Graphene quantum dot-gold hybrid nanoparticles integrated aptasensor for ultra-sensitive detection of vitamin D₃ towards point-of-care application

Highlights

• Detection of vitamin D₃ using GQD-Au hybrid nanoparticles integrated aptasensor is presented.

• The developed aptasensor has a linear range of 1 nM – 500 nM & LOD of 0.28 ng mL^−1.

• The developed aptasensor has a sensitivity of 0.90 Ω nM⁻¹ mm⁻² and a response time < 1 min.

• The shelf life of the developed aptasensor is nearly 35 days.

• A prototype with the developed aptasensor integrated with controlled electronics is established.

Abstract

Vitamin D is a sunshine vitamin required by the body for various physiological activities. Deficiency of vitamin D (≤29 ng mL⁻¹) can cause dental diseases, sarcopenia, osteoporosis, depression, type 2 diabetes, cancer, etc. Additionally, elevated levels of vitamin D (>150 ng mL⁻¹) can result in numerous infirmities such as anorexia, irregular heartbeat, hypercalcemia, fatigue, etc. Hence, a periodic detection can help maintain an appropriate level (≥30 ng mL⁻¹) of vitamin D in blood serum. Conventional techniques used for the detection of vitamin D are expensive, time consuming, require skilled work force and a specialised laboratory. Herein, we report a portable electrochemical aptasensor for the detection of vitamin D₃ using graphene quantum dot-gold (GQD-Au) hybrid nanoparticles. The developed aptasensor has a linear range of 1 nM – 500 nM, limit of detection (LOD) of 0.70 nM (0.28 ng mL⁻¹), limit of quantification (LOQ) of 2.09 nM (0.84 ng mL⁻¹), sensitivity of 0.90 Ω nM⁻¹ mm⁻² and a response time < 1 min. The sensor shows high specificity towards vitamin D₃, a good stability, shelf life of over 35 days and nearly 98% recovery with serum samples. The developed sensor has been integrated with controlled electronics, thus establishing a portable prototype.

Introduction

Vitamin D (sunshine vitamin) is an essential fat-soluble vitamin required for a variety of vital physiological activities. Individuals with deficient levels of vitamin D are growing at an alarming rate, around the globe. Due to numerous environmental and socio-economic factors, people are unable to derive sufficient quantity of vitamin D required for healthy functioning of their bodies. In addition, due to indirect symptoms individuals are unaware to get themselves monitored.

Maintaining optimum levels of vitamin D (≥30 ng mL⁻¹) is essential for staying free from maladies. Deficiency of vitamin D (≤29 ng mL⁻¹) can lead to ocular infection, dental and periodontal diseases, respiratory viral infections [1], sarcopenia [2], osteoporosis [3], type 2 diabetes [4], cancer [5], [6], depression, alcoholism and schizophrenia [7], [8]. In addition, toxicity of vitamin D₃ (>150 ng mL⁻¹) can lead to hypercalcemia [9], constipation, anorexia, muscle pain, joint pain and irregular heartbeat [10].

Vitamin D can be found in different forms however, only vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol) are significantly present in the body. Amongst vitamin D₂ and D₃, the latter has a higher biological activity in maintaining the overall vitamin D status. This is mainly because it has a superior affinity towards vitamin D binding receptors and is a preferred substrate for the hydroxylase enzyme present in the liver (which is responsible for the conversion of vitamin D to 25-hydroxyvitamin D) [11] and has a higher half-life [12].

Some of the conventional techniques utilized for the detection of vitamin D₃ include radioimmunoassay [13], chemiluminescence immunoassay [14], high-performance liquid chromatography (HPLC) [15] and enzyme-linked immunosorbent assay (ELISA) [16]. The disadvantages of such techniques are that these are time-consuming, expensive, use elaborate machinery and require high volumes of patient samples along with skilled lab technicians for analysis. Therefore, it is has become the need of the hour to develop point-of-care (POC) devices that are portable, user friendly and extremely cost effective in order to perform relevant medical interventions to overcome insufficiency, deficiency and toxicity of vitamin D₃ in humans.

In contrast to the conventional diagnostic techniques, electrochemical biosensors have been established as an economic, rapid, highly sensitive and simple tool, which facilitates POC monitoring and diagnosis of medical ailments [17]. Previously reported biosensors for the determination of vitamin D₃ levels utilize numerous bio-recognition elements such as enzymes [18] and antibodies [19] immobilized onto glassy carbon electrode (GCE) and indium tin oxide (ITO) respectively. Reports have also shown the determination of vitamin D₃ using the principle of Surface Plasmon Resonance (SPR) [20]. However, the drawbacks of such electrochemical biosensors include high cost, instability at room temperature (rapid denaturation) and a shorter shelf life.

The use of Micrux Single Electrodes as sensing platform for smart sensing devices pose a multitude of advantages such as low sample volumes, enhancement in signal-to-noise ratio, etc. [21]. Aptamers are groups of single-stranded nucleic acids that have an ability to specifically bind to a wide range of targets [22] and have been widely utilized as recognition elements. Use of aptamers in biosensors provide several advantages such as they facilitate the generation of signals when an analyte of choice interacts with the aptamer at the electrode surface thereby aiding in the quantification of analytes.

Nanomaterials are found to have unique properties owing to high surface to volume ratio that lead to both physical and chemical differences. Therefore, a wide range of nanomaterials have been employed in the development of biosensors. From the plethora of nanomaterials, carbon and its allotropes (especially graphene quantum dots (GQDs)) have become the state-of-the-art material for electrochemical sensing application due to advantages such as low cost in comparison to metals, facile and green fabrication techniques, easy functionalization, and high electron transport properties [23], [24]. Modification of GQDs with noble metal nanoparticles (e.g. Au, Ag, Pt, etc.) allow effective and site-specific conjugation of various bio-recognition elements [25]. GQD-metal hybrid nanoparticles exhibit superior electrochemical, electronic properties and have been extensively used in electrochemical sensors. Au nanoparticles and graphene quantum dots (GQDs) co-modified glassy carbon electrode have been designed for catechol sensing [26]. A synergistic cooperation between Au nanoparticles and carbon dots is reported to increase specific surface area and enhance electronic and catalytic properties of glassy carbon electrode for sensing applications. A bimetallic nano composite incorporating Au-Pt nanoparticles along with GQDs has been fabricated [27] for simultaneous determination of ascorbic acid, dopamine (DA), uric Acid and tryptophan. Pd nano particles decorated with N-doped graphene quantum dots@N-doped carbon hollow nano spheres demonstrated high electrochemical performance due to the synergistic effect derived from their unique structure and extraordinary electrocatalytic properties [28].

Herein, we report for the first time an electrochemical aptasensor for the detection of vitamin D₃ using GQD-Au hybrid nanoparticles. The hybrid structure provides suitable functional groups that facilitate faster electron transfer kinetics for electrochemical sensing of vitamin D₃ and superior optical and electronic properties. The developed aptasensor has also been integrated with miniaturized electronics for rapid and timely theragnostic.