Dengue virus type 2 NS1 (DENV2 NS1) is a specific and sensitive protein biomarker for dengue fever diagnosis. In this study we used polyvalent phage display to identify unique affinity peptides that can bind NS1 protein. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to investigate the binding interactions. The potential affinity peptide-displayed phage from these methods was selected; its sequence was EHDRMHAYYLTR (R3#10). Amino acid sequence analysis showed that the peptide was rich in basic residues (two His and Arg). Among all the peptides tested, R3#10 showed the greatest decrease in current in CV and increase in impedance in EIS upon binding to NS1 proteins. EIS revealed that R3#10 phage clones were more specific towards NS1 proteins, as compared to bovine serum albumin or the M13 wild type used as control. Detection of NS1 proteins is in accordance with the electron-transfer resistance (Rct) value of the sensor layer, which is confirmed by EIS, and the Kd value of the R3#10 peptide while binding to the phage particles was measured. To the best of our knowledge, this is the first example of identification and characterization of NS1 binding affinity peptides using phage display technology and electrochemical methods. We concluded that these new peptide-displayed phages or free peptides from phages may have potential applications in dengue diagnosis.

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Therefore, more sensitive and early diagnostic methods for CRC are urgently needed. In this study, an efficient electrochemical biosensor for early diagnosis of adenoma-to-carcinoma progression that employs a series of chemically modified affinity peptides was developed. A series of amino acid-substituted and cysteine-incorporated synthetic peptides with flexible linkers was chemically synthesized and immobilized to a gold sensor layer; performance of the sensor was monitored using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Potential affinity peptides (LRG1 BP1–BP4) specific for the LRG1 biomarker as a target protein were chosen according to a quantitative current decrease and dynamic impedance increase by CV and EIS, respectively. Using EIS, the Kd value of the LRG1 BP3 peptide was found to be 8.3 ± 2.7nM. The applicability of the sensor to detect LRG1 proteins was confirmed in human plasma from colorectal adenomas and carcinomas (n = 20 in each group). The detection of LRG1 in accordance with the ΔRct value (electron-transfer resistance at the electrode surface) of the sensor layer incorporating LRG1 BP3 peptides showed a statistically significant difference (p < 0.001) between adenomas and carcinomas, indicating that the potential use of this biosensing platform for detecting the CRC biomarker, as well as for monitoring the colorectal adenoma-to-carcinoma transition in an electrochemically miniaturized biosensor (e-chem biosensor) in point-of-care testing, is possible.

An electrochemical peptide sensor employing a sensitive synthetic peptide was designed for the diagnosis of sepsis.

The exopolysaccharides (EPS) production in an edible mushroom Collybia maculata was substantially increased by supplementation of organic solvents into the medium. Among the organic solvents tested as stimulating agents, 0.3% (v/v) toluene gave maximum EPS production (3.81g/l) when supplemented at the late growth phase, by which the EPS production increased by 86% in flask cultures. This result was similarly reproduced in a bioreactor, where the maximum EPS concentration indicated 3.94g/l after 4.5d. A prolonged culture time after supplementation of toluene (preferably 12h in this study) resulted in additional enhancement in EPS production (max. 4.12g/l). The compositional analysis of the EPS produced before and after toluene treatment revealed that the EPS did not contain significant amounts of other intracellular polymeric substances. The microscopic observations evidenced that a distinct morphological difference existed between the cells before and after toluene treatment. The altered outer cell structure, as observed from transmission electron microscope (TEM) appears to be the most likely explanation for the increased EPS production in toluene-treated culture. The solvent-assisted fermentation strategy presented in this study may be worth attempting with other mushroom fermentation processes for enhancing production of EPS, particularly those with industrial potential.

Two different exopolysaccharides (EPSs) were obtained by submerged mycelial culture of an edible mushroom, Collybia maculata TG-1, and their chemical structures were studied by gas chromatography (GC), Fourier transform-infrared (FT-IR) spectroscopy, methylation analysis, and NMR spectroscopy. A compositional analysis result by GC indicated that both EPSs (designated as Fr-I and Fr-II) were galactomannans consisting of mainly mannose and galactose. FT-IR spectroscopy was used for obtaining vibrational spectra of the EPSs. In the anomeric region (950–700cm−1), both EPSs exhibited the characteristic absorption at 810cm−1 corresponding to the existence of mannose. The obvious absorption peaks at 910 and 880cm−1 in both EPSs revealed the co-existence of α and β configurations. In a 13C NMR analysis, two anomeric peaks appeared at 102.0 and 99.6ppm, which were assigned to the mannose (C-1) and galactose (C-1) residues, respectively. In a GC–MS analysis, the methlylation data confirmed the presence of a (1→3)-linked β-d-mannopyranosyl backbone mainly substituted O-6 by galactopyranosyl residues. The size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) system showed that the weight-average molecular mass of the Fr-I and Fr-II were 7.95×104 and 2.09×104g/mol, respectively. Moreover, the SEC/MALLS revealed that the molecular conformation of the Fr-I was a random coil, with Fr-II being a rigid rod in aqueous solution.

Optimization of submerged culture conditions for the production of mycelial growth and exopolysaccharides (EPSs) by Collybia maculata was investigated. The optimum temperature and the initial pH for EPS production in a shake-flask culture of C. maculata were found to be 20°C and 5.5, respectively. Among the various medium’s constituents examined, glucose, Martone A-1, K2HPO4, and CaCl2 were the most suitable carbon, nitrogen, and mineral sources for EPS production, respectively. The optimum concentration of the medium’s ingredients determined using the orthogonal matrix method was as follows: 30 g/L of glucose, 20 g/L of Martone A-1, 1g/L of K2HPO4, and 1g/L of CaCl2. Under the optimized culture conditions, the maximum concentration of EPSs in a 5-L stirred-tank reactor was 2.4 g/L, which was approximately five times higher than that in the basal medium. A comparative fermentation result showed that the EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor despite the lower mycelial growth rate. The specific productivities and the yield coefficients in the airlift reactor were higher than those in the stirred-tank reactor even though the volumetric productivities were higher in the stirred-tank reactor than in the airlift reactor.

The effect of medium components (carbon, nitrogen, and mineral sources) and environmental factors (initial pH and temperature) for mycelial growth and exopolysaccharide (EPS) production in Sarcodon aspratus (Berk) S.lto TG-3 was investigated. The optimal temperature (25°C) and initial pH (5.0) for the EPS production in shake flask cultures of S. aspratus were determined using the two-dimensional contour plot. The most suitable carbon, nitrogen, and mineral sources for EPS production were glucose, yeast extract, CaCl 2 and KH 2 PO 4 , respectively. Notably, the EPS production was significantly enhanced by supplementation of calcium ion. Subsequently, the optimum concentration of glucose (30gl −1 ), yeast extract (15gl −1 ), CaCl 2 (1.1gl −1 ), and KH 2 PO 4 (1.2gl −1 ) were determined using the orthogonal matrix method. The effects of nutritional requirement on the mycelial growth of S. aspratus were in regular sequence of glucose>KH 2 PO 4 >yeast extract>CaCl 2 , and those on EPS production were in the order of glucose>yeast extract>CaCl 2 >KH 2 PO 4 . Under the optimal culture conditions, the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68gl −1 after 4days of fermentation, which was 6-fold higher than that at a basal medium. The two-dimensional contour plot and orthogonal matrix method allowed us to find the relationship between environmental factors and nutritional requirement by determining optimal operating conditions for maximum EPS production in S. asparatus . The statistical experiments used in this work can be useful strategies for optimization of submerged culture processes for other mushrooms.