Development of peptide biosensor for the detection of dengue fever biomarker, nonstructural 1.

Dengue virus (DENV) nonstructural 1 (NS1) protein is a specific and sensitive biomarker for the diagnosis of dengue. In this study, an efficient electrochemical biosensor that uses chemically modified affinity peptides was developed for the detection of dengue virus NS1. A series of amino acid-substituted synthetic peptides was rationally designed, chemically synthesized and covalently immobilized to a gold sensor surface. The sensor performance was monitored via square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). Potential affinity peptides specific for NS1 were chosen according to the dynamic current decrease in SWV experiments. Using circular dichroism, the molar ellipticity of peptides (DGV BP1-BP5) was determined, indicating that they had a mostly similar in random coil structure, not totally identical. Using SWV, DGV BP1 was selected as a promising recognition peptide and limit of detection for NS1 was found to be 1.49 µg/mL by the 3-sigma rule. DGV BP1 showed good specificity and stability for NS1, with low signal interference. The validation of the sensor to detect NS1 proteins was confirmed with four dengue virus culture broth (from serotype 1 to 4) as proof-of-concept. The detection performance of our sensor incorporating DGV BP1 peptides showed a statistically significant difference. These results indicate that this strategy can potentially be used to detect the dengue virus antigen, NS1, and to diagnosis dengue fever within a miniaturized portable device in point-of-care testing.

An electrochemical peptide sensor for detection of dengue fever biomarker NS1.

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.

Selection of affinity peptides for interference-free detection of cholera toxin.

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.

Electrochemical peptide sensor for diagnosing adenoma-carcinoma transition in colon cancer.

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.

High sensitive and selective electrochemical biosensor: Label-free detection of human norovirus using affinity peptide as molecular binder.

Norovirus is known as the major cause of highly infection for gastrointestinal tracts. In this study, robust and highly sensitive biosensors for detecting human norovirus by employing a recognition affinity peptide-based electrochemical platform were described. A series of amino acid-substituted and cysteine-incorporated recognition peptides isolated from evolutionary phage display technique was chemically synthesized and immobilized to a gold sensor layer, the detection performance of the gold-immobilized synthetic peptide-based sensor system was assessed using QCM, CV and EIS. Using EIS, the limit of detection with Noro-1 as a molecular binder was found to be 99.8nM for recombinant noroviral capsid proteins (rP2) and 7.8copies/mL for human norovirus, thereby demonstrating a high degree of sensitivity for their corresponding targets. These results suggest that a biosensor which consists of affinity peptides as a molecular binder and miniaturized microdevices as diagnostic tool could be served as a new type of biosensing platform for point-of-care testing.

Localization of CKII beta subunits in Lewy bodies of Parkinson's disease.

We reported previously that phosphorylation by casein kinase II (CKII) regulates the interaction between alpha-synuclein and its binding partner synphilin-1, and that both CKII alpha and beta subunits co-localize with alpha-synuclein in cytoplasmic inclusions in transfected cells. In this study, we extended these observations to the brains of patients with Parkinson's disease (PD) and examined whether CKII subunits are present in Lewy bodies. Immunohistochemical studies on PD brains harboring Lewy bodies revealed a positive stain for CKII beta but not for CKII alpha. In addition, CKII beta subunits co-localized with alpha-synuclein in most Lewy bodies. These findings suggest that CKII beta subunits may play a role in the formation of intracytoplasmic inclusions in human alpha-synucleinopathies either through phosphorylation events or through a separate mechanism linked to the beta subunit itself.