Immunoreaction-triggered diagnostic device using reduced graphene oxide/CuO NPs/chitosan ternary nanocomposite, toward enhanced electrochemical detection of albumin

Highlights

• Reduced Graphene Oxide/CuO NPs/Chitosan Nanocomposite was successfully applied for detection of the Human Serum Albumin (HSA).

• The constructed immunoassay exhibited a linear detection range from 10 to 450 ng.mL⁻¹, with the limit of detection (LOD) 2.6 ng.mL⁻¹.

• The immunosensor performed excellently on the detection of clinical serum samples of HSA.

• The constructed diagnostic device is better than other reported studies in term of LOD and dynamic linear range.

Abstract

Determination of Human Serum Albumin (HSA), an important biomarker, is needed for early diagnosis and therapy monitoring in a variety of diseases. A novel electrochemical immunosensor was constructed to detect HSA in both standard solutions and human serum samples. In so doing, glassy carbon electrode (GCE) was modified in three steps. First, graphene oxide (GO) was drop-casted on the GCE surface and electrochemically reduced. Second, in situ electrochemical deposition (ECD) of cupric oxide nanoparticles (CuO NPs) in two consecutive steps has been done. Finally, anti-HSA antibodies have been immobilized via amino groups of chitosan which drop-casted earlier. The surface morphology and nature of the modified electrode was characterized using common electrochemical and imaging techniques. Using differential pulse voltammetry (DPV), the response of the immunosensor was linear in the range of 10–450 ng.mL⁻¹ HSA, with the limit of detection (LOD) 2.6 ng.mL⁻¹. Moreover, the selectivity of the modified electrode was investigated to some common interferences. Under optimum conditions, the immunosensor displayed excellent stability, reproducibility, selectivity and repeatability. The findings indicated that fabricated electrode is a promising candidate for quantifying HSA that is applicable in diagnostics and therapeutics.

Introduction

Human serum albumin (HSA) is a well-known protein in human plasma which constitutes nearly 50% of the plasma proteins [1]. In clinical studies, the sensitive and precise detection of HSA can be promised complementary for diagnosis of some diseases such as coronary heart disease [2], multiple myeloma [3], liver failure, cirrhosis, chronic hepatitis [4], and kidney damage [5]. Hence, various methods have been applied to determine HSA. One of the well-known methods for HSA determination is colorimetry which is based on binding of Bromocresol Green dye to the HSA [6]. However, there are some disadvantages of using this method such as non-equilibrium and non-specific binding between the dye and analytes as well as time-dependent process [7,8]. Another method is fluorescence spectroscopy, however, it lacks specify, since the emission of HSA interferes with other proteins emission [9,10]. Molecularly imprinted polymers (MIPs), are the other fabrications that have been used as chemosensory for detection of HSA. Generally, complicated fabrication could be one of the most important obstacles in analysis of proteins [11,12]. In compare to various analytical methods, electrochemical procedures could provide us many benefits such as selectivity, simplicity, rapidness, and low-cost of analysis for detection of various analytes including proteins, drugs, and etc. [[13], [14], [15], [16]].

In the past few years, a large number of studies have been focused on electrochemically detection of HSA using various nanomaterials such as functionalized carboxylic graphene/AuNPs [17], single-walled carbon nanotube (SWCNT) [18], polystyrene core with silver nanoshells (PS/Ag) [19], functionalized nanoporous gold [20], immunoglobulin-coated magnetoelastic [21], and Au nanoparticles (AuNPs)-polythionine-methylene blue (PTH-MB) [22] for improvement of analytical properties of the sensing systems.

On the other hand, there has been a dramatic increase in the number of studies which paid attention on graphene structure and its potential application [23,24], due to interesting mechanical and electronic properties of graphene as a 2D conducting nano-sized structure [25,26]. Therefore, it has been used in many field of research like energy storage, preparing of sheet structure materials, water purification, sensors, and biological applications [[27], [28], [29], [30]]. Graphene, in its oxidized form (GO), contains various functional groups such as carboxylic acid, hydroxyl, and epoxy functional groups, in compare to graphene, GO has low conductivity due to impurities of sp³ hybridized functional groups [31,32]. Reduced GO (RGO), is a form of GO which comprises both sp² graphene nano-sized area and low sp³ area (approximately below 10%) [33]. Although the electrical conductivity of the RGO is greater than GO, reacting with other organic molecules or intercalation of metal particles/ions further increases the conductivity of RGO which provides a wide field of research in electrochemical applications especially sensors. Cupric oxide (CuO) is a p-type semiconductor with unique features such as non-toxicity, earth abundance, and environmental compatibility [34,35]. Recently, Cu₂O NPs and CuO NPs have received much attention in various fields such as electrocatalysis [36], photocatalysis [37], supercapacitors [38], optical sensors [39], phase extraction [40], biosensors [41], and storage systems [42]. Hence, CuO NPs is one of the more practical materials of biosensor designing. In the recent years, nanocomposites containing variety of copper nanomaterials and RGO have been applied applied for detecting of glucose [43], catechol organic pollutant [44], tryptophan [45], and Malathion [46]. Growth of the CuO and rGO can greatly improve the charge transfer between the modified layer and GCE surface, probably due to the synergistic effect of the CuO nanoparticles and RGO nanosheets. In addition to the above mentioned advantages, availability and cost-effective of CuO NPs, encouraged us to design a diagnostic device using the CuO NPs instead of some expensive nanoparticles such as silver, gold or so on.

Chitosan (deacetylated chitin (CS)) is a natural polysaccharide containing amino and hydroxyl polar sits which can behave as electron donors and it has the electrical capability related to polarizability of the branched amino groups [47,48]. Due to biocompatibility, non-toxicity, and biodegradability, CS is widely applied in biomedical area including drug delivery, tissue engineering, implant material, cancer therapy, and bio-sensing [49,50]. In addition to these favorable properties, CS represents the strong ability to coordinate to transition metal ions or stabilized them, which was making it useful in sensor filed [49,51]. This ability is related to the presence of amino groups in CS structure. Also, existence these amino groups, make it a versatile support material for immobilization of antibodies with covalent attachment [52].

The aim of this study is to design an electrochemical immunosensor based on CS/CuO NPs/ERGO/GCE for sensitive HSA determination. To the best of our knowledge, there is no report on using the synergistic effect of CS/CuO NPs/ERGO nanocomposite toward fabrication of HSA immunoassay. The properties and characterization of this modified electrode were studied using Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS), Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM), and Energy-dispersive X-ray spectroscopy (EDS) techniques. In addition, stability, reproducibility, and selectivity of the modified electrode to other proteins including BSA, urea, uric acid, glucose, and creatinine have been evaluated. Finally, the applicability of the immunosensor was examined in human serum samples and compared with a clinical method (bromocresol green; BCG).