Development of proof of concept for prostate cancer detection: an electrochemical immunosensor based on fullerene-C₆₀ and copper nanoparticles composite film as diagnostic tool

Highlights

• A novel proof of concept was developed for the detection of prostate-specific antigen (PSA) in serum samples.

• HQ@CuNPs-fullerene-C₆₀/GCE sensing platform was designed to fabricate immunosensor.

• The fabricated immunosensor exhibited excellent sensitivity, special selectivity, and long-term stability.

• The immunosensor showed excellent limit of detection as 0.002 ng/mL.

• The immunosensor displayed a promising capacity for applying in clinical detection.

Abstract

Screening of Prostate-specific antigen (PSA) in human blood is the most common approach to diagnose prostate cancer. The joint application of biology and electrochemistry has shown a tremendous rise in research towards the development of electrochemical diagnostic tools for various diseases. The present study demonstrates the development of an effective immunosensing platform incorporating hydroquinone (HQ) immobilized, fullerene-C₆₀ and copper nanoparticles (CuNPs) composite film on glassy carbon electrode (HQ@CuNPs-reduced-fullerene-C₆₀/GCE) for the selective, quick and trace detection of PSA. In order to fabricate immunosensor sequential immobilization of primary antibody (Ab₁), blocking agent (bovine serum albumin (BSA)), antigen (prostate-specific antigen (PSA)) and secondary antibody (Ab₂) tagged with horseradish peroxide (HRP) was carried out on HQ@CuNPs-reduced-fullerene-C₆₀/GCE. Electrochemical characterization and the signal response of immunosensor were tested using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Due to the synergetic effect of fullerene-C₆₀ and CuNPs, the novel nanocomposite film exhibited excellent catalytic activity towards hydrogen peroxide (H₂O₂) reduction for greatly amplified immunosensing signals. HQ@CuNPs-fullerene-C₆₀/GCE exhibited a well-defined redox peak and accelerated electrochemical reduction of H₂O₂ without any interference of dissolved oxygen and false-positive result in phosphate buffer solution (PBS) at pH 7.0. The parameters influencing the electrochemical response were optimized. Under the optimized conditions, wide linearity between PSA concentrations and current responses ranging from 0.005 ng/mL to 20 ng/mL with the lower detection limit of 0.002 ng/mL was obtained at the proposed immunosensor. The clinical applicability of the proposed immunosensor was successfully tested in serum and urine samples. Results revealed that the proposed immunosensor may create new boundaries in the identification of PSA in human blood samples.

Introduction

Providing diagnostic testing with high precision and sensitivity at the bedside of the patients is the need for clinical diagnostics and research as well. Therefore affordable, portable user-friendly, rapid and sensitive detection point of care (POC) testing devices are required to be developed [1]. Cancer is the leading cause of mortality and prostate cancer is a third major cause of death in men due to cancer [[2], [3], [4]]. However, currently, there is still no effective diagnostic tool for the early detection of prostate cancer. Early and accurate detection can provide the possibility to prolong survival time and increase the chances of successful treatment [[5], [6], [7]]. Prostate-specific antigen (PSA) is the most significant and effective protein biomarker for the screening of prostate cancer [8]. While diagnosing the patients for prostate cancer, total PSA concentration in human serum is determined. Increasing concentration of PSA beyond 4.0 ng/mL confirms the positive result of prostate cancer [9]. For the patients, who have been operated for prostate cancer, it is regarded as a serious sign when the level of PSA increases beyond 0.5 ng/mL [10]. In regard to these issues, sensitive and accurate detection of PSA becomes crucially necessary for early and quick detection and proper monitoring during treatment.

Nowadays, most of the preliminary diagnosis is being pursued and carried using extremely sophisticated techniques, such as computed and positron-emission tomography, ultrasound-based imaging, and nuclear magnetic resonance [11]. They entirely depend upon the growth and attainment of size of detectable cells. These methods are limited to the mass of billions of cells, but failure in the identification of advanced stage [12]. Apart from these, several advances in bioanalytical methods have been developed, such polymerase chain reaction (PCR) [13], enzyme-linked immunosorbent assay (ELISA) [14], mass spectroscopy (MS) [15], chromatography [16], electrophoresis [17], and surface plasmon-based methods [18]. These methods make the identification of a thousand times lesser-sized cells possible than previous conventional primary diagnosis. But the main disadvantages with these techniques are that they require sophisticated laboratory space, sample transportation, increased waiting times, increased administration and medical cost, required professional skilled technicians for the sample processing and handling conditions, and storage time, so sometimes it may produce inadequate results and finally increase the number of biopsies. Such problems may be overcome by using a simple and specific biosensing tool in the real-time analysis [19]. Though various types of biosensors have been developed, electrochemical immunosensors stand as a promising tool with several significant features such as high specificity, simplicity and biocompatibility, large surface area, less expensive with existing methods and easy to handle [20,21]. While developing immunosensing platforms, the stability of the immobilized proteins and tremendously increased signal intensity are the two key factors [22]. For efficient immobilization of bio proteins, better sensitivity, fast and facile detection of biomarkers various nanomaterials [8,[23], [24], [25], [26]] have been synthesized. Several metal nanoparticles are being used for the development of modified electrodes as an electrochemical sensor but copper nanoparticles (CuNPs) offer excellent properties due to small diameter, large specific surface area, and the ability for fast electron transfer. It is less expensive than other noble metals therefore, the application of copper nanoparticles in the development of modified electrodes is very promising [[27], [28], [29]].

Literature reveals that carbon-based materials have been extensively studied in a variety of applications [[30], [31], [32], [33], [34], [35]]. Various carbon-based materials such as carbon nanotubes (CNTs), graphene, and fullerene have drawn the attention of researchers for several important applications due to the high surface area, good electrical conductivity, strong mechanical strength, biocompatibility and low manufacturing cost. Recently, researchers have paid attention to fullerene-C₆₀ due to its excellent electrochemical properties. The previous studies revealed that partially reduce fullerene-C₆₀ films offer remarkably enhanced electrochemical behaviors in the aqueous solution therefore it has been used for modification electrodes for various electroactive molecules [[36], [37], [38]]. However, fullerene-C₆₀ modified electrodes for the detection of analyte molecules are limited due to its sensitivity issue compared with other nanomaterials' modified electrodes. This could be overcome by incorporating metal nanoparticles over the fullerene-C₆₀ film. However, metal nanoparticles-decorated fullerene-C₆₀ modified electrodes for electrochemical applications are rarely reported in the literature [[39], [40], [41], [42]]. To the best of our knowledge this the first report on novel CuNP-reduced-fullerene-C₆₀ composite film based immunosensor for PSA detection.

Present research work focuses upon the development of an accurate and simple immunosensing platform employing CuNP-reduced-fullerene-C₆₀ nanocomposite film modified on the glassy carbon electrode (GCE). Excellent electron transfer kinetics and attachment of immobilized antibodies could increase at composite film due to enhanced biocompatibility and electrochemical properties. Due to a large amount of HRP-tagged secondary immobilized on CuNP-reduced-fullerene-C₆₀ nanocomposite film, the electrochemical signal of fabricated immunosensor was greatly amplified achieving sensitive detection of PSA. Meanwhile proposed immunosensor also showed good reproducibility, long term stability, wide concentration range and lower detection limit. Therefore, the proposed immunosensor exhibited excellent potential for applications in the monitoring of prostate cancer.