Cupric oxide (CuO) is a member of first row transition metal oxides with unique properties and advantages such as its inexpensive nature and abundance on Earth [1
]. CuO, with its controlled shape and dimension, has received more attention due to its potential applications in various fields such as catalysis [3
], batteries [9
], solar cells [13
], supercapacitors [15
], sensors [17
], and photodetectors [20
]. Besides this, CuO as nanostructured materials can reveal size dependent physical and chemical properties, along with high surface area and quantum confinement [22
]. Active research activities have been carried out for the synthesis of nanostructured CuO materials with well-defined morphology and size [2
]. Thus, numerous morphologies of CuO are produced including nanoparticles, nanoneedles, nanowhiskers, nanowires, nanoshuttles, nanorods, nanotubes, nanoleaves, and nanoribbons via wet chemistry and physical methods [23
]. Moreover, the complex nanostructures of CuO are also synthesized including nanoellipsoids [32
], peanut-like nanostructures [33
], nano-dendrites [34
], prickly/layered microspheres [35
], and dandelion-like hollow morphology [36
]. The wet chemical method has more importance over other existing methods due to its low cost, simplicity, and gives a high yield of nanostructured material. Due to fascinating electrochemical properties of CuO, it is able to be a main component of electrochemical sensors, especially potentiometric sensors [37
Uric acid (UA) is the major product of purine metabolism and its release in urine is because of purines that are formed in the catabolism of the dietary and endogenous nucleic acid. The formation of uric acid in excess can result in precipitation in the kidney and it hinders urine excretion. The possibility of gout may be observed due to the metabolism of uric acid [38
]. Several studies have shown that the formation of excessive uric acid in human blood is highly risky and can cause cardiovascular diseases [39
]. Therefore, the estimation of uric acid in human physiological fluids is very important for the early diagnosis of patients who are victim to a wide range of abnormalities due to variations in purine metabolism. Currently, several uric acid biosensors are reported from different research groups [40
]. Many of these biosensors are based on an amperometry technique [45
]. These biosensors suffer severe disadvantages that hinder their practical applications due to their working potential at 0.7 V [49
]. The relatively high electrode potential makes other competing species oxidize on the surface of electrode [50
]. This kind of limitation and interference can be avoided by using the potentiometric configuration that works at a negligible bias voltage as previously reported in the several studies [51
]. The vitamin B12
has been used as a reducing and capping agent for the preparation of noble metal nanoparticles [55
The template assisted nanostructured materials have the advantage of fast growth nucleation and in getting controlled morphology of nanomaterials. The vitamin B12 has biocompatibility with metal oxide nanostructures, which is being presented in this work as an evident for the growth of other metal oxides. It is for the first time that vitamin B12 is used as a growth directing agent for tuning the morphology of CuO nanostructures. The present study is focused on the preparation of heart/dumbbell-like CuO nanostructures using vitamin B12 with excellent functional properties during the development of a potentiometric uric acid biosensor for the first time.
In this research work, vitamin B12 is used as growth directing agent to control the morphology of CuO nanostructures using a low-temperature aqueous chemical growth method. The CuO nanostructured material is characterized by SEM, XRD, and XPS techniques. The functional properties of nanostructured CuO are demonstrated in the development of a sensitive, selective, stable, reproducible, and repeatable uric acid biosensor. The proposed potentiometric configuration was selectivity used in the determination of uric acid from the real samples, which confirms the practicality of the presented analytical device.
2. Experimental Section
2.1. Chemicals Used
Copper nitrate pentahydrate (Cu (NO3)2·5H2O, 25% ammonia, vitamin B12, uricase (E.C. 126.96.36.199), 25 units/1.5 mg from Arthrobacter globiformis, uric acid, d-glucose, ascorbic acid, glutaraldehyde, hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), and potasium dihydrogen phosphate (KH2PO4) were purchased from Sigma Aldrich, Jilin, china. A phosphate buffer solution of 10 mM was made by mixing appropriate quantities of NaCl, KCl, Na2HPO4, and KH2PO4 in deionized water and a fixed pH of 7.4 for the phosphate buffer solution was obtained by adding a certain volume of 1 M NaOH, and 1 M HCl. A fresh uric acid solution was prepared in the phosphate buffer solution and kept at 4 °C. The low concentration solutions were prepared using a dilution method. All the chemicals used were of analytical grade and used without further any purification.
2.2. Synthesis of CuO Nanostructures Using a Low-Temperature Aqueous Chemical Growth Method with Vitamin B12 on Gold Coated Glass Substrates
To modify the surface of the gold-coated glass substrates with CuO nanostructures, a two-step methodology was followed. First, glass substrates were cleaned with acetone and deionized water in an ultrasonic bath, then dried with flowing nitrogen gas. Afterwards, the glass substrates were fixed inside the vacuum chamber of a Satis, Norrköping, Sweden (CR 725) evaporator. A thin layer of 10 nm of chromium was deposited on the glass substrates as an adhesive layer, followed by the 100 nm thickness deposition of the gold layer. In the second step, gold-coated glass substrates were again sonicated in acetone in an ultrasonic bath for 15 min and dried using flowing nitrogen gas, then a seed layer of CuO nanoparticles was spin-coated using a spin coater at 2500 rpm for 30 s and seed coated substrates were annealed at 120 °C for 30 min in order to get a firm binding of seed particles on the substrates. Afterwards, a 25 mM copper nitrate pentahydrate solution was prepared in 100 mL deionized water and 5 mL of 25% ammonia was added in the solution. In order to facilitate the growth process and to get CuO nanostructured material of desired properties, 0.5 g of vitamin B12 was used as a soft template. Then, the seed-coated gold-coated glass substrates were fixed in a Teflon sample holder and kept in the growth solution. The beaker was tightly sealed using aluminum foil and kept at 95 °C for 24 h. Afterwards, the CuO-modified substrates were collected and washed with the deionized water in order to remove the residual particles from the surface of the nanostructured CuO and dried with flowing nitrogen gas.
2.3. Material Characterization
The morphology and structural investigations of nanostructured CuO were performed by SEM at a 15 kV accelerating voltage. The crystal structure was studied using X-ray powder diffraction (XRD) with a Phillips (PW 1729, Tokyo, Japan) powder diffractometer associated with CuKα radiation (λ = 1.5418 Å at a generator voltage of 40 kV and a current of 40 mA). The XPS experiments were done using an ESC (A200, Sweden) spectrometer in highly vacuum of pressure of 10−10 mbar. The measurement chamber was equipped with a monochromatic Al (Kα) X-ray source employing photons of frequency (hν = 1486.6 eV).
2.4. The Immobilization of Uricase Enzymes on the Nanostructured CuO and Potentiometric Measurement
To immobilize uricase enzymes on nanostructured CuO, first uricase solution was prepared in a phosphate buffer solution of pH 7.4 using (3 mg/mL uricase) and 100 μL of glutaraldehyde was used as a cross-linker to avoid the self-enzyme reaction. Then, the CuO material was dipped in the enzyme solution for 5 min and the immobilized electrodes were dried in air at room temperature for 1 h. After the immobilization, the electrodes were kept at 4 °C when not in use. The potentiometric measurements for the sensing of uric acid were done against Ag/AgCl as a reference electrode using a Metrohm pH meter (Model 744, Beijing, China) by employing the uricase-immobilized nanostructured CuO material as a working electrode. All the experiments were performed at room temperature and all solutions were prepared in a 10 mM phosphate buffer solution of pH 7.4. The biosensor can be reused after rinsing with the buffer solution.
In this study, heart/dumbbell-like CuO nanostructured material was synthesized uisng vitamin B12 as a growth-directing agent and template for the facilitation of the growth process using a low-temperature aqueous chemical growth method. CuO nanostructures were investigated uisng SEM, XPS, and XRD techniques. Using these nanostructures of CuO, uricase enzyme was immobilized on them and used for the development of a stable, sensitive, selective, reproducible, and repeatable uric acid biosensor. The nanostructures of CuO are porous, which allowed the uricase molecules to reside within those pores to further allow the fast oxidation of uric acid, and finally we have a successful and an alternative analytical device for the monitoring of uric acid. The obtained results were unique and can be capitalized to commercialize the uric acid biosensor because the fabrication process is simple, cost effective, and scalable. We propose the functional properties of the prepared nanostructured CuO material in the field of lithium ion batteries, solar cells, and supercapacitors based on its functional properties.