Ultrasensitive detection of interleukin 1α using 3-phosphonopropionic acid modified FTO surface as an effective platform for disposable biosensor fabrication

Highlights

• An EIS immunosensor based on carboxyalkylphosphonic acid covered FTO electrode was developed for the determination of IL-1α.

• The prepared immunosensor was characterized by several electrochemical and morphological techniques.

• The prepared immunosensor showed high stability, good repeatability, excellent reproducibility and reusability.

• The proposed biosensor could detect the IL-1α in clinical samples.

Abstract

In this study, we utilized a carboxyalkylphosphonic acid covered fluorine doped tin oxide (FTO) as an electrode material for fabrication of Interleukin 1α (IL-1α) immunosensor. For this aim, anti-IL-1α antibodies were attached on the 3-phosphonopropionic acid (PHP) modified FTO surface covalently. Electrochemical (electrochemical impedance spectroscopy and cyclic voltammetry) and morphological (scanning electron microscopy and atomic force microscopy) characterizations were performed to monitor the successful fabrication of immunoelectrodes. After incubation of anti-IL-1α antibody immobilized FTO electrodes in IL-1α antigen solutions, increases were seen in impedimetric responses. IL-1α antigen was determined in a linear detection range from 0.02 to 2 pg/mL by EIS. The detection limit of the suggested immunosensor was 6 fg/mL. The applicability of the designed biosensor was tested by using human serum and saliva samples and acceptable results were obtained. In addition, high sensitivity, good specificity, low detection limit made the proposed immunosensor a potential technique for IL-1α antigen determination in routine clinical analysis.

Introduction

Cancer is a complex and harmful disease and it affects the human health in worldwide with a high rate of mortality. According to latest cancer statistics, the growth rate of cancer cases will increase in next few years due to some factors such as work, diet and environment. Biomarkers are medical signs and indicate the health status of patients. The accurate detection of tumor biomarkers provides essential information about disease conditions. Therefore, the new method development is necessary to detect tumor biomarkers accurately and quickly in human fluids for early cancer diagnosis and treatment. Saliva is an important biofluid and contains water (about 99%), electrolytes (sodium, potassium, calcium, chloride, magnesium, bicarbonate, phosphate), glucose, enzymes, immunoglobulins, glycoproteins, and polypeptides. These components are important for human health and therefore, saliva is a possible diagnostic tool for disease diagnosis and treatment monitoring. Saliva collection provides several advantages over blood, such as being easy, simple and non-invasive. The biomarker level in saliva is very low. Therefore, the development of sensitive techniques and biosensing methods is important for disease diagnosis and monitoring. There are several commercially available protein assay tools, but these tools are suitable for laboratory testing and non-suitable for point-of-care applications. In addition, these tools are currently used for detection of glucose, different hormones, bacteria [1], [2], [3], [4].

Interleukin 1 is a multifunctional cytokine and has a role in development of inflammatory and immunological responses [5]. IL 1 gene family on chromosome 2q14.2 includes the three related genes (IL-1α, IL 1β and IL-RN), which encode IL-1α, IL 1β and their endogenous receptor antagonist (IL-IRA), respectively [6]. These cytokines are produced in several cell types such as monocytes, macrophages and epithelial cells [7]. IL-1α is a membrane-anchored molecule and has an autocrine signaling mechanism [8]. Furthermore, IL-1α increases the permeability of the endothelial cell monolayers [9]. The role of IL 1α in carcinogenesis has been investigated extensively. Experimental results illustrate the vital role of IL-1α in breast [6], oral squamous cell [10], [11], head and neck squamous cell [12], [13] and tongue cancer [14]. The levels of IL-1α antigen in oral squamous cell cancer patient were 0–137 pg/mL and 175–1000 pg/mL in human serum and saliva, respectively [15].

As mentioned above, IL-1α is a very important biomarker in the biological and pathological events. Therefore, enzyme-linked immunosorbent assay (ELISA) kits and biosensors for specific detection of IL-1α have been developed. There are not many biosensors developed in the literature for the quantification of IL-lα. Because of this, this developed immunosensor is vital for the quantification of IL-1α level in biological fluids. Tao et al. (2006) developed protein imprinted xerogels with integrated emission sites (PIXIES) without utilizing biorecognition molecule for IL-1α detection. To fabricate the PIXIES sol–gel derived xerogels, molecular imprinting, and a luminescent reporter element were used. Interaction between the template site and IL-1α changed the physicochemical properties which were detected with a luminescent probe molecule [16]. An optical biosensor was developed for the investigation of binding kinetics between interleukin 1 receptor and IL-1α. IL-1α was attached on the carboxymethyl dextran surface via amide bonds formed between amino groups of IL-1α antigen and carboxyl groups of carboxymethyl dextran. The linear range and detection limit of this optic biosensor was 100–1600 nM and 100 nM, respectively [17]. A quantum dot (QD) based electrochemical immunoassay was fabricated for IL-1α detection. QD conjugated anti-IL-1α antibody was utilized as a label in biorecognition event and the principle of this immunoassay based on a sandwich immunoreaction. Electrochemical voltammetric stripping analysis was employed to determine the level of IL-1α antigen. The linear range and detection limit were found as 0.5–50 ng/mL and 0.3 ng/mL, respectively. The developed immunoassay gave reproducible results and it was suitable for biomarker detections [18].

Transparent conductive oxides (TCO) are semi-conductive materials and have been extensively utilized in recent years in different fields such as solar cells, electrochemistry, display applications, electrochromic mirrors [19]. They have a special role in electrochemistry, and they are utilized as transparent electrodes for optoelectrochemical and electrochemical experiments [20]. Fluorine-doped tin oxide (F-doped SnO₂, FTO) glass is one of the commercially available conductive oxide (TCO) substrates and it has great potential to replace the currently most popular TCO, tin-doped indium oxide (ITO). ITO is more expensive than FTO owing to the scarcity of indium on Earth. Moreover, FTO is more stable than ITO in thermal conditions. It has high optical transparency in the visible range of spectrum and good electrical conductivity. FTO has many applications as transparent conducting electrode in flat panel displays, LCD’s, electro chromic instruments, organic light emitting diodes and transparent conductive transistors. In recent years, FTO has been used in solar cells, optoelectronic devices, thiols oxidation, lipoic acid quantification, photoelectrochemical biosensors and hydrogen peroxide biosensing [21], [22]. Additionally, FTO provides direct observations of electrode morphology, with confocal microscopes before and after attachment of biorecognition element and desired analyte [23]. In recent years, gold, glassy carbon electrode and indium tin oxide have been used as working electrodes in different applications. However, these working electrodes are more expensive than FTO electrodes. Therefore, we select FTO glass as a working electrode for development of IL-1α biosensor. FTO-glass substrates show high electrical conductivity, high chemical and mechanical stability, good transparency. In addition, they eliminate the problem of diffusion as seen in case of ITO sheets [24].

A critical stage while fabrication of an immunosensor is the immobilization of antibodies on the electrode. The amount of immobilized antibody will affect the accuracy, sensitivity and stability of an immunosensor. Several modification routes have been developed for electrode surface preparation [25], [26]. Electrodeposition [27], [28], [29], electropolymerization [30], [31], anodic treatment [23] techniques have been utilized for FTO biosensor construction. The unique properties of the phosphonates to form a self-assembled monolayer (SAM) on different metal oxides like Al₂O₃, SiO₂, Fe₂O₃, Fe₃O₄, In₂O₃, SnO₂, ITO etc., provide a modified platform for biorecognition elements such as enzymes, antibodies, aptamers immobilization [32], [33]. Silanes, carboxylic acids, and phosphonic acids have been utilized to construct the SAMs on metal oxides. Generally, silanes are usually utilized to form multilayers on metal oxide surfaces. Carboxylate SAMs on metal oxide surfaces are not stable because they rely on the electrostatic interactions between the adsorbate and the surface. However, phosphonic acids form monolayers on metal oxide surfaces in either aqueous or organic solutions, and the phosphonate SAMs are more stable than either silane or carboxylate SAMs. Therefore, phosphonate SAMs become more popular than silane and carboxylate SAMs due to their good stability [34]. In addition, phosphonic acid binds more strongly than carboxylic acids to metal oxides by forming well-packed SAMs [35]. Compared to some types of silane SAMs, the phosphonate SAMs have a better surface coverage and hydrolytic stability. The phosphonate SAMs are more stable than thiol SAMs on gold under ambient conditions for a greater time due to higher bond energy (P-O bond energy: 80 kcal/mol; S-Au bond energy: 40 kcal/mol). These features make phosphonate SAMs very promising platforms for different applications [36].

The aim of the present work was to fabricate a new electrochemical immunosensor for IL-1α biomarker detection. The proposed immunosensor had a simple fabrication protocol without need to use any high-cost matrix material. PHP, a carboxyalkylphosphonic acid, was used as matrix material for immobilization of IL-1α antibodies, which were the biorecognition elements of IL-1α antigens. PHP formed a stable SAMs on the FTO electrode and it was used as an immobilization matrix to prepare a sensitive immunosensor for the first time. PHP was not only a good biorecognition element immobilization matrix material for the fabrication of biosensor, but also it was a low-cost interface material and reduced the cost of the immunosensor. Additionally, this study illustrated the applicability of phosphonic acid-based surface on biosensing technology. Moreover, FTO electrode was cheaper than traditional electrodes such as gold, platinum, glassy carbon electrode and screen-printed electrode. FTO electrode had good electrical conductivity and high stability. Because of these promising properties of FTO electrode, it was chosen as an immobilization platform. In order to quantify the IL-1α antigen, EIS technique was utilized as an analytical technique. The variations formed on the FTO electrode surface were followed by EIS and cyclic voltammetry (CV) measurements. During the electrochemical analyses potassium ferricyanide/ferrocyanide ([Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻) was employed as an electrochemical redox couple. After immobilization of IL-1α antigen on the FTO surface, an increase was measured in impedance response because of the immunocomplex formation between anti-IL-1α antibody and IL-1α antigen. The suggested biosensor had high sensitivity, excellent selectivity, excellent repeatability and reproducibility, and acceptable recovery. Its high sensitivity was originated from specific interaction between immobilized anti-IL-1α antibody and IL-1α antigen.