Solution-free and simplified H&E staining using a hydrogel-based stamping technology.

Hematoxylin and eosin (H&E) staining has been widely used as a fundamental and essential tool for diagnosing diseases and understanding biological phenomena by observing cellular arrangements and tissue morphological changes. However, conventional staining methods commonly involve solution-based, complex, multistep processes that are susceptible to user-handling errors. Moreover, inconsistent staining results owing to staining artifacts pose real challenges for accurate diagnosis. This study introduces a solution-free H&E staining method based on agarose hydrogel patches that is expected to represent a valuable tool to overcome the limitations of the solution-based approach. Using two agarose gel-based hydrogel patches containing hematoxylin and eosin dyes, H&E staining can be performed through serial stamping processes, minimizing color variation from handling errors. This method allows easy adjustments of the staining color by controlling the stamping time, effectively addressing variations in staining results caused by various artifacts, such as tissue processing and thickness. Moreover, the solution-free approach eliminates the need for water, making it applicable even in environmentally limited middle- and low-income countries, while still achieving a staining quality equivalent to that of the conventional method. In summary, this hydrogel-based H&E staining method can be used by researchers and medical professionals in resource-limited settings as a powerful tool to diagnose and understand biological phenomena.

Streamlined Specimen Purification for Rapid COVID-19 Diagnosis Using Positively Charged Polymer Thin Film-Coated Surfaces and Chamber Digital PCR.

The ongoing coronavirus disease 2019 (COVID-19) pandemic demands rapid and straightforward diagnostic tools to prevent early-stage viral transmission. Although nasopharyngeal swabs are a widely used patient sample collection method for diagnosing COVID-19, using these samples for diagnosis without RNA extraction increases the risk of obtaining false-positive and -negative results. Thus, multiple purification steps are necessary, which are time-consuming, generate significant waste, and result in substantial sample loss. To address these issues, we developed surface-modified polymerase chain reaction (PCR) tubes using the tertiary aminated polymer poly(2-dimethylaminomethylstyrene) (pDMAMS) via initiated chemical vapor deposition. Introducing the clinical samples into the pDMAMS-coated tubes resulted in approximately 100% RNA capture efficiency within 25 min, which occurred through electrostatic interactions between the positively charged pDMAMS surface and the negatively charged RNA. The captured RNA is then detected via chamber digital PCR, enabling a sensitive, accurate, and rapid diagnosis. Our platform provides a simple and efficient RNA extraction and detection strategy that allows detection from 22 nasopharyngeal swabs and 21 saliva specimens with 0% false negatives. The proposed method can facilitate the diagnosis of COVID-19 and contribute to the prevention of early-stage transmission.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

Electrochemical One-Step Immunoassay Based on Switching Peptides and Pyrolyzed Carbon Electrodes.

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (Cdl), high electron transfer rate (kapp), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.

Multiplex SNP Genotyping Using SWITCH: Sequence-Specific Nanoparticle with Interpretative Toehold-Mediated Sequence Decoding in Hydrogel.

Single nucleotide polymorphisms (SNPs) that can alter phenotypes of individuals play a pivotal role in disease development and, more importantly, responses to therapy. However, SNP genotyping has been challenging due to the similarity of SNP alleles and their low concentration in biological samples. Sequence-specific nanoparticle with interpretative toehold-mediated sequence decoding in hydrogel (SWITCH) for multiplex SNP genotyping is presented. The encoding with gold nanoparticle probes transduces each SNP target to ≈1000 invaders with prominently different sequences between wild and mutant types, featuring polymerase chain reaction (PCR)-free amplification. Subsequently, the toehold-mediated DNA replacement in hydrogel microparticles decodes the invaders via SNP-specific fluorescence signals. The 4-plex detection of the warfarin-associated SNP targets spiked in commercially validated human serum (S1-100ML, Merck) is successfully demonstrated with excellent specificity. This work is the first technology development presenting PCR-free, multiplex SNP genotyping with a single reporting fluorophore, to the best of knowledge.

Switching-peptides for one-step immunoassay and its application to the diagnosis of human hepatitis B.

Herein, we present switching-peptides for a one-step immunoassay, without the need for additional antibody treatment or washing steps to detect antigen-antibody interactions. Fluorescently labeled switching-peptides were dissociated from the immobilized antibody soon after the antigens were bound to the binding pockets. In this study, four different parts of the antibody (IgG) frame regions were chemically synthesized, and these peptides were bound to immobilized antibodies as switching-peptides. We presented the design principle of switching-peptides and used Pymol software, based on the changes in thermodynamic parameters, to study the interaction between antibodies and switching-peptides. The binding properties of switching-peptides were analyzed based on Förster resonance energy transfer between switching-peptides as well as between switching-peptides and antibodies (IgGs) isolated from different animals. The binding constants of the four switching-peptides to antibodies were estimated to be in the range of 1.48-3.29 µM. Finally, the feasibility of using switching-peptides for the quantitative one-step immunoassay was demonstrated by human hepatitis B surface antigen (hHBsAg) detection and statistical comparison of the assay results with those of conventional ELISA. The limit of detection for HBsAg was determined to be 56 ng/mL, and the dynamic range was estimated to be 136 ng/mL-33 µg/mL. These results demonstrate the feasibility of the one-step immunoassay for HBsAg.

Comparison the sixth and seventh editions of the AJCC staging system for T1 gastric cancer: a long-term follow-up study of 2124 patients.

BACKGROUND/AIM: The aim of this study was to establish an appropriate TNM staging system for early gastric cancer. METHODOLOGY: We evaluated 2124 patients who had undergone gastrectomy for early gastric cancer between 1989 and 2001. RESULTS: Using the seventh edition of the American Joint Committee on Cancer (AJCC) staging system, we found no significant differences in tumor recurrence and survival between N1 and N2 cancers or between N3a and N3b cancers, whereas the survival curves for N2 and N3 cancers were quite different. Similarly, using the classification in the sixth edition of the AJCC staging system, we found no significant difference in survival between the N2 and N3 cancer groups, whereas the survival curves for N1 versus N2 or N3 cancers were quite different. CONCLUSIONS: The classifications in the sixth and seventh editions of the AJCC staging system have a limitation for T1 gastric cancer (early gastric cancer).

Pressure-tunable guided-mode resonance sensor for single-wavelength characterization.

A method of tuning a one-dimensional guided-mode resonance grating through the use of an air-pressure-responsive membrane is demonstrated here using finite-element method simulation. The device consists of a titanium dioxide (TiO(2)) grating structure embedded in a flexible polydimethylsiloxane membrane. This grating has a resonant response to TM-polarized light at a wavelength dependent upon the refractive index of the surrounding medium. By varying the pressure by 5500 Pa, lateral strain may be applied to the grating; this allows resonances to be produced for medium refractive indices ranging from 1.33 to 1.50 for a fixed-wavelength 850 nm light source.