Advances in AFM Imaging Applications for Characterizing the Biophysical Properties of Amyloid Fibrils

Although the formation mechanism of amyloid fibrils in bodies is still debated, it has recently been reported how amyloid fibrils can be formed in vitro. Accordingly, we have gained a better understanding of the self-assembly mechanism and intrinsic properties of amyloid fibrils. Because the structure of amyloid fibrils consists of nanoscaled insoluble strands (a few nanometers in diameter and micrometers long), a special tool is needed to study amyloid fibrils at length. Atomic force microscopy (AFM) is supposed to be a versatile toolkit to probe such a tiny biomolecule. The physical/chemical properties of amyloid fibrils have been explored by AFM. In particular, AFM enables the visualization of amyloid fibrillation with different incubation times as well as the concentrations of the formed amyloid fibrils as affected by fibril diameters and lengths. Very recently, the minute structural changes and/or electrical properties of amyloid fibrils have been made by using advanced AFM techniques including dynamic liquid AFM, PeakForce QNM (quantitative nanomechanical mapping), and Kelvin probe force microscopy (KPFM). Herein, we summarize the biophysical properties of amyloid fibrils that are newly discovered with the help of those advanced AFM techniques and suggest our perspectives and future directions for the study of amyloid fibrils

Application of Red Cell Membrane in Nanobiotechnology

Red cells are full of unique biological properties such as immune evasion and molecular-specific permeability. These properties originate from various membrane proteins on the surface of the cell membrane. For this reason, red cell membrane is coated on nanomaterials or sensors to bestow the functionalities of the membrane proteins. In this chapter, various types of membrane proteins of red cell and its functions are described. Also, the following two experimental procedures are summarized: (I) the extraction of red cell membrane containing membrane proteins and (II) coating of the extracted cell membrane onto the nanoparticles and solid surface of sensors. Finally, the applications of red cell membrane in drug delivery system and biosensor are discussed

Aptamer-functionalized nano-pattern based on carbon nanotube for sensitive, selective protein detection

We have developed a horizontally aligned carbon nanotube sensor that enables not only the specific detection of biomolecules with ultra-sensitivity, but also the quantitative characterization of binding affinity between biomolecules and/or interaction between a carbon nanotube and a biomolecule, for future applications in early diagnostics. In particular, we have fabricated horizontally aligned carbon nanotubes, which were functionalized with specific aptamers that are able to specifically bind to biomolecules (i.e. thrombin). Our detection system is based on scanning probe microscopy (SPM) imaging for horizontally aligned aptamer-conjugated carbon nanotubes (ACNTs) that specifically react with target biomolecules at an ultra-low concentration. It is shown that the binding affinity between thrombin molecule and ACNT can be quantitatively characterized using SPM imaging. It is also found that the smart carbon nanotube sensor coupled with SPM imaging permits us to achieve the high detection sensitivity even up to similar to 1 pM, which is much higher than that of other bioassay methods. Moreover, we have shown that our method enables a quantitative study on small molecule-mediated inhibition of specific biomolecular interactions. In addition, we have shown that our ACNT-based system allows for the quantitative study of the effect of chemical environment (e.g. pH and ion concentration) on the binding affinity. Our study sheds light on carbon nanotube sensor coupled with SPM imaging, which opens a new avenue to early diagnostics and drug screening with high sensitivity.close2

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

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

Detection of Chlorpyrifos Using Bio-Inspired Silver Nanograss

Chlorpyrifos (CPF) is widely used as an organophosphorus insecticide; however, owing to developmental neurotoxicity, genotoxicity, and other adverse effects, it is harmful not only to livestock but also to humans. Therefore, the use of CPF was recently regulated, and its sensitive detection is crucial, as it causes serious toxicity, even in the case of residual pesticides. Because it is hard to detect the chlorpyrifos directly using spectroscopy (especially in SERS) without chemical reagents, we aimed to develop a SERS platform that could detect the chlorpyrifos directly in the water. In this study, we utilized the intrinsic properties of natural lawns that grow randomly and intertwine with each other to have a large surface area to promote photosynthesis. To detect CPF sensitively, we facilitated the rapid fabrication of biomimetic Ag nanograss (Ag-NG) as a surface-enhanced Raman spectroscopy (SERS) substrate using the electrochemical over-deposition method. The efficiency of the SERS method was confirmed through experiments and finite element method (FEM)-based electromagnetic simulations. In addition, the sensitive detection of CPF was enhanced by pretreatment optimization of the application of the SERS technique (limit of detection: 500 nM). The Ag-NG has potential as a SERS platform that could precisely detect organic compounds, as well as various toxic substances

Colorimetric Sensing of Lactate in Human Sweat Using Polyaniline Nanoparticles-Based Sensor Platform and Colorimeter

In emergency medicine, the lactate level is commonly used as an indicator of the severity and response to the treatment of hypoperfusion-related diseases. Clinical lactate measurements generally require 3 h for clinical determination. To improve the current gold standard methods, the development of sensor devices that can reduce detection time while maintaining sensitivity and providing portability is gaining great attention. This study aimed to develop a polyaniline (PAni)-based single-sensor platform for sensing lactate in human sweat using a CIELAB color system-based colorimetric device. To establish a lactate sensing platform, PAni nanoparticles were synthesized and adsorbed on the filter paper surface using solvent shift and dip-coating methods, respectively. PAni is characterized by a chemical change accompanied by a color change according to the surrounding environment. To quantify the color change of PAni, a CIELAB color system-based colorimetric device was fabricated. The color change of PAni was measured according to the chemical state using a combination of a PAni-based filter paper sensor platform and a colorimetric device, based on the lactate concentration in deionized water. Finally, human sweat was spiked with lactate to measure the color change of the PAni-based filter paper sensor platform. Under these conditions, the combination of polyaniline-based sensor platforms and colorimetric systems has a limit of detection (LOD) and limit of quantitation (LOQ) of 1 mM, linearity of 0.9684, and stability of 14%. Tbe confirmed that the color of the substrate changes after about 30 s, and through this, the physical fatigue of the individual can be determined. In conclusion, it was confirmed through this study that a combination of the PAni paper sensor platform and colorimeter can detect clinically meaningful lactate concentration

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