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

Highlights

• An ultrasensitive and label-free electrochemical biosensor for human norovirus was developed.

• The electrochemical detection was performance by CV, QCM and EIS.

• The detection limit was found to be 7.8 copies/mL.

Abstract

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 99.8 nM for recombinant noroviral capsid proteins (rP2) and 7.8 copies/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.

Introduction

Norovirus is the most common cause of foodborne-disease outbreaks worldwide. Norovirus exposure occurs mainly from contaminated water or polluted food, and infection can rapidly spread from person to person through fecal-oral routes (Beier et al., 2014, Bu et al., 2008, Chung et al., 2015). Infection with norovirus often occurs with relatively low amounts of virus, as low as 1×10² copies/mL (Yakes et al., 2013), however, there are no reports of development of vaccines or tremendous anti-noroviral agents. Considering these issues, a platform that yields rapid, sensitive, and accurate detection of norovirus is desirable (Han et al., 2014, Kim et al., 2015). Currently, there are several methods utilized for efficient detection of the relative levels of norovirus in a sample, such as nucleic acid amplification (Ishida et al., 2008), antibody-based immunoassay (Hong et al., 2015, Yoda et al., 2000), real-time PCR (Suffredini et al., 2011), and other methods (Kwon et al., 2010, Yakes et al., 2013). However, these methods require multiple steps, a long time for sample preparation, and relatively expensive analytical instruments (Park et al., 2010). One of the major issues with these methods is that they are not ready-to-use with high sensitivity and are not suitable for point-of-care clinical applications.

Many studies have focused on providing promising alternative methods for more sensitive and accurate detection of norovirus, such as aptamer-based electrochemical biosensing platforms (Beier et al., 2014, Giamberardino et al., 2013) and miniaturized microfluidic biochips (Chung et al., 2015). In fact, the miniaturized electrochemical detection technique has been widely used for the creation of straightforward label-free diagnostic biosensors capable of real-time monitoring (Wu et al., 2010a, Wu et al., 2010b, Wu et al., 2011, Zhang et al., 2014). The advantages of using an electrochemical method are that it is cost-effective, produces a fast response, is label-free, and is easy to integrate into miniaturized microdevices like portable biosensors (Hong et al., 2015, Shin et al., 2011, Wu et al., 2011, Yea et al., 2016). For example, quartz crystal microbalance (QCM) has been widely used in biosensor development (Geng et al., 2008, Ogi et al., 2011, Wu et al., 2011). With this system, two parameters including frequency (related to mass and thickness) and dissipation (related to rigidity) are measured simultaneously in real time on the sensor surface (Wu et al., 2011). In addition, both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are widely used to measure either the current or the impedance obtained by varying the applied potential (Geng et al., 2008, Wu et al., 2011) upon the binding of targets. Therefore, there is no doubt that electrochemical-based bioanalytical techniques are powerful tools for studying molecular binding events such as peptide-protein interactions on a gold surface.

In aforementioned above, current antibody-based immunoassay has been used for clinical applications. This method relies on antibodies as affinity element and is relatively expensive and multi-step processing for sample preparation. Polyclonal antibodies are relatively cheaper, but heterogeneous to binding properties, while monoclonal antibodies are expensive to produce, but homogeneous. In modern clinical practice, more rapid, sensitive and cost-effective diagnoses for norovirus are crucial. Although the progress has been made in ready-to-use applications, improved biosensing platforms are still needed. Several studies have addressed these pitfalls on the affinity element with the use of peptide (Park et al., 2010, Wu et al., 2010a, Wu et al., 2010b), peptide nucleic acid (Mateo-Marti et al., 2007, Wang et al., 2009), aptamer (Kwon et al., 2010) and nanobody (Chen et al., 2016). In comparison to the molecular weight or size of antibodies, peptides are relatively small, and it can be cost-effectively synthesized. The molecular structures of antibodies are standard units with different isotypes, however, peptides are linear or cyclic, indicating that peptides are more amenable over antibodies to engineering at the molecular level (Hwang et al., 2015). As we previously described (Hwang et al., 2015), unique and short linear peptides specific for recombinant noroviral capsid proteins (rP2) was identified with evolutionary phage display and characterized by ELISA assay. As we previously described (Hwang et al., 2015), P-domain in noroviral capsid proteins connects with the internal S-domain, and it is exposed on the outermost viral surface. In addition, P-domain is involved in norovirus-host cell binding and possesses immunogenic activity (Bereszczak et al., 2012, Bu et al., 2008), indicating that P2 domain has enough potential to be a good candidate for recognizing and detection of norovirus. Based on the our results, the binding affinity of the peptide-displayed phage particles was found to be a nanomolar range against rP2 proteins. Therefore, we have chosen the best peptides specific for noroviral proteins and further explored the performance of the biosensor for electrochemical detection for human norovirus. In this study, we demonstrate an effective approach by creating a novel peptide-based small-molecule electrochemical biosensor for the detection of human norovirus.