Cholesterol biosensing based on highly immobilized ChOx on ZnO hollow nanospheres

Abstract

We report a high-performance cholesterol biosensor using low-temperature solution grown ZnO hollow nanospheres (ZHNSs). The fabricated cholesterol biosensor showed a rapid response time of ∼2 s, a detection limit of 0.4 mM, a wide linear detection range (0.2–15.6 mM), and high sensitivity (99.8 μA mM⁻¹ cm⁻²). Owing to good selectivity and long-term stability, the biosensor manifests a cost-effective and highly potential platform for cholesterol detection.

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Cholesterol level evaluation in humans and animals, and foodstuffs has great importance in medicine and other industries. Apparently, growing incidences of cardiac arrest and cardiovascular diseases in the contemporary society have necessitated the development of advanced cholesterol biosensors.¹ Cholesterol concentration in blood is the basic parameter for diagnosis and disease prevention. Regarding this, patients' blood samples are drawn, sent to the clinical laboratory to assay blood cholesterol levels, employing expensive and sophisticated instruments. Among various cholesterol biosensors, amperometric biosensors are the most studied ones owing to better selectivity, rapid response, and low fabrication cost.² Where, cholesterol oxidase (ChOx) enzyme is used as a receptor that catalyses cholesterol in the presence of oxygen and provides convenient, rapid specific measurements. In particular, development of high-performance cholesterol biosensor requires high enzyme loading and its stabilization on the respective electrode/matrix. Thus, the selection of an appropriate matrix to immobilize enzyme becomes highly critical for fabrication of practically usable biosensors.

In search of suitable matrix, varieties of nanomaterial have been exploited for ChOx immobilization and biosensor fabrication,³ since they provide large specific surface area for higher enzyme loading and compatible microenvironment for enzyme bioactivity retention.⁴ Among all, ZnO has been efficiently used in various arena including drug delivery systems, chemical and biological sensors, photocatalyst, LEDs and so on⁵ because of its nanostructures availability, high catalytic efficiency, nontoxicity, chemical stability and strong adsorption ability.⁶ By design, ZHNSs with hollow interior and porous nature, low density and high surface area opens wide possibilities for high medicine loadings, fillers for modifying resin properties, superior device performance by enhancing enzyme loading with high catalytic activities, and prolonging the catalytic reactions time span.^4b,7 Of particular interest, current researches revealed that ZHNSs showed remarkable photocatalytic activity and excellent glucose sensing owing to its hollow nature providing high specific surface area,⁸ which also are the prime factors for the enhancement of amperometric biosensors. Motivated by those characteristics, we herein demonstrate the first-time fabrication of highly efficient cholesterol biosensor using low-temperature solution grown ZHNSs.

For cholesterol biosensor fabrication (Fig. 1a), first, ZHNSs were synthesized using two-step process.⁹ In step I, ZNSs were grown by dissolving Zn(NO₃)₂·6H₂O (0.0035 M) and HMTA (0.0035 M) in 50 mL DI water with subsequent addition of C₆H₅Na₃O₇·2H₂O (0.002 M) followed by refluxing with Si substrate at 80 °C for 1 h. In step II, ZHNSs were synthesized by taking Zn(NO₃)₂·6H₂O (0.005 M), HMTA (0.005 M) and C₆H₅Na₃O₇·2H₂O (0.001 M) in 50 mL DI water; the solution pH = 9 was maintained by NH₄OH addition and heated at 80 °C for 1 h in a three-necked refluxing reactor with the step I resulted products. Finally, the resulted products were rinsed with DI water and air-dried. Further, the ZHNSs were mixed with conducting binders (butylcarbitol acetate) in a weight ratio of 80 : 20 (ZHNSs : binder) and the prepared slurry was casted on sputtered Ag/glass electrode (0.8 cm²). Prior to modifications, Ag electrode was polished with 0.05 μm Al slurry and sonicated in DI water. After drying, ChOx (1.0 mg mL⁻¹ in 0.1 M PBS) was immobilized onto the ZHNSs/Ag/glass electrode surface by physical adsorption and air-dried. Finally, 5 μL Nafion solution (0.5 wt%) was dropped onto the electrode to prevent possible enzyme leakage and stored at 4 °C when not in use.

Fig. 1 (a) Schematic of cholesterol biosensing device fabrication. (b) Low- (c) high-resolution FESEM with its EDX pattern (inset of b). (d) TEM image of ZHNSs synthesized at 80 °C and (e) HRTEM image of single ZHNS.

Field emission scanning electron microscopy (FESEM) images in Fig. 1b and c displays a well-dispersed large quantity of products along with the regular spherical shape of ZHNSs and its hollow nature. Transmission electron microscopy (TEM) images (Fig. 1d and e), further confirms the porous nature of nanospheres from both outside and inside. EDX spectrum (Fig. 1b, inset) demonstrates that the products were made up of zinc (Zn) and oxygen (O) only, with an atomic ratio of Zn to O is about 1 : 1. Crystallinity of ZHNSs was studied by XRD (Fig. 2a), which showed that all the diffraction peaks were indexed to wurtzite ZnO (JCPDS no. 36-1451). No other reflection related with any impurity was detected in the pattern, confirming the purity and high-quality of the as-synthesized products. Furthermore, the optical property was characterized by UV-visible spectroscopy (Fig. 2b), which displays a single and well-defined absorption band at 361 nm, a characteristic band for the wurtzite hexagonal structure of ZnO. From Fig. 2c & d, it can be seen that the ZHNSs possess high surface area, large pore volume, and narrow pore size distribution evaluated from N₂ adsorption–desorption isotherms analysis. At high P/P₀ between 0.4 and 1.0, the samples exhibit a type H1 hyteresis loop (IUPAC classification) indicating the porous nature of ZHNSs. The BET (Brunauer–Emmett–Teller) surface area was found to be 56 m² g⁻¹. The pore size distribution curve of ZHNSs suggests a narrow pore size distribution ranging from 2 to 25 nm with an average pore diameter of 4.1 nm.

Fig. 2 (a) XRD, (b) UV-visible absorbance spectra, (c) nitrogen adsorption–desorption isotherm and (d) BJH pore size distribution curve of the ZHNSs.

The detailed electrochemical response of fabricated biosensor was carried out by cyclic voltammetry (CV). Fig. 3 shows the CV sweep curve of the biosensor in the presence of 2.0 mM cholesterol at a scan rate of 100 mV s⁻¹ in the potential range of −0.3 to +0.80 V with respect to the Ag/AgCl (sat'd KCl) reference electrode. This electrode exhibits a pair of well-defined redox peaks at +0.38 V (oxidation) and −0.10 V (reduction) due to H₂O₂ generation during cholesterol oxidation by ChOx and H₂O₂ reduction, respectively. No peaks were observed in the absence of cholesterol (lower inset of Fig. 3), demonstrating much-improved electroactivity because of good conductivity, specific surface area and higher enzyme immobilization on the ZHNSs. Moreover, the SEM image taken after ChOx immobilization (upper inset of Fig. 3) shows a good distribution of enzyme on the ZHNSs surface, further confirming the efficiency of ZnO matrix for enzyme immobilization. The blurriness of the image is mainly due to difficulty to focus while capturing the image, attributed to the layers of coated enzyme. From the image, it can be seen that most of the ChOx globules were not only attached to the outer surface but also found to be filled in the hollow space of ZHNSs. Moreover, the surface charge of ZHS is the additive property which facilities specific electrostatic interaction with the negatively charged ChOx enzyme (low isoelectric point) to encapsulate high amount of enzyme molecules. Therefore, the unique hollow architecture and porous nature of ZHNSs is particularly promising for enzyme immobilization and maintaining its bioactivity, assigned to the large specific surface area and enhanced absorption capability. Fig. 4a shows the CV curves for the modified electrode in the presence of 2.0 mM cholesterol measured at different scan rates. The redox peak current increase as the scan rate increases and the currents are linear against the square root of the scan rate in the range of 25–200 mV s⁻¹ (Fig. 4b), indicating a typical diffusion controlled process.

Fig. 3 CV response of Nafion/ChOx/ZHNSs/Ag/glass electrode in the presence of 2.0 mM cholesterol at the scan rate of 100 mV s⁻¹. The upper and lower insets show the SEM image taken after ChOx immobilization and the CV response in the absence of cholesterol, respectively.

Fig. 4 (a) CV at different scan rates in PBS (0.1 M; pH = 7.0) containing 2.0 mM cholesterol, and (b) linear relationship of I_p and square root of scan rate (ν).

A typical steady-state amperometric response of fabricated biosensor was shown in Fig. 5a, with the successive addition of cholesterol in 0.1 M PBS (pH = 7.0) at an applied potential of +0.38 V under stirring condition. Additionally, a clear magnified plot of the amperometric response in the range of 0.01 to 1.61 mM was displayed in Fig. 5b. A rapid and sensitive response to each injection of cholesterol was obtained with a response time of ∼2 s for the enzyme electrode to reach 97% steady state current. From the graph, it can be clearly seen that the response current increases with the increase in cholesterol concentration and gets saturated at higher cholesterol concentration, suggesting the saturation of enzymes active sites at those cholesterol levels. From the calibration plot (response current vs. cholesterol concentration; Fig. 5c), biosensor showed the linear range from 0.2 mM to 15.6 mM (R² = 0.9986), detection limit of 0.4 mM (S/N = 3) and high sensitivity of 99.8 μA mM⁻¹ cm⁻². The Lineweaver–Burk plot (Fig. 5d) gives an apparent Michaelis–Menten constant (K^app_M) of ∼6.9 mM. Owing to high specific surface area, ZHNSs provide a favourable microenvironment for larger ChOx loadings. Moreover, the high conductivity of ZHNSs provides high electron communication features that enhance the direct electron transfer between the active sites of enzyme and the electrodes leading to higher sensitivity and wider linear range. Compared to previously reported ZnO nanostructures based cholesterol biosensors, the obtained sensitivity and detection concentration range of our fabricated ZHNSs based electrode were superior.^4a,10 (For instance, sensitivity and linear range were 79.4 μA mM⁻¹ cm⁻² and 1.0 × 10⁻⁶ to 13.0 mM;^4a 2.296 μA mM⁻¹ cm⁻² and 0.13 to 10.36 mM;^10a 14.1 μA mM⁻¹ cm⁻² and 0.125 to 7.76 mM;^10b 3.6 μA mM⁻¹ cm⁻² and 0.13 to 7.77 mM;^10c 26.8 μA mM⁻¹ cm⁻² and 1.0 × 10⁻⁷ to 7.593 × 10⁻⁴ mM (ref. 10d).)

Fig. 5 (a) Amperometric response of the Nafion/ChOx/ZHNSs/Ag/glass electrode to cholesterol in 0.1 M PBS at +0.38 V; (b) magnified amperometric response in the range of 0.01 to 1.6 mM; (c) calibration curve i.e. response current vs. cholesterol concentration; (d) the Lineweaver–Burk plot. Inset in (c) shows the magnified calibration curve.

To investigate selectivity of the fabricated biosensor, amperometric response was measured in PBS at an applied potential of +0.38 V by introducing 2.0 mM cholesterol and 0.2 mM of each electroactive species (such as ascorbic acid (AA), glucose, uric acid (UA), dopamine (DA)). Fig. 6 shows the absence of any obvious effect of these interfering species on the biosensor response, indicating that our fabricated electrode is favourable for selective detection of cholesterol in the presence of interfering species. Further, the sensor's reproducibility and long-term stability was evaluated. Five different bioelectrodes were fabricated in similar fabrication conditions and their response were recorded (Fig. 7a). As shown in the figure, our biosensor demonstrates good reproducibility for cholesterol detection. The repeatability of cholesterol biosensor was checked by keeping the electrode at 4 °C followed by regular response assessment. The resultant cholesterol biosensor was found to possess a high stability for cholesterol detection by retaining about 98% of its original response towards cholesterol after 20 days of storage. In further usage, the bioelectrode showed slight decrease in its current response due to the denaturation of the immobilized enzyme biomolecules (Fig. 7b). Such high stability of the modified electrodes is ascribed to the ZHNSs providing friendly microenvironment for maintaining bioactivity of ChOx.

Fig. 6 Selectivity test of the fabricated cholesterol biosensor towards successive addition of 2.0 mM cholesterol and 0.2 mM of each electroactive species (such as ascorbic acid (AA), glucose, uric acid (UA), dopamine (DA)).

Fig. 7 (a) Reproducibility studies of the fabricated cholesterol biosensors under similar conditions; (b) stability studies of the bioelectrode in 1.0 mM cholesterol.

To evaluate our fabricated biosensor's applicability, the concentration of cholesterol in human blood serum sample was determined by the standard addition method. In this test, known concentration of cholesterol (5.2 mM) in the human serum (Sigma-Aldrich, H4522) was diluted with PBS. Then, known concentration of pure cholesterol was added to the diluted human serum samples to prepare three samples. (Each sample was detected three times.) The cholesterol recovery percentage was found to be in the range of 94.5–102.4%, indicating good accuracy and great potential of fabricated biosensor for the analysis of cholesterol in real clinical samples.

Conclusions

A superior potential of ZHNSs for effective fabrication of cholesterol biosensor was demonstrated where, ZHNSs not only provide a suitable environment for enzyme immobilization, but also establish effective electronic communication between ChOx and the electrode. As a result, the fabricated biosensor shows a high sensitivity, wide linear range and fast response time with better selectivity against electroactive species. Keeping in view the sensor applicability, cholesterol detection was performed in human serum sample which showed results in accordance with the calculated ones. Thus, this biosensor provides a useful tool for cholesterol detection and could be used for real clinical samples analysis.

Acknowledgements

This work was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2012-0001039).

Notes and references

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Footnote

† These authors contributed equally.

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