Efficient label-free chemiluminescent immunosensor based on dual functional cupric oxide nanorods as peroxidase mimics

Highlights

• Dual-functional copper oxide nanorods (CuONRs) as peroxidase mimics were synthesized.

• The CuONRs possess excellent chemiluminescent (CL) catalytic activity with high stability.

• A novel and sensitive label-free CL immunesensor was proposed based on these mimics.

• This nanozyme-based immunosensor is potential for fast and efficient biosensing applications.

Abstract

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1–60 ng mL⁻¹ and a low detection limit of 0.05 ng mL⁻¹. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

Introduction

Artificial enzymes have gained significant interest in recent years (Bonar-law and Sanders, 1995, Gao et al., 2007, Lin et al., 2014a) due to the numerous intrinsic defects natural enzymes suffer from, which include limited sources, poor stability, and high sensitivity to environmental changes leading to denaturation and inactivation (Shoji and Freund, 2001, Nelson and Cox, 2005, Wei and Wang, 2008). As a result, the design of artificial, biomimetic materials for biosensor applications has rapidly emerged as an active research field (Genfa and Dasgupta, 1992, Liu et al., 1999, Xu et al., 2013, Souza et al., 2013, Lin et al., 2014b). For example, significant advances in nanotechnology (Jiang et al., 2015) have boosted the emergence of numerous functional nanomaterials with peroxidase-like characteristics, such as V₂O₅ nanowires (André et al., 2011), WS₂ nanosheets (Lin et al., 2014c), Cu nanoclusters (Hu et al., 2013), ceria nanoparticles (NPs) (Asati et al., 2009), carbon NPs (Wang et al., 2011), Pt NPs (Gao et al., 2013), Au NPs (Zhao et al., 2016), Ag NPs (McKeating et al., 2013), Fe₃O₄ NPs (Gao et al., 2007, Dong et al., 2012, Guan et al., 2012), and CoFe₂O₄ NPs (Shi et al., 2011). These nanozymes, which possess both intrinsic enzymatic activity and high stability, have been utilized to catalyze analyte-triggered and H₂O₂-mediated colorimetric reactions, which have been applied within a variety of bioanalyses and clinical disease diagnostics. Cupric oxide (CuO) NPs, which are inexpensive, abundant, and easily prepared, have recently been reported to exhibit excellent peroxidase-like activity and high stability over a wide range of pH and temperatures (Chen et al., 2012, Hong et al., 2013), though to the best of our knowledge, have not been utilized for their enzyme-mimetic capabilities within an immunosensing platform.

Label-free detection platforms, which include surface plasmon resonance (SPR) (Zou et al., 2015), quartz crystal microbalance (QCM) (Tang et al., 2013), atomic force microscopy (AFM) (Zhang et al., 2014), and electrochemical impedance spectroscopy (EIS) (Date et al., 2014), show tremendous promise for quantitative protein detection. Label-free processes are usually simple, cost-effective, less time-consuming, and amenable to real-time analysis, thereby improving assay efficiency (Okuno et al., 2007, Zhao et al., 2009a, Qi and Li, 2011, Pang et al., 2015). However, the aforementioned systems usually require expensive and complicated instruments, which greatly restrict their popularization and practical use. Chemiluminescence (CL) detection, on the other hand, is a versatile analytical technique that has been widely exploited in immunoassay development for its multiple attractive features, which include remarkable sensitivity, wide dynamic range, and cheap instrumentation with simple operation (Adam et al., 2005, Chen et al., 2013, Zhao et al., 2009b). However, traditional CL immunoassays usually employ labeled detection, and the development of a label-free CL method remains an ongoing challenge. In our previous work, we developed a label-free CL immunosensing platform based on co-immobilization of capture antibodies and peroxidase enzymes on a solid support (Yang et al., 2015, Luong and Vashist, 2017). However, the natural enzymes used in this system are relatively expensive and are sensitive to structural/activity changes during immersion in the CL substrate solution, which may lead to an unstable signal. As a potential solution, nanozymes have recently been utilized to catalyze luminol/H₂O₂ reactions for CL detection of biological substances (Triantis et al., 2008, Liu et al., 2011, Qi and Li, 2011). Such constructs could offer an alternative pathway toward the design of more stable and cost-efficient label-free CL immunosensing platforms by means of peroxidase-mimetic nanomaterials.

Carcinoembryoic antigen (CEA) is a cell surface glycoprotein related to lung, liver, pancreas, breast, cervix, and prostate cancer (Gao et al., 2015, Xu et al., 2017), and its concentration in healthy adults is usually less than the value of 5.0 ng mL⁻¹. Abnormally elevated serum AFP usually occurs in several malignant diseases or non-cancerous diseases. Therefore, the quantitative detection of this tumor biomarker is of great significance in clinical tumor diagnosis and evaluating curative effects (Fu et al., 2006). In this work, a novel and facile label-free CL immunosensor based on dual-functional CuO nanorods (CuONRs) is proposed for the highly sensitive detection of CEA. CuONRs, which were synthesized by a simple hydrothermal method, served not only as a peroxidase mimic to catalyze the CL reaction, but also as a solid support for the immobilization of biomolecules and recognition elements. The construction and design of our nanoparticle-mediated, label-free CL assay is illustrated in Scheme 1. A CuONR-chitosan solution was first coated onto an epoxy-modified glass slide to form a solid CuONRs-chitosan support. Streptavidin was then used to functionalize the composite for highly selective capture of biotinylated antibodies. The immunocomplexes formed on the sensing interface after online incubation are shown to hinder the diffusion of the CL substrate molecules to the CuONR surface. These restrictions effectively inhibit the nanozyme-catalyzed CL reaction, thereby leading to a decrease in CL signal with increasing analyte concentration, without the need for target labeling or enhancement schemes. This research opens a promising avenue for the development of robust and efficient label-free CL immunoassay methods.