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.

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.

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.

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.

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.

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.

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.

Polyaniline Nanoskein: Synthetic Method, Characterization, and Redox Sensing.

Polyaniline nanoskein (PANS), which have polyaniline nanofibers, was developed. PANS was formulated via sequential extracting, heating, and swelling processes. The compositions of PANS have been analyzed using X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller analysis, and the results of which indicate that PANS is composed of solely organic materials. Moreover, PANS has been shown convertible absorbance characteristics according to surrounding acidic environments, and using these characteristics, the possibility of PANS for sensing of surrounding redox state changes is presented.