Biosensors Nanotechnology

By Ashutosh Tiwari

This book provides detailed reviews of a range of nanostructures used in the construction of biosensors as well as the applications of these biosensor nanotechnologies in the biological, chemical, and environmental monitoring fields

Biological sensing is a fundamental tool for understanding living systems, but also finds practical application in medicine, drug discovery, process control, food safety, environmental monitoring, defense, and personal security. Moreover, a deeper understanding of the bio/electronic interface leads us towards new horizons in areas such as bionics, power generation, and computing. Advances in telecommunications, expert systems, and distributed diagnostics prompt us to question the current ways we deliver healthcare, while robust industrial sensors enable new paradigms in R&D and production.

Despite these advances, there is a glaring absence of suitably robust and convenient sensors for body chemistries. This book examines some of the emerging technologies that are fueling scientific discovery and underpinning new products to enhance the length and quality of our lives.

The 14 chapters written by leading experts cover such topics as:

• ZnO and graphene microelectrode applications in biosensing
• Assembly of polymers/metal nanoparticles
• Gold nanoparticle-based electrochemical biosensors
• Impedimetric DNA sensing employing nanomaterials
• Graphene and carbon nanotube-based biosensors
• Computational nanochemistry study of the BFPF green fluorescent protein chromophore
• Biosynthesis of metal nanoparticles
• Bioconjugated-nanoporous gold films in electrochemical biosensors
• The combination of molecular imprinting and nanotechnology
• Principles and properties of multiferroics and ceramics

• Language: English
• Publisher: Wiley
• Release date: Jun 26, 2014
• ISBN: 9781118773819

Book preview

Preface

Biosensors and biosensing technologies have grown from a tiny, niche activity in the 1980s into a major, worldwide industry. Nanomaterials have played key roles in this development, not only in pharmaceuticals and healthcare, but also in sectors such as telecommunications, paper and textiles. Biological sensing is a fundamental tool for understanding living systems, but also finds practical application in medicine, drug discovery, process control, food safety, environmental monitoring, defense and personal security. Moreover, a deeper understanding of the bio/electronic interface leads us towards new horizons in areas such as bionics, power generation and computing. Advances in telecommunications, expert systems and distributed diagnostics prompt us to question the current ways we deliver healthcare, while robust industrial sensors enable new paradigms in R&D and production.

Personalization of everything from medicine to environmental control gives new impetus to consumer choice and ownership of information, and will inevitably generate new payment structures and business models. Wearable, mobile and integrated sensors are becoming commonplace, but most current products have taken the easy path of incorporating physical sensors for parameters such as temperature, pressure, orientation or position. There is a glaring absence of suitably robust and convenient sensors for body chemistries and therein lies the real opportunities for progress. This book examines some of the emerging technologies that are fuelling scientific discovery and underpinning new products to enhance the length and quality of our lives. This new field combines nanoscale materials with biosensor technology and is receiving considerable attention. Nanostructures have been used to achieve direct wiring of biosensing elements to electrode surfaces, to promote bioreactors, to impose Nan barcodes on biomaterials, and to amplify the signal from misrecognition events. Biosensors based on nonmaterial have widespread potential applications in medical diagnostics and environmental monitoring due to their sensitivity, specificity, speed of response, simplicity and cost-effectiveness.

This book tracks the pursuit of these objectives and provides detailed reviews of a range of nanostructures used in the construction of biosensors, including nanoparticles, nanowires, nanotubes, nanoribbons, nanorods, nanobelts and nanosheets,.Applications of these biosensor nanotechnologies span biological and chemical analyses for food safety, biomedical diagnostics, clinical detection and environmental monitoring. This volume in the Advanced Materials Book Series includes fourteen chapters divided into three main areas. In Part 1, New Materials and Methods, renowned experts cover such topics as ZnO and graphene microelectrode applications in biosensing, assembly of polymers/metal nanoparticles, gold nanoparticle-based electrochemical biosensors, impedimetric DNA sensing employing nanomaterials, graphene and carbon nanotube-based biosensors and state-of-the-art of nanomedicine. Part 2, Principles and Prospective, begins witha computational nanochemistry study of the BFPF green fluorescent protein chromophore, and then moves on to discuss biosynthesis of metal nanoparticles, ionic discotic liquid crystals and the role of advanced materials as nanosensors in water treatment. Presented in Part 3, Advanced Structures and Properties, experts in the fielddiscuss bioconjugated-nanoporous gold films in electrochemical biosensors, the combination of molecular imprinting and nanotechnology, principles and properties of multiferroics and ceramics.

The book is written for a wide readership, including university students and researchers from diverse backgrounds such as chemistry, materials science, physics, pharmacy, medical science and biomedical engineering. It can be used not only as a textbook for undergraduate and graduate students, but also as a review and reference book for researchers in the materials science, bioengineering, medical, pharmacy, biotechnology and nanotechnology arenas. We hope that the chapters of this book will provide the reader with valuable insight into the cutting-edge nanotechnology of this major new area of biosensors.

Editors

Ashutosh Tiwari

Anthony PF Turner

Part 1

NEW MATERIALS AND METHODS

Chapter 1

ZnO and Graphene Microelectrode Applications in Biosensing

Susana Campuzano¹, María Pedrero¹, Georgia-Paraskevi Nikoleli², José M. Pingarrón¹, Dimitrios P. Nikolelis*,³, Nikolaos Tzamtzis² and Vasillios N. Psychoyios²

¹Department of Analytical Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain

²Laboratory of Inorganic and Analytical Chemistry, School of Chemical Engineering, Dept. 1, Chemical Sciences, National Technical University of Athens, Athens, Greece

³Laboratory of Environmental Chemistry, Department of Chemistry, University of Athens, Athens, Greece

*Corresponding author: [e-mail]

Abstract

Graphene nanomaterials have been the focus of tremendous attention not only in the field of basic research but also in technological applications, owing to their unique physicochemical dimensions such as good sensing ability, and excellent mechanical, thermal and electrical properties. On the other hand, ZnO nanomaterials have attracted considerable interest in relation to sensors due to their many advantages, including large surface-to-volume ratio, excellent biological compatibility, high electron-transfer rates, non-toxicity and biosafety. The development of biosensors can potentially be an interesting application for the utilization of these nanomaterials tremendously large surface-area-to-volume ratio, which is a dominating and promising parameter with the potential to solve biocompatibility and biofouling problems. The present chapter describes recent examples in the development of miniaturized amperometric and potentiometric biosensors by integrating enzymes and one of these two nanomaterials. The latest advances relating to the application of these biosensors to rapidly detect biomedically relevant substrates such as glucose, urea, uric acid, cholesterol, etc., with enormous prospects in clinical medicine applications are reviewed throughout. The presented biosensors exhibit good reproducibility, reusability, selectivity, rapid response times, long shelf life and high sensitivity, and do not suffer from interference by coexisting oxidable substances. These electrochemical nanobiosensors prepared through the integration of biomolecules with graphene or ZnO nanostructures have demonstrated that, besides enhancing the biosensing capabilities compared with conventional platforms, bring out new approaches such as miniaturization, reagentless biosensing and single-molecule detection. This chapter highlights the significant milestones achieved and further elucidates the emerging future prospects in this area.

Keywords: Biosensors, electroanalysis, ZnO and graphene microstructures, microelectrodes

1.1 Biosensors Based on Nanostructured Materials

Biosensors have become important and practical tools in the field of healthcare, chemical and biological analysis, environmental monitoring, food safety control, and homeland security. The performance of biosensors depends on their components, among which the matrix material, i.e., the layer between the recognition layer of biomolecule and transducer, plays a crucial role in defining the stability, sensitivity and shelf life of a biosensor [1]. Among biosensors, electrochemical ones are of particular interest due to several combined advantages such as low detection limits, short response times, long-term stability, power requirements, low cost, ease of operation, and miniaturization capability. A current goal for these types of biosensors is their translation to point-of-care diagnostic devices. Much effort has been put into improving key performance parameters, such as sensitivity, specificity, recognition rates, stability and multiplexing capabilities for parallel recognition, to allow this possibility.

The emergence of nanotechnology has opened new horizons for electrochemical biosensors. It is believed that highly sensitive and selective biosensors can be realized through the integration of biomolecules and nanomaterial-based sensor platforms. Over the last fifteen years, efforts have focused on the use of nanotechnology to develop nanostructured materials (e.g., graphene and ZnO nanowires, nanotubes, nanowalls and nanorods) as biomolecule immobilizing matrices/supports to improve electrochemical detection [2]. Nanoscale structures like these offer many unique features and show great promise for faster response and higher sensitivity at the device interface than planar sensor configurations. Their nanometer dimensions, being in the scale of the target analyte, show an increased sensing surface and strong binding properties, thus allowing a higher sensitivity. The interest in developing these nanostructures for biosensing applications has resulted from the development of new synthesis methods and improved characterization techniques, allowing for new functionalities to be created [2].

Because of their interesting advantages among the nanomaterials that have been developed, this chapter describes the increasing application of graphene and ZnO nanostructures to the fabrication of highly sensitive electrochemical biosensors. Latest advances (from 2004 onwards) in electrochemical biosensors based on the distinct advantages and practical sensing utility of these two nanostructured materials are discussed and illustrated in the following sections in connection to enzyme electrodes for the determination of analytes of clinical relevance. Although several strategies have been described for using these nanomaterials in such bioaffinity and biocatalytic sensing [3, 4], both for amplification tagging or modifying electrode transducers, this chapter will focus only on their applications as surface modifiers. The broad capabilities of such modern nanomaterials-based bioelectrodes for biocatalytic electrochemical detection (mainly amperometric and potentiometric) of numerous biologically important analytes, and for other bioelectronic affinity assays (e.g., DNA hybridization assays), will be discussed along with future prospects and challenges.

1.2 Graphene Nanomaterials Used in Electrochemical Biosensor Fabrication

Graphene and its derived structures (graphene oxide, graphene platelets, graphene nanoflakes) have become popular materials for fabricating electrode matrixes for sensing and biosensing [5]. Graphene is the mother of all graphitic forms, including zero-dimensional fullerenes, one-dimensional carbon nanotubes, and three-dimensional graphite [6].

Graphene, defined as a single-layer two-dimensional sp²-hybridized carbon, is currently, without any doubt, the most intensively studied material. This single-atom-thick sheet of carbon atoms arrayed in a honeycomb pattern is the world’s thinnest, strongest, and stiffest material, as well as an excellent conductor of both heat and electricity [7]. It is often categorized by the number of stacked layers: single layer, few layer (2–10 layers), and multilayer, which is also known as thin graphite. Ideally, for graphene to preserve its distinct properties, its use should be narrowed to single- or few-layer morphologies [5].

Graphene’s considerable attention as a next generation electronic material derives from its unique electronic, optical, mechanical, thermal, and electrochemical properties [5]. It being electronically a very good low-noise material, graphene can be employed in the achievement of molecular sensing [8].

Graphene is attractive for electrochemistry because it is a conductive yet transparent material, with a low cost and low environmental impact, a wide electrochemical potential window, low electrical resistance in comparison to glassy carbon (GC), atomic thickness and two well defined redox peaks linearly aligned with the square root of the scan rate magnitude, suggesting that its redox processes are primarily diffusion controlled. Peak-to-peak values under cyclic voltammetry are low, suggesting rapid electron transfer kinetics, and its apparent electron transfer rate is orders of magnitude higher than that of GC. This rate of electron transfer has been shown to be surface dependent and can be increased significantly by the creation of specific surface functional groups [8]. The high density of edge-plane defect sites on graphene provides multiple electrochemically active sites. Its entire volume is exposed to the surroundings due to its 2D structure, making it very efficient in detecting adsorbed molecules. Graphene-based electrodes also exhibit high enzyme loading due to their high surface area. This, in turn, can facilitate high sensitivity, excellent electron transfer promoting ability for some enzymes, and excellent catalytic behavior towards many biomolecules [8, 9]. Graphene-based devices also possess the required biocompatibility to be amenable for in situ biosensing.

Graphene exhibits the advantages of a large surface area (2,630 m² g−1 for single-layer graphene) similar to that of carbon nanotubes (CNTs), and a small size of each individual unit, also exhibiting some other merits like low cost, two external surfaces, facile fabrication and modification and absence of metallic impurities, which may yield unexpected and uncontrolled electrocatalytic effects and toxicological hazards [5, 8, 9].

It has also been reported that the edges of graphene sheets possess a variety of oxygenated species that can support efficient electrical wiring of the redox centers of several heme-containing metalloproteins to the electrode and also enhance the adsorption and desorption of molecules [8, 9].

Graphene-based nanomaterials can be classified in relation to the method of production. They can be produced by chemical vapor deposition (CVD) growth, by mechanical exfoliation of graphite, or by exfoliation of graphite oxide. Neither CVD-produced graphene nor mechanically exfoliated graphene contain large quantities of defects or functionalities. Bulk quantities of graphene-based nanomaterials are typically prepared by different methods, such as the thermal exfoliation of graphite oxide which leads to a material called thermally reduced graphene (GO) or, for example, sono-assisted exfoliation of graphite oxide to graphene oxide (GO), which can be further reduced chemically or electrochemically. The products are typically referred to as chemically reduced GO (CRGO) or electrochemically reduced GO (ERGO). The TRGO contains large amounts of defects and significantly differs from pristine graphene, which has a perfect honeycomb lattice structure. The GO has a structure that is not fully planar because the sp² carbon network is heavily damaged. It contains large amounts of oxygen-containing groups, which can be beneficial to the functionalization through the action of the biomolecules for biorecognition events during biosensing. Reduced forms of GO have a partly restored sp² lattice but still hold some fraction of oxygen-containing groups [10]. Therefore, one could have a large graphene toolbox to choose the right type of graphene for the right application and transduction mechanism [11]. Most of graphene used in electrochemistry is graphene produced from GO chemical/thermal reduction, which is also called functionalized graphene sheets or chemically reduced GO, and usually has abundant structural defects and functional groups which are advantageous for electrochemical applications. It has been demonstrated that ERGO exhibits much better performance for electrochemical applications than CRGO. Moreover, Chua et al. [12] demonstrated that not all graphene materials are beneficial for the detection in lab-on-chip devices. Their findings could provide valuable insights into the future applicability of graphene materials towards practical applications.

The future development of electrochemical graphene-based nanobio-devices should be based on the better understanding of some electrochemical details, such as the role of the defects and oxygen-containing groups at the edges of graphene sheets, the interaction mechanism of biomolecules with graphene surface, and the role of doping heteroatoms in graphene. Furthermore, it is important to remark that novel methods for well-controlled synthesis and processing of graphene should be developed. Although graphene has been synthesized with various strategies, the economical production approach with high yield is still not widely available.

1.3 ZnO Nanostructures Used in the Fabrication of Electrochemical Biosensors

Recently, nanostructured metal oxides (NMOs) based on metals such as zinc, iron, cerium, tin, zirconium, copper, titanium, and nickel, have aroused much interest as immobilizing matrices for the development of improved electrochemical biosensors [13]. They have been found to exhibit interesting nanomorphological, functional biocompatible, non-toxic and catalytic properties, providing an effective surface for biomolecule immobilization with the desired orientation, better conformation and high biological activity, resulting in enhanced biosensing characteristics [13, 14]. The NMOs with desired functionalities and surface charge properties provide interesting platforms for interfacing biorecognition elements with transducers for signal amplification. These materials also exhibit enhanced electron-transfer kinetics and strong adsorption capability, providing suitable microenvironments for the stable immobilization of biomolecules, resulting in improved biosensing performances.

To fabricate an efficient biosensor, it is crucial to select an NMO that is suitable for the immobilization of the desired biomolecule. The interface formed due to binding between an NMO and a biomolecule is known to significantly affect the performance of the biosensor. The formation and properties of a nanobiointerface depend on the nature of the NMO; parameters like effective surface area, surface charge, energy, roughness and porosity, valence/conductance states, functional groups, physical states and hygroscopic nature, all affect the formation of a biointerface [13].

Among the NMOs, ZnO nanostructures have unique physical and chemical advantages, including high surface-to-volume ratio, which provides greater enzyme loading, and a favorable microenvironment, which can preserve the activity of the immobilized biomolecules, non-toxicity, chemical stability with a high isoelectric point (~9.5), which facilitates the physical immobilization of biomolecules, electrochemical activity, high electron communication features, with high ionic bonding (60%) and abundance in nature [14–16]. Indeed, ZnO nanostructures, due to their excellent electron transfer rate, can evoke the hidden electrochemical ability of biomolecules, and facilitate the direct electrochemistry of enzymes whose redox capability is not highlighted because their redox centers are insulated [1]. They have shown binding of biomolecules in desired orientation with improved conformation and high biological activity, resulting in enhanced sensing characteristics. Furthermore, their compatibility with complementary metal oxide semiconductor technology for constructing integrated circuits makes them suitable candidates for small integrated biosensor devices [1]. All these advantageous properties make them one of the most promising materials for biosensing applications and for intracellular electrochemical measurements. One can engineer the diameter of these nanostructures comparable to the size of the biological and chemical species being sensed, which intuitively could be excellent primary transducers for producing electrical signals [17].

Interestingly, ZnO can be grown to form highly anisotropic nanostructures on various substrates, including sapphire, glass, silicon and conductive surfaces (e.g., indium-tin-oxide [ITO], gold) with different morphologies [1].

Moreover, the numerous choices for ZnO fabrication and also their different growth parameters have led to a rather rich ZnO nanoworld consisting of nanostructures with different shapes. This polymorphic capability of ZnO for the synthesis of nanostructured materials offers a great potential for fundamental studies in the roles of dimensionality and size-based physical properties. The ease of fabrication using low-cost processes, which can yield a wide range of nanostructures, makes ZnO-based matrices a promising platform for low-cost biosensors [1]. Researchers have reported a myriad of ZnO nanostructures for biosensor applications synthesized through various physical and chemical routes: nanowires (ZnONWs), nanorods (ZnONRs), nanowalls, nanobelts, nanonails, nanoneedles, nanotubes (ZnONTs), nanocombs, nanoforks, nanofibers (ZnONFs), nanoflakes, nano-waxberries, nanobundles, nanospheres (ZnONSs), nanocomposites, nanotetrapods, nanoparticles (ZnONPs), nanorod spheres, nanoflowers, and nanosheets/disks. Nanoporous and nanostructured ZnO films have also been used for biosensor applications [1]. These various ZnO nanostructures in different shapes are also favorable for surface functioning if needed [1, 14, 15]. These nanostructures result in the formation of different structures exhibiting diverse properties, which might further influence the microenvironments after an enzyme is immobilized. For example, small dimensional ZnONTs arrays have a higher surface area, subsurface oxygen vacancies and provide a larger effective surface area with higher surface-to-volume ratio as compared to ZnONW arrays, thus enabling sensors with higher sensitivity [18]. Comparative studies have also demonstrated that nanosheet-based ZnO microspheres are more effective in facilitating the electron transfer of immobilized enzymes than solid ZnO microspheres, which may result from the unique nanostructures and larger surface area of the porous ZnO [19], and that the nanostructure of a prickly ZnO/Cu nanocomposite offers significant advantages over ZnONRs in facilitating direct electron transfer [20].

The following section of this chapter will describe the state-of-the-art of the utilization of graphene and ZnO nanostructures as modified transducers and for enzyme immobilization in electrochemical biosensors for various applications (i.e., clinical, food, environmental). In particular, graphene and ZnO nanostructures-based biosensors, classified according to different electrochemical detection techniques and targets, will be thoroughly discussed.

1.4 Miniaturized Graphene and ZnO Nanostructured Electrochemical Biosensors for Food and Clinical Applications

Graphene and ZnO with various nanostructures prepared by different fabrication techniques have been widely used for enzyme immobilization in recent years.

In this chapter, we will summarize and discuss some of the most interesting approaches that have been adopted for improving the performance of graphene and ZnO nanomaterials-based miniaturized electrochemical biosensors for clinical applications. These nanostructured matrices have been used for the binding of various biosensing molecules, such as glucose oxidase (GOx) [17, 18, 20–31], glutamate dehydrogenase [32], cholesterol oxidase (ChOx) [33–37], uricase [38–41], horseradish peroxidase (HRP) [19, 34, 43–45], urease (Urs) [46–48], alcohol dehydrogenase (ADH) [26, 49, 50], lactate oxidase [51, 52], ascorbate oxidase [53], galactose oxidase [53] and catalase (CAT) [54] for the detection of their respective analytes in various device configurations. Interesting reported applications of some miniaturized potentiometric nanosensors for the detections of ions (H+, Ca²+, Mg²+, K+ and Na+) relevant in clinics [17, 55–57] and single-stranded (ss)DNA monitoring [58] will also be discussed.

1.4.1 Amperometric Biosensors

A glucose biosensor based on GOx immobilized through electrostatic interaction on ZnONR array grown by hydrothermal decomposition was developed by Wei et al. [21]. At an applied potential of +0.8 V versus an Ag/AgCl reference electrode, ZnONRs-based biosensor presented a high and reproducible sensitivity of 23.1 μA cm−2 mM−1 with a response time of less than 5 s, a linear range from 0.01 to 3.45 mM and a limit of detection (LOD) of 0.01 mM. The KMapp value of 2.9 mM demonstrated a high affinity between glucose and GOx immobilized on ZnONRs. These features demonstrated that the hydrothermal deposition method provides a cheap yet efficient method to grow ZnO nanostructures for biosensor application. It would probably provide an economic way to meet the industrial requirements of low-cost processing technique for large-scale production.

A novel amperometric glucose biosensor based on ZnO:Co nanoclusters (doping 2% Co in ZnO), synthesized by nanocluster-beam deposition with an averaged particle size of 5 nm and porous structure, has been developed [22]. The GOx was immobilized into the ZnO:Co nanocluster assembled thin film through Nafion-assisted crosslinking technique. Due to the high specific active sites and high electrocatalytic activity of the ZnO:Co nanoclusters, the constructed glucose biosensor showed a high sensitivity of 13.3 μA mA−1 cm−2. The LOD was estimated to be 20 μM and the KMapp was found to be 21 mM, indicating the high affinity of the enzyme on ZnO:Co nanoclusters to glucose. Although the results show that the ZnO:Co nanocluster-assembled thin films with nanoporous structure and nanocrystallites have potential applications as platforms to immobilize enzyme in biosensors, the interference of some species, such as uric and ascorbic acids, cannot be completely removed for the biosensor at the operating potential (+0.8 V versus an Ag/AgCl reference electrode), limiting the applicability of this biosensor to environmental and industrial monitoring.

Dai et al. developed an amperometric glucose biosensor based on direct electrochemistry of GOx immobilized by simple adsorption on tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructures [23]. The prepared TPSP-ZnO has a large surface area and exhibits favorable biocompatibility for facilitating the electron transfer between protein and electrode surface. The immobilized GOx at a TPSP-ZnO-modified glassy carbon electrode (GCE) shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOx to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2 mM with a low LOD (0.01mM), at an applied potential of −0.50 V (versus a saturated calomel reference electrode), and can operate under air without the exclusion of the dissolved O2. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Thus, the TPSP-ZnO nanostructure provides a good matrix for protein immobilization, promoting the direct electron transfer of proteins and developing biosensors.

An amperometric glucose biosensor has been fabricated by immobilization of GOx onto ZnONT arrays by crosslinking method [24]. The ZnONT arrays have been synthesized by chemical etching of ZnONR electrochemically deposited on the Au surface. Due to the good biocompatibility and intrinsic porous structure of ZnONTs, the fabricated glucose biosensor shows very sensitive response (sensitivity = 21.7 μA mM−1 cm−2, KMapp =19 mM) and can detect glucose as low as 1 μM without any electron mediators. The biosensor shows a fast response to glucose (3 s) and has quite a wide linear range from 50 μM to 12 mM. It also possesses good anti-interference ability and long-term stability. All these advantageous features can make the designed biosensor applicable in medical, food or other areas. Moreover, the investigation also shows that the ZnONTs may be applied as a potential novel immobilization material for a variety of biosensor designs.

Shan et al. constructed a novel polyvinylpyrrolidone (PVP)-protected graphene/polyethylenimine-functionalized ionic liquid (PFIL)/GOx electrochemical biosensor, which achieved the direct electron transfer of GOx, maintained its bioactivity and showed potential application for the fabrication of novel glucose biosensors with linear glucose response up to 14 mM [25].

A highly sensitive amperometric glucose biosensor based on a single ZnONF (φ = 350–195 nm) of PVP/zinc acetate composite synthesized by electrospinning technique has been presented by Ahmad et al. [27]. A single NF on a gold electrode is functionalized with GOx by physical adsorption. Furthermore, the performance of the biosensor showed high and reproducible sensitivity of 70.2 μA. mM−1 cm−2 with a response time of less than 4 s, a linear range from 0.25 to 19 mM and a low LOD of 1 μM. Furthermore, it has been revealed that the biosensor exhibits a good anti-interference ability and favorable stability over relatively long-term storage (more than 4 months). All these results strongly suggest that a single ZnONF could provide a new platform for biosensor design and other biological applications.

A novel, highly efficient needle-type glucose sensor based on functionalized graphene has been developed [10] (Figure 1.1). The immobilization of GOx has been apprehended by the direct interaction between carboxyl acid groups of the RGO and amines of GOx together with the electrostatic interactions existing between the positively charged polymeric ionic liquid (PIL) and GOx. This combined system can provide a favorable microenvironment for the GOx to retain its good bioactivity. The enzyme-coated graphene biosensor exhibited glucose-dependent electrochemical measurements against an Ag/AgCl reference electrode. The resulting electrochemical sensor exhibits a broad linear range up to 100 mM glucose concentration with a sensitivity of 5.59 μA decade−1 and a stable output response. This glucose biosensor based on functionalized graphene can be seen as an effective candidate for the detection of sugar concentration, paving the way for its potential application in clinical diagnosis.

Figure 1.1 Schematic illustration of the glucose sensing setup using a working electrode comprised of graphene nanosheets (GSs) coated with GOx, along with the possible electrochemical reaction near the electrode.

(Reprinted with permission from [10]; Copyright © 2012 Journal of Biosensors and Bioelectronics).

A novel ZnO/Cu nanocomposite platform has also been developed for direct electrochemistry of enzymes and biosensing applications [20]. The ZnO/Cu nanocomposite was grown prickly directly on the electrode via a corrosion method and without using any organic reagent, generating a nanocomposite with a large specific surface area, favorable to immobilize the biomolecules and construct biosensors. This ZnO/Cu nanocomposite was employed for immobilization of GOx, constructing a glucose biosensor where direct electron transfer of GOx was achieved with a high heterogeneous electron transfer rate constant of (0.67 ± 0.06) s−1. The prepared reagentless mediator-free third-generation biosensor displayed good sensitivity (97 nA mM−1), wide linear range (1–15 mM), low LOD (0.04 mM), and fast response for the detection of glucose. The prickly ZnO/Cu nano-composite proved to be a promising matrix for direct electrochemistry of proteins and biosensors.

A composite film based on the dispersion of nanosized flower-like ZnO in a chitosan solution was applied as matrix for HRP immobilization for electro-biosensing [42]. Using hydroquinone as the mediator, this amperometric H2O2 biosensor showed a fast response of less than 5 s with the linear range of 1.0×10⁵ to 1.8×10³ M and a LOD of 2.0 μM. The biosensor exhibited satisfactory reproducibility and stability and retained about 78% of its original response after 40 days storage.

A nanostructured inorganic-organic hybrid material based on porous nanosheet-based ZnO microspheres (Figure 1.2) combined with Nafion (ZnO–Nafion composite) has been used for the construction of direct amperometric biosensors [19]. This ZnO-based composite demonstrated to be a biocompatible immobilization matrix for enzymes with good enzymatic stability and bioactivity, facilitating direct electron transfer of the metalloenzymes. The prepared mediator-free third-generation biosensor displayed good sensitivity and reproducibility for the detection of H2O2 and NaNO2, with wide linear ranges (1–410 and 10–2700 μM, respectively), low LODs, fast responses and good long-term stability. The entrapped hemoglobin exhibited high peroxidase-like activity for the catalytic reduction of H2O2 with a KMapp of 143 μM. The nanosheet-based ZnO was described as a promising matrix for the fabrication of direct electrochemical biosensors applicable in biomedical detection and environmental analysis.

Figure 1.2 SEM images of as-prepared porous nanosheet-based ZnO microspheres with low (left) and high magnification (right).

(Reprinted with permission from [19]; Copyright © 2008 Elsevier).

An amperometric H2O2 biosensor based on flowerlike ZnO–gold nanoparticles (GNPs)–Nafion nanocomposite has been developed [43]. The flowerlike ZnO–GNPs showed a synergistic effect, while the ZnO–GNPs–Nafion–HRP modified GCE promoted the direct electron transfer of HRP immobilized in the film effectively, giving an enhanced electrocatalytic activity towards the reduction H2O2. The calculated KMapp was 1.76 mM, which is much lower than that reported previously, indicating a high catalytic activity of HRP. The catalysis currents increased linearly with the H2O2 concentration in a wide range from 1.5×10−5 to 1.1×10−3 M, and a LOD of 9.0×10−6 M was obtained, demonstrating that the formed film provided a favorable microenvironment for the enzyme to retain its activity. Moreover, the modified electrode displayed a rapid response to H2O2 and possessed good stability and reproducibility.

An amperometric biosensor for H2O2 based on layer-by-layer immobilized HRP on ZnONRs was developed by Gu et al. [44]. The ZnONRs were fabricated on a gold wire end coated by a thin layer of Zn-Au alloy to improve the nucleation for growth of ZnO nanostructures and the performance of the biosensor, which was constructed by alternatively immobilizing poly(sodium 4-styrenesulfonate) (PSS) and HRP on the ZnONRs. The multilayered HRP sensors exhibited bioactivity for H2O2 detection without an electron transfer mediator, a wide linear range, a low LOD and a response time of less than 5 s. The sensitivity of the biosensor increased with the immobilized HRP layers from the lowest value of 36.28 μA mM−1 for a monolayer.

A hierarchical enzyme–graphene nanocomposite for H2O2 amperometric detection has been fabricated through electrostatic self-assembly of HRP and sodium dodecyl benzene sulphonate (SDBS) functionalized GSs [45]. The SDBS-functionalized GSs can not only provide large, open and accessible two-dimensional surfaces for tethering of the enzymes, but also flexible distance and restack by adapting to the dimensions of the biomolecules through electrostatic self-assembly. This attribute is of great importance for retaining the native conformations of the guest enzymes. The HRP–GSs composites display excellent electrocatalytic performance toward the reduction of H2O2 with fast response, wide linear range, high sensitivity, low LOD and good stability (the signal gain displays no substantial decrease (>85%) after two-month’s storage at 4°C in a refrigerator). These desirable electrochemical performances are attributed to an excellent biocompatibility and superb electron transport efficiency of GSs, as well as to a high HRP loading and synergistic catalytic effect of the HRP–GSs bionanocomposites toward H2O2. As graphene can be readily non-covalently functionalized by designer aromatic molecules with different electrostatic properties, the proposed self-assembly strategy was described as affording a facile and effective platform for the assembly of various biomolecules into hierarchically ordered bionanocomposites in biosensing and biocatalytic applications.

An ultrasensitive cholesterol amperometric biosensor based on the immobilization of ChOx onto ZnONPs [33] showed a very high and reproducible sensitivity of 23.7 μA mM−1 cm−2, LOD of 0.37 nM, a response time lower than 5 s, a linear range from 1.0 to 500.0 nM, and a relatively low value of KMapp of 4.7 mM. These results demonstrated that due to the simple synthesis and electrode fabrication, ultra-sensitivity, low LOD, and fast response, the as-grown, well-crystallized ZnONPs open a way for the fabrication of highly efficient cholesterol biosensors

Dey et al. described the development of a highly sensitive amperometric biosensor based on Pt nanoparticle-decorated, chemically-synthesized graphene (GNS-nPt) as immobilization matrix for the sensing of H2O2 and cholesterol [34]. The biosensing platform was developed by immobilizing ChOx and cholesterol esterase on the surface of this graphene-Ptnanoparticle hybrid material (Figure 1.3). The sensing platform demonstrated high sensitivity and showed a linear response towards H2O2 up to 12 mM, with a LOD of 0.5 nM in the absence of any redox mediator or enzyme at a >100 mV less positive potential with respect to the bulk Pt electrode. The bienzyme integrated nanostructured platform showed a high sensitivity of (2.07 ± 0.1) μA μM−1 cm−2, a LOD of 0.2 μM, high stability, selectivity toward cholesterol, and a fast response time. The high sensitivity and low LOD can be accounted for by the low background current and high electronic conductivity of graphene, together with the good catalytic activity of the nanoparticles. The KMapp was calculated to be 5 mM. Because the biosensor is highly sensitive and graphene is known to be biocompatible, it can be used for real sample analysis. The analytical performance of the hybrid material was further evaluated using screen-printed electrodes with 50 μL of electrolyte. The Pt nanoparticle-decorated graphene is a promising material for the electroanalysis of biologically important analytes.

Figure 1.3 Schematic illustrating the biosensing of cholesterol ester with the GNS-nPt-based biosensor.

(Reprinted with permission from [34]; Copyright © 2010 American Chemical Society).

A highly sensitive cholesterol biosensor was successfully fabricated by modifying a GCE with electrodeposited Pt-incorporated fullerene-like ZnONSs (PtZnONSs) (φ = 50–200 nm) [35]. The PtZnONSs/GCE was functionalized with ChOx by physical adsorption. The enzyme electrode exhibited a very high and reproducible sensitivity of 1886.4 mA M−1 cm−2 to cholesterol with a response time lower than 5 s and a linear range from 0.5 to 15 μM. Furthermore, the biosensor exhibited a good anti-interference ability and favorable stability over relatively long-term storage (more than 5 weeks). It was found that the combination of ZnO and Pt nanoparticles (PtZnONSs) facilitates the low potential amperometric detection of cholesterol and enhances the anti-interference ability of the biosensor. Furthermore, it was revealed that ZnO improves the electrocatalytic activity of Pt nanoparticles, which in turn enhances the sensitivity of the biosensor for cholesterol detection.

Another amperometric layer-by-layer biosensor for cholesterol based on electrochemical microelectrode with graphene films synthesized by thermal CVD method coated on PANi/Fe3O4 films has been recently developed [37]. The integrated array was fabricated by using micro-electro-mechanical systems (MEMS) technology in which a Fe3O4-doped polyaniline (PANi) film was electropolymerized on Pt/Gr electrodes. The ChOx was immobilized onto the working electrode with glutaraldehyde agent. By taking advantage of graphene-patterning, the layer-by-layer fabricated electrode exhibited excellent analytical quantification in the wide cholesterol concentration range from 2 to 20 mM, with high sensitivity (74 μA mM−1 cm−2) and fast response time (< 5 s).

Shan et al. achieved low-potential β-nicotinamide adenine dinucleotide (NADH) detection and biosensing for ethanol at an ionic liquid-functionalized graphene (IL-graphene) and chitosan-modified GCE (Figure 1.4) [49]. Chitosan with abundant amino groups was chosen to immobilize the IL-graphene and the enzyme due to its good biocompatibility and excellent film-forming ability for the solubility in slightly acidic solution, attributed to its protonation and insolubility in solutions with pH above its pKa (6.3). The IL-graphene/chitosan-modified GCE showed a more stable and low-potential amperometric detection of NADH when compared with the bare electrode. The IL-graphene/chitosan film offered a remarkable decrease in the overvoltage for the NADH oxidation and eliminated surface fouling effects. Furthermore, the IL-graphene/chitosan-modified GCE exhibited a good linearity, from 0.25 to 2 mM, and a high sensitivity of 37.43 μA mM−1 cm−2. The ability of IL-graphene to promote the electron transfer between NADH and the electrode substrate exhibited a novel and promising biocompatible platform for the development of dehydrogenase-based amperometric biosensors. Using ADH as a model enzyme, a rapid and highly sensitive amperometric biosensor for ethanol, with a low LOD (5 μM), was constructed by immobilizing ADH on the GCE surface in the IL-graphene/chitosan coating process through a simple casting method. The IL-graphene-based sensor for NADH and dehydrogenase substrates exhibited very good analytical performance with low cost, convenient preparation, and sensitive, rapid, and reproducible detection. Moreover, the proposed biosensor was used to determine ethanol in real samples with the results in good agreement with those certified by the supplier, thus demonstrating that such IL-functionalized graphene nanocomposite provided a biocompatible platform for the fabrication of sensitive electrochemical biosensors and biomolecular diagnostics with great potential for practical applications.

Figure 1.4 Schematic representation of the bioelectrocatalytic sensing of ethanol using a IL-graphene/chitosan/ADH modified GCE.

(Reprinted with permission from [49]; Copyright © 2010 Elsevier).

The GSs modified GCEs (GSs/GCE) have been presented and applied for the electrochemical biosensing of NADH and ethanol [50]. Based on the highly enhanced electrochemical activity of NADH, ADH was immobilized on the graphene modified electrode, displaying a more desirable analytical performance in the amperometric detection of ethanol, compared with the conventional graphite-functionalized and bare GCE-based bioelectrodes. It also exhibited good performance with faster, highly selective and sensitive response, and a wide linear range and low LOD for ethanol detection. Moreover, the accurate determination of ethanol in real samples demonstrated the great potential of this proposed biosensor for practical applications. Above all, GS, with favorable electrochemical activity, was considered as opening up a new challenge to explore a range of electrochemical sensing and biosensing applications.

A highly sensitive amperometric biosensor has been developed for L-lactic acid detection based on the lactate oxidase immobilization on the surface of ZnO nanotetrapods (Figure 1.5) by electrostatic adsorption [51]. Unlike traditional detectors, the special four-leg individual ZnO nanostructure, as an adsorption layer, provided a 3D spatial network structure and multiterminal charge transfer channels (six electron conduction ways, in theory). Furthermore, a large amount of ZnO tetrapods were randomly stacked to naturally form a three-dimensional network that facilitated the exchange of electrons and ions in the phosphate buffer solution. This simple and low-cost ZnO nanotetrapod L-lactic acid biosensor presented a linear response from 3.6 μM to 0.6 mM, a high sensitivity of 28.0 μA cm−2 mM−1, a LOD of 1.2 μM, and a low KMapp of 0.58 mM. The accuracy of the biosensor was achieved by making use of the good biocompatibility of ZnO nanotetrapods, which maintains the activity of enzymes, and the unique multiterminal electron transmittal feature.

Figure 1.5 Schematic diagram illustrating the selective intracellular Ca²+ measurement setup showing a typical microscope image of a single human fat cell adipocytes during measurement.

(Reprinted with permission from [56]; Copyright © 2009 American Institute of Physics).

A reagentless uric acid (UA) biosensor based on uricase immobilized on ZnONRs was developed [38]. It was shown that both conductive and biomimetic properties of ZnONRs played important roles in the electrochemical behavior of the adsorbed enzyme. The ZnO nanorods derived electrode retained the enzyme bioactivity, could enhance the electron transfer between the enzyme and the electrode and showed excellent thermal stability (up to 85°C), an electrocatalytic activity to the oxidation of UA without the presence of an electron mediator and anti-interference ability. The electrocatalytic response showed a linear dependence on the uric acid concentration ranging from 5.0×10−6 to 1.0×10−3 M with an LOD of 2.0×10−6 M and a KMapp of 0.238 mM, demonstrating a high affinity.

The ZnONRs grown onto indium-tin-oxide (ITO)-coated glass surface using zinc nitrate hexahydrate/hexamethylenetetramine (HMT) in aqueous phase have been utilized in the development of a urea biosensor [46]. The Urs was immobilized onto ZnONRs/ITO at physiological pH via electrostatic interactions between Urs and ZnO to fabricate an Urs/ZnONRs/ITO bioelectrode. A linear amperometric response was obtained on the Urs/ZnONRs/ITO biolectrode for urea concentrations in the range of 1–20 mM with a sensitivity of 0.4 μA mM−1, a response time of 3 s, a LOD of 0.13 mM, and a KMapp of 9.09 mM. Further, studies indicate the selectivity of bioelectrode against glucose and ascorbic acid. Results indicate that ZnONRs provide suitable microenvironment for Urs immobilization and can be utilized in biosensor design and other biological applications.

Functionalized multilayered graphene (MLG), because of its very large 2D electrical conductivity and large surface area, has been used for the fabrication of a novel amperometric urea biosensor [47]. A thin film of functionalized MLG was fabricated onto an ITO substrate by electro-phoretic deposition (EPD) technique and was used to immobilize urease and GLDH using ethyl(dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (NHS) chemistry. This biosensor showed linearity in the 10–100 mg dL−1 concentration range, sensitivity of 5.43 μA mg−1 dL cm−2, a low LOD of 3.9 mg dL−1, and a response time of 10 s, demonstrating that MLG is a promising material for electrochemical biosensing applications of other clinically important bioanalytes such as glucose, cholesterol, triglycerides, etc.

An amperometric biosensor based on CAT and a modified carbon paste electrode (CPE) with ZnONPs have been applied to dopamine (DA) detection [54]. The ZnONPs could play a key role in facilitating the electron transfer between CAT and CPE. The CAT/ZnONPs/CPE showed a good sensitive state towards oxidation of DA in the range from 5 to 41 μM. The designed biosensor showed a good stability and retained 91% activity after 21 days.

Zhou et al. proposed the application of a CRGO with the nature of a 2D single sheet modified glassy carbon electrode (CRGO/GCE) for the preparation of an electrochemical sensing and biosensing platform [26]. The electrocatalytic behaviors of eleven kinds of organic and inorganic electroactive compounds (i.e., probe molecule (potassium ferricyanide), free bases of DNA (guanine [G], adenine [A], thymine [T], and cytosine [C]), oxidase/dehydrogenase-related molecules (H2O2/NADH), neurotransmitters (DA), and other biological molecules (ascorbic acid [AA], UA, and acetaminophen [APAP]) were employed to study their electrochemical responses at the CRGO/GCE, which show more favorable electron transfer kinetics than graphite modified GC (graphite/GCE) and GCE electrodes. The presented results demonstrated the possibility to detect a single-nucleotide polymorphism (SNP) site for short oligomers with a particular sequence at the CRGO/GCE electrode without any hybridization or labeling processes, or the use of electrochemical mediators or indicators, suggesting the potential applications of CRGO in the label-free electrochemical detection of DNA hybridization or DNA damage for further research. Based on the greatly enhanced electrochemical reactivity of H2O2 and NADH at the CRGO/GCE, CRGO/GCE-based bioelectrodes (in connection with GOx and ADH) showed a better analytical performance for the detection of glucose and ethanol compared with graphite/GC- or GC-based bioelectrodes. The CRGO with the nature of a single sheet showing favorable electrochemical activity should be extremely attractive for a wide range of electrochemical sensing and biosensing applications, ranging from amperometric sensors to amperometric enzyme biosensors and label-free DNA biosensors.

1.4.2 Potentiometric Sensors

Fulati et al. [55] used the ZnONTs and ZnONRs grown on gold thin film to create pH sensor devices. The developed ZnO nanotube and nanorod pH sensors display good reproducibility, repeatability and long-term stability and exhibit a pH-dependent electrochemical potential difference versus an Ag/AgCl reference electrode over a large dynamic pH range. They found that ZnO nanotubes provide sensitivity as high as twice that of the ZnONRs, which can be ascribed to the fact that small dimensional ZnONTs have a higher level of surface and subsurface oxygen vacancies and provide a larger effective surface area with higher surface-to-volume ratio as compared to ZnONRs. A good linear electrochemical potential response was observed and their devices showed good sensitivity and reproducibility. These results demonstrated that the ZnONT arrays may find potential application as a novel material for measurements of intracellular biochemical species within single living cells, since nanoscale ZnONT structures can miniaturize the size of the sensor in a significant way.

Hexagonal ZnONRs coated with a polymeric membrane with selective ionophores and grown on a silver-coated tip of a borosilicate glass capillary, were used as selective potentiometric sensors of intracellular free Ca²+ and Mg²+ [56, 57]. The respective membrane-covered ZnONRs exhibited a Ca²+ or Mg²+-dependent electrochemical potential difference versus an Ag/AgCl reference microelectrode within the concentration ranges 100 nM–10 mM and 500 nM–100 mM, respectively. The developed nanosensors, with a simple fabrication method, achieved excellent performance in terms of sensitivity, stability, selectivity, reproducibility and anti-interference. These nanoelectrode devices, which were successfully applied to the determination of intracellular Ca²+ and Mg²+ in two types of cells (human adipocytes and frog oocytes) (see experimental setup in Figure 1.5), pave the way to enable analytical measurements in single living cells and to sense other biochemical species at the intracellular level.

Functionalized ZnONR-based potentiometric microsensors have been developed to measure intracellular metal ions (Ca²+, Mg²+, K+ and Na+) and glucose [17]. ZnO nanorods, grown on the tip of borosilicate glass capillaries (0.7 μm in diameter), and functionalized by using a metal-ion selective plastic membrane or GOx showed high sensitivity and good biocompatibility for intracellular environments and were capable of penetrating the cell membrane. Human adipocytes and frog oocytes were used for determinations of intracellular free metal ions and glucose concentrations. The performance of the ZnO nanostructure-based intracellular sensor could be improved through engineering of morphology, effective surface area, functionality, and adsorption/desorption capability.

A functionalized ZnONRs-based selective electrochemical sensor for intracellular glucose measurements was developed by Asif et al. [28]. To adjust the sensor for intracellular glucose measurements, hexagonal ZnONRs grown on the tip of a silver-covered borosilicate glass capillary (0.7 μm diameter) were then coated with GOx. The proposed intracellular potentiometric biosensor showed a fast response with a time constant of less than 1 s and showed quite a wide linear range from 0.5 to 1000 μM. It was used to measure intracellular glucose concentration in human adipocytes and Xenopus laevis oocytes and to demonstrate that insulin increased the intracellular glucose concentration in both cells. These results demonstrated the capability to perform biologically relevant measurements of glucose within living cells. The ZnONRs-based glucose electrode thus holds promise for minimally invasive dynamic analyses of single cells. All of these advantageous features can make the proposed nanoelectrode bio-device applicable in medical, food or other areas. Moreover, the fabrication method is simple and can be extended to immobilize other enzymes and other bioactive molecules with small IEPs for a variety of biosensor designs.

Ali et al. [18] also developed a potentiometric nanosensor for glucose based on functionalized highly-oriented single-crystal ZnONT arrays (Figure 1.6). The ZnONT arrays were prepared by a trimming of ZnONRs along the c-axis on the gold coated glass substrate having a diameter of 100–200 nm and a length of ~1 μm, using low temperature aqueous chemical growth process. The GOx was immobilized by physical adsorption in conjunction with a Nafion coating on the prepared ZnONT arrays. The electrochemical response of the sensor was found to be linear over a relatively wide logarithmic concentration range from 0.5×10−6 M to 12×10−3 M. The proposed sensor showed a high sensitivity of 69.12 mV decade−1. A fast response time, lower than 4 s, with good selectivity, reproducibility, stability and negligible response to common interferences such as ascorbic acid and uric acid prevailed. The great performance of the ZnONT arrays-based sensor can be attributed to its unique properties like the vast surface-to-volume ratio due to the porous structure of ZnONT arrays, which can provide a favorable microenvironment for the immobilization of GOx, the enzyme catalysis of the glucose oxidation on the electrode, and an excellent electrical contact between the gold electrode and the ZnONTs. In addition, due to the large surface-to-volume ratio of the porous structures of the ZnONTs, the sensor electrode enhances the sensitivity for analytes, as demonstrated by the detection of glucose without the presence of a mediator. The good performance of the proposed sensor also makes it suitable for externally integrating/interfacing nano-sensing element to commercial (low threshold) field-effect transistor (FET) devices, giving the advantages of simplicity and low cost for the enzymatic detection of biochemically important substances. All these advantageous features can make the proposed biosensor applicable in wireless physiological parameters monitoring, environmental, food or other areas.

Figure 1.6 Schematic diagram showing the measuring setup and sensing mechanism of the glucose

(Reprinted with permission from [18]; Copyright © 2011 Institution of Engineering and Technology [IET]).

Recently, Ali et al. [29] designed a prototype wireless remote glucose monitoring system interfaced with a ZnONW arrays-based glucose sensor. The GOx was immobilized onto ZnONWs in conjunction with a Nafion membrane coating, using an existing general packet radio services (GPRS)/global system for mobile communication (GSM) network. They demonstrated the remote monitoring of patients’ glucose levels with existing GPRS/GSM network infrastructures using their proposed functionalized ZnONW arrays sensors integrated with standard readily available mobile phones. The proposed potentiometric ZnO nanosensor device showed good linearity and negligible interference of anionic species like uric and ascorbic acids. The calibration curve for glucose is linear from 0.5 μM to 10 mM, with an LOD of 0.5 μM. This proposed system can provide a means of using emerging nanosensors/nanodevices for monitoring multiple health parameters outside the traditional hospital environment and efficiently transferring data to physicians for immediate consultation in case of urgent need. Such an application can reduce health care costs and allow caregivers to monitor and support their patients remotely, especially those located in rural areas. In the future, similar techniques with various ZnO nanostructure-based platforms can provide nanosensor/nanodevices for monitoring multiple health parameters outside central labs.