorbed colorimetric paper sensor, herein termed Glu@CPS, is created by immobilizing ferrocene and glucose onto paper substrates that have been functionalized with PAni nanoparticles. Glu@CPS not only demonstrated rapid detection (within 5 min) but also exhibited remarkable selectivity for GOx and a limit of detection as low as 1.25 µM. Moreover, Glu@CPS demonstrated consistent accuracy in the measurement of GOx activity, exhibiting no deviations even after being stored at ambient temperature for a duration of one month. To further corroborate the effectiveness of this method, we applied Glu@CPS in the detection of GOx activity in a moldy red wine. The results highlight the promising potential of Glu@CPS as a convenient and precise platform for GOx activity measurement in diverse applications including food quality control, environmental monitoring, and early detection of fungal contamination.

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.

Fabrication of fully aligned self-assembled cell-laden collagen filaments for tissue engineering via a hybrid bioprinting process.

Cell-laden structures play a pivotal role in various tissue engineering applications, particularly in tissue restoration. Interactions between cells within bioprinted structures are crucial for successful tissue development and regulation of stem cell fate through intricate cell-to-cell signaling pathways. In this study, we developed a new technique that combines polyethylene glycol (PEG)-infused submerged bioprinting with a stretching procedure. This approach facilitated the generation of fully aligned collagen structures consisting of myoblasts and a low concentration (2 wt%) of collagen to efficiently encourage muscle tissue regeneration. By adjusting several processing parameters, we obtained biologically safe and mechanically stable cell-laden collagen filaments with uniaxial alignment. Notably, the cell filaments exhibited markedly elevated cellular activities compared to those exhibited by conventional bioprinted filaments, even at similar cell densities. Moreover, when we implanted structures containing adipose stem cells into mice, we observed a significantly increased level of myogenesis compared to that in normally bioprinted struts. Thus, this promising approach has the potential to revolutionize tissue engineering by fostering enhanced cellular interactions and promoting improved outcomes in regenerative medicine.

Dual regulatory effects of neferine on amyloid-ß and tau aggregation studied by in silico, in vitro, and lab-on-a-chip technology.

Alzheimer's disease (AD) is characterized by the presence of two critical pathogenic factors: amyloid-ß (Aß) and tau. Aß and tau become neurotoxic aggregates via self-assembly, and these aggregates contribute to the pathogenesis of AD. Therefore, there has been growing interest in therapeutic strategies that simultaneously target Aß and tau aggregates. Although neferine has attracted attention as a suitable candidate agent for alleviating AD pathology, there has been no study investigating whether neferine affects the modulation of Aß or tau aggregation/dissociation. Herein, we investigated the dual regulatory effects of neferine on Aß and tau aggregation/dissociation. We predicted the binding characteristics of neferine to Aß and tau using molecular docking simulations. Next, thioflavin T and atomic force microscope analyses were used to evaluate the effects of neferine on the aggregation or dissociation of Aß42 and tau K18. We verified the effect of neferine on Aß fibril degradation using a microfluidic device. In addition, molecular dynamics simulation was used to predict a conformational change in the Aß42-neferine complex. Moreover, we examined the neuroprotective effect of neferine against neurotoxicity induced by Aß and tau and their fibrils in HT22 cells. Finally, we foresaw the pharmacokinetic properties of neferine. These results demonstrated that neferine, which has attracted attention as a potential treatment for AD, can directly affect Aß and tau pathology.

Cellular subpopulations identified using an ensemble average of multiple dielectrophoresis measurements.

While the average value measurement approach can successfully analyze and predict the general behavior and biophysical properties of an isogenic cell population, it fails when significant differences among individual cells are generated in the population by intracellular changes such as the cell cycle, or different cellular responses to certain stimuli. Detecting such single-cell differences in a cell population has remained elusive. Here, we describe an easy-to-implement and generalizable platform that measures the dielectrophoretic cross-over frequency of individual cells by decreasing measurement noise with a stochastic method and computing ensemble average statistics. This platform enables multiple, real-time, label-free detection of individual cells with significant dielectric variations over time within an isogenic cell population. Using a stochastic method in combination with the platform, we distinguished cell subpopulations from a mixture of drug-untreated and -treated isogenic cells. Furthermore, we demonstrate that our platform can identify drug-treated isogenic cells with different recovery rates.

Use of biosilica to improve loading and delivery of bone morphogenic protein 2.

The clinical utility of bone morphogenetic protein 2 (BMP2) is limited because of the poor attraction between BMP2 and carriers, resulting in low loading efficiency and initial burst release. Here, the high binding affinity of BMP2 to the biosilica surface was utilized to overcome this limitation. Atomic force microscopy revealed that BMP2 bound nearly 8- and 2-fold more strongly to biosilica-coated hydroxyapatite than to uncoated and plain silica-coated hydroxyapatite, respectively. To achieve controlled release, collagen was introduced between the silica layers on hydroxyapatite, which was optimized by adjusting the collagen concentration and number of layers. The optimal biosilica/collagen formulation induced sustained BMP2 release without compromising loading efficiency. BMP2 combined with the mentioned formulation led to an increase in osteogenesis, as compared to the combination of BMP2 with either biosilica-coated or non-coated hydroxyapatite in vitro. In rat calvarial defect models, the biosilica/collagen-coated hydroxyapatite with 1 µg BMP2 showed 26 % more bone regeneration than the same dose of BMP2-loaded hydroxyapatite and 10.6 % more than hydroxyapatite with 2.5-fold dose of BMP2. Using BMP2 affinity carriers coated with biosilica/collagen allows for more efficacious in situ loading and delivery of BMP2, making them suitable for the clinical application of growth factors through a soaking method.

Hydrophobic Barriers for Directing Physarum polycephalum Propulsion and Navigation.

Physarum polycephalum (P. polycephalum) is a unicellular protist with unique properties, such as learning and remembering in its cultured environment without a brain or central nervous system. The organism has been extensively used in morphology, taxis, and positive feedback dynamics studies. However, the lack of standardization of materials and substrate designs used in P. polycephalum studies has significantly limited conducting such studies, increasing the cost and time. In this study, we introduce a method to control the direction and migration of P. polycephalum by drawing hydrophobic lines and patterns. Our study succeeded in controlling the movement of P. polycephalum by setting a variety of hydrophobic designs such as complete barrier, single-slit barrier, taper barrier, dumbbell barrier, and one-side-opened rectangular barrier, suggesting the effectiveness of the hydrophobic barrier in regulating the propulsion and navigation of the organisms. Moreover, we demonstrated that utilizing such geometric constraints can reduce the experimental time required for toxicity testing based on P. polycephalum by more than 300%. Our techniques open new possibilities for studying the biophysical properties and behaviors of P. polycephalum, while also facilitating toxicity testing.

PCR-like performance of rapid test with permselective tunable nanotrap.

Highly sensitive rapid testing for COVID-19 is essential for minimizing virus transmission, especially before the onset of symptoms and in asymptomatic cases. Here, we report bioengineered enrichment tools for lateral flow assays (LFAs) with enhanced sensitivity and specificity (BEETLES2), achieving enrichment of SARS-CoV-2 viruses, nucleocapsid (N) proteins and immunoglobulin G (IgG) with 3-minute operation. The limit of detection is improved up to 20-fold. We apply this method to clinical samples, including 83% with either intermediate (35%) or low viral loads (48%), collected from 62 individuals (n = 42 for positive and n = 20 for healthy controls). We observe diagnostic sensitivity, specificity, and accuracy of 88.1%, 100%, and 91.9%, respectively, compared with commercial LFAs alone achieving 14.29%, 100%, and 41.94%, respectively. BEETLES2, with permselectivity and tunability, can enrich the SARS-CoV-2 virus, N proteins, and IgG in the nasopharyngeal/oropharyngeal swab, saliva, and blood serum, enabling reliable and sensitive point-of-care testing, facilitating fast early diagnosis.

Ultra-sensitive label-free SERS biosensor with high-throughput screened DNA aptamer for universal detection of SARS-CoV-2 variants from clinical samples.

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10-18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

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.

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.

Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

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.

A regression-based machine learning approach for pH and glucose detection with redox-sensitive colorimetric paper sensors.

Colorimetric paper sensors are used in various fields due to their convenience and intuitive manner. However, these sensors present low accuracy in practical use because it is difficult to distinguish color changes for a minute amount of analyte with the naked eye. Herein, we demonstrate that a machine learning (ML)-based paper sensor platform accurately determines the color changes. We fabricated a colorimetric paper sensor by adsorbing polyaniline nanoparticles (PAni-NPs), whose color changes from blue to green when the ambient pH decreases. Adding glucose oxidase (GOx) to the paper sensor enables colorimetric glucose detection. Target analytes (10 µL) were aliquoted onto the paper sensors, and their images were taken with a smartphone under the same conditions in a darkroom. The red-green-blue (RGB) data from the images were extracted and used to train and test three regression models: support vector regression (SVR), decision tree regression (DTR), and random forest regression (RFR). Of the three regression models, RFR performed the best at estimating pH levels (R2 = 0.957) ranging from pH 2 to 10 and glucose concentrations (R2 = 0.922) ranging from 0 to 10 mg mL-1.

Fast Responsive, Reversible Colorimetric Nanoparticle-Hydrogel Complexes for pH Monitoring.

Hydrogels containing redox-sensitive colorimetric nanoparticles (NPs) have been used to sense ambient pH in many fields owing to their simple and fast visualization capabilities. However, real-time pH monitoring still has limitations due to its poor response rate and irreversibility. Herein, we developed a fast responsive colorimetric hydrogel called ferrocene adsorption colorimetric hydrogel (FACH). Ferrocene, an organometallic compound, plays a vital role as an electron transfer mediator (i.e., redox catalyst) within the hydrogel network. FACH shows fast color change performance with high reactivity and penetrability to ambient pH changes. In detail, FACH shows distinct color change within 2 min under various pH conditions from four to eight, with good reliability. The speed for color change of FACH is approximately six times faster than that of previously developed colorimetric hydrogels, suggesting the fastest hydrogel-based colorimetric pH sensor. Furthermore, FACH shows reversibility and repeatability of the redox process, indicating scalable utility as a sustainable pH monitoring platform.

Hemolysis-Inspired, Highly Sensitive, Label-Free IgM Detection Using Erythrocyte Membrane-Functionalized Nanomechanical Resonators.

Immunoglobulin detection is important for immunoassays, such as diagnosing infectious diseases, evaluating immune status, and determining neutralizing antibody concentrations. However, since most immunoassays rely on labeling methods, there are limitations on determining the limit of detection (LOD) of biosensors. In addition, although the antigen must be immobilized via complex chemical treatment, it is difficult to precisely control the immobilization concentration. This reduces the reproducibility of the biosensor. In this study, we propose a label-free method for antibody detection using microcantilever-based nanomechanical resonators functionalized with erythrocyte membrane (EM). This label-free method focuses on the phenomenon of antibody binding to oligosaccharides (blood type antigen) on the surface of the erythrocyte. We established a method for extracting the EM from erythrocytes and fabricated an EM-functionalized microcantilever (MC), termed EMMC, by surface-coating EM layers on the MC. When the EMMC was treated with immunoglobulin M (IgM), the bioassay was successfully performed in the linear range from 2.2 pM to 22 nM, and the LOD was 2.0 pM. The EMMC also exhibited excellent selectivity compared to other biomolecules such as serum albumin, γ-globulin, and IgM with different paratopes. These results demonstrate that EMMC-based nanotechnology may be utilized in criminal investigations to identify blood types with minimal amounts of blood or to evaluate individual immunity through virus-neutralizing antibody detection.

Nanoscale biophysical properties of small extracellular vesicles from senescent cells using atomic force microscopy, surface potential microscopy, and Raman spectroscopy.

Cells secrete extracellular vesicles (EVs) carrying cell-of-origin markers to communicate with surrounding cells. EVs regulate physiological processes ranging from intercellular signaling to waste management. However, when senescent cells (SnCs) secrete EVs, the EVs, which are newly regarded as senescence-associated secretory phenotype (SASP) factors, can evoke inflammation, senescence induction, and metabolic disorders in neighboring cells. Unlike other soluble SASP factors, the biophysical properties of EVs, including small EVs (sEVs), derived from SnCs have not yet been investigated. In this study, sEVs were extracted from a human IMR90 lung fibroblast in vitro senescence model. Their biomechanical properties were mapped using atomic force microscopy-based quantitative nanomechanical techniques, surface potential microscopy, and Raman spectroscopy. The surfaces of sEVs derived from SnCs are slightly stiffer but their cores are softer than those of sEVs secreted from non-senescent cells (non-SnCs). This inversely proportional relationship between deformation and stiffness, attributed to a decrease in the concentration of genetic and protein materials inside the vesicles and the adsorption of positively charged SASP factors onto the vesicle surfaces, respectively, was found to be a peculiar characteristic of SnC-derived sEVs. Our results demonstrate that the biomechanical properties of SnC-derived sEVs differ from those of non-SnC-derived sEVs and provide insight into the mechanisms underlying their formation and composition.

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.

Amyloid Formation in Nanoliter Droplets.

Processes that monitor the nucleation of amyloids and characterize the formation of amyloid fibrils are vital to medicine and pharmacology. In this study, we observe the nucleation and formation of lysozyme amyloid fibrils using a facile microfluidic system to generate nanoliter droplets that can control the flow rate and movement of monomer-in-oil emulsion droplets in a T-junction microchannel. Using a fluorescence assay, we monitor the nucleation and growth process of amyloids based on the volume of droplets. Using the microfluidic system, we demonstrate that the lag phase, which is vital to amyloid nucleation and growth, is reduced at a lower droplet volume. Furthermore, we report a peculiar phenomenon of high amyloid formation at the edge of a bullet-shaped droplet, which is likely due to the high local monomer concentration. Moreover, we discovered that amyloid fibrils synthesized in the nanoliter droplets are shorter and thicker than fibrils synthesized from a bulk solution via the conventional heating method. Herein, a facile procedure to observe and characterize the nucleation and growth of amyloid fibrils using nanoliter droplets is presented, which is beneficial for investigating new features of amyloid fibril formation as an unconventional synthetic method for amyloid fibrils.