Recent Advances in Biosensor Technology: Volume 1

Nanomaterials for Biosensing Applications

Abhay Dev Tripathi¹, Soumya Katiyar¹, Avinash K. Chaurasia², Abha Mishra¹, *

¹ School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi-221005, India

² School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi – 221005, India

Abstract

A biosensor is a device that detects the presence of analytes with its biological receptor entity, having unique specificities corresponding to their analytes. Most of these analytes are usually physical in nature, such as DNA, proteins, antibodies, and antigens, but they may also be simple compounds, including glucose, H2O2, toxins, and so on. Biosensors’ significance rises in providing real-time quantitative and qualitative information on analyte composition. The sensing mechanism involves the transduction of target binding interactions into optical, electrochemical signals, etc ., which can be amplified and detected.

Nanomaterials (NMs) have shown significant potential in biological sensing—these allow close interactions with target biomolecules due to their extremely small size and suitable surface modifications. Nanomaterials appear to be potential possibilities because of their capacity to immobilize a greater number of bioreceptor units in confined devices and even act as a transduction element, allowing for enhanced sensitivity and reduced detection limits down to specific molecules. Nanomaterials have been widely used for in vitro detection of disease-related molecular biomarkers and imaging, contrasts to map out the distribution of biomarkers in vivo. This chapter summarizes nanomaterials such as gold nanoparticles, quantum dots, polymeric nanoparticles, carbon nanotubes, nanodiamonds, and graphene nanostructured materials that are currently being researched or utilized as biosensors.

Keywords: Biosensors, Carbon nanostructures, Graphene nanostructure, Nanodiamonds, Nanomaterials, Quantum dots.

* Corresponding author Abha Mishra: Biomolecular Engineering Laboratory, School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi - 221005, India; E-mail: [email protected]

INTRODUCTION

Nanomaterials (NMs) have piqued the interest of many people because of the increasing preference to regulate highly favoured molecular systems not only in

the human body but also in the environment. The interface of nanomaterials with bioactive molecules such as proteins, enzymes, and nucleic acid has arisen as a multidisciplinary area described as nanotechnology which refers to the scientific ways by which nanoparticles or nanomaterials are integrated to generate instruments for investigating biological mechanisms [1].

According to the European Commission's 2011 suggestion, nanomaterials (NMs) are a natural, incidental, or manufactured material containing particles, in an unbound state, as an aggregate, or as an agglomerate, and where, for 50% or more of the particles in the number size distribution, one or more external dimensions are in the size range 1 nm-100 nm [2]. NMs have distinctive characteristics, including a high specific surface/volume ratio, high sensitivity, excellent electrical properties, and outstanding magnetic and catalytic capabilities, among several others [3]. Adsorption and catalytic activity are very efficient due to active binding sites and an abundant supply of reactive surface functional groups of NMs. As a result, NMs may be employed in a variety of industries, including biosensors, medicines, cosmetics, agriculture, and energy, among others [4]. The increased total surface area of all nanomaterials allows for the immobilization of a more significant number of bio-recognition units. Nano-biochip materials, nanoscale biocompatible materials, nanomotors, nanocomposites, interface biomaterials, nano biosensors, and nano-drug-delivery platforms offer immense potential for industrial, security, food, forensic analysis, and therapeutic applications.

NMs are classified into three types depending on the materials used in their production, including (i) carbon-based nanostructures (e.g., Carbon nanotubes or CNTs, Graphene, Nanodiamonds, Fullerenes, etc.), (ii) organic (e.g., Quantum dots, Nanofilms, Nanogels, Dendrimers, etc.) or (iii) inorganic (e.g., Magnetic nanoparticles, Ag/Au nanoparticles, Nanoshells, Nanowires, etc.). Carbon-based nanostructures (such as carbon nanotubes or graphene) seem to be the most often employed NMs in biological investigations due to their diverse surface properties, and electrical and optical properties [5]. Among metallic NPs, Gold NPs are promising candidates because of their excellent oxidative stability and low toxic effects as contrasted to others, such as Ag, which oxidize and demonstrate cytotoxicity in vivo [6]. The large specific surface area of all NMs allows for the immobilization of an increased number of biorecognition units. Nevertheless, one of several ongoing hurdles is the immobilization technique employed to bind the specific analyte intimately onto such nanostructured materials. As a consequence, one of the most important elements in constructing a via ble biosensing system is the approach utilized to encapsulate the enzymes. The elements of NMs appropriateness in better transducer circuits are the size and shape-based energy of system distributions. For example, nanorods (NRs), nanotubes (NTs) or cylindrical architectures facilitate many contacts simultaneously at the same time, decreasing the overall reaction time and even expense. In this manner, even little changes in the typical reaction might be efficiently noticed.

A biological or biomimetic receiver element with distinct specificities toward related bioanalytics defines a biosensor system. Over the past ten years, substantial work has been spent on pioneering and continuing to develop biosensors with better specificity, responsiveness, affordability, simplicity, and detection time accuracy. In summary, a biosensing system is composed of a selective bioreceptor element (DNA, peptides, cells, aptamers, etc.) for analyte acquisition, a physical transducer (e.g., optical, electrochemical, thermal, acoustic, etc.), and signal processing unit for the electronic assessment of the accompanying interactions. Among the most essential difficulties in biosensing systems is achieving excellent sensitivity while maintaining an incredibly simple format to use, and the selection of an appropriate biological recognition interface is vital to this goal. Nanomaterials, like most other technical segments, have proved their inherent suitability for biological sensing applications. The main purpose of incorporating NMs into a biosensing operation is to optimize and improve responsiveness with the lowest detection limit in the shortest period. Due to their fast reaction times, nano biosensors are becoming more desirable for fast and real-time analyte monitoring and identification. Minimal LOD biosensors are applied to detect bioanalytics at trace amounts or volumes. The LOD is the lowest analyte concentration that a biosensing unit can recognize but not quantify, meanwhile, the LOQ is the lowest analyte concentration that a biosensor can quantify with therapeutic high precision and specificity. The appropriate employment of such nanostructured devices resulted in demonstrably improved performances, higher efficacy with improved sensitivities, and a lower sample amount requirement. Approaches towards engineering the NMs for a predictable output by manipulation of their interacting coordinates are presently being rapidly optimized for biosensing applications. The final attribute of NMs' usage in biosensing is unquestionably their large surface area, which confers stronger surface functionalization capacities, allowing for the tracking of any stimulus of the reactions in biological and environmental settings. The following surface modification methods are significant for attaching bio-physiological constituents to NM surfaces, including thiol-based NM, streptavidin-biotin association, π-π interactions, and EDC-NHS reactions.

The foremost objective of this chapter is to discuss an assessment of developments in the fields of innovative NM-based biosensor systems. We explain the production of carbon-based nanostructured materials, metals/metal oxides, and nanoparticle-based sensor systems, as well as their current and future applications for accurate and consistent monitoring of bioanalytics with higher sensitivity and specificity. Additionally, we identify the limitations related to many of these NMs in order to encourage widespread interest from researchers in the fabrication of novel nanomaterial-based biosensor devices.

NANOMATERIALS IN BIOSENSING APPLICATION

Nanomaterials as a biosensing tool have shown great potential as these allow close interactions with target biomolecules due to their nanostructured size and their capacity to immobilize a greater number of bioreceptor units in confined devices and suitable surface modifications (Fig. 1).

Fig. (1))

Schematic representation for a biosensor using nanomaterials and nanostructures: Cytosensors, Nanoparticle tagged DNA/RNA for detection of several proteins involved in binding with these materials, Enzyme based sensors, Immunosensors.

NMs can be generally divided into three types based on their chemical composition, namely carbon allotropes-based nanomaterials with only carbon atoms, inorganic nanomaterials with metallic or non-metallic elements, and organic nanoparticles with mostly polymeric nanomaterials. Below is the descriptive list of the categories (Fig. 2).

Carbon Nanostructures

The extraordinary attributes of nano-structured carbons, including carbon nanotubes/nanodots, graphene, or nanodiamonds, have led to their widespread application as electrical or electrochemical transducers in biosensing systems. The rising use of these NMs in bio-physiological sensing systems is attributed to their numerous incomparable and distinct physical properties. Carbon NMs are gaining popularity for biosensing purposes due to their high surface area, stable thermal optical, flexibility, electrical, and physical characteristics [7, 8]. The exceptional applicability of all these NMs in biosensing is mostly due to tetravalent interaction in carbon, and also catenation-facilitated expanded binding capacity, which has shown to be extremely effective in medical diagnostics and real-time assessment [9]. The biosensor based on all these NMs not only has good specificity and various innovative functional processes but also has a better resolution (for localized monitoring) and may be used in real-time assessment, even without the need for labels or markers. The following section of the chapter highlights significant structural characteristics of carbon-nanostructured-based materials, as well as some current sensing breakthroughs.

Fig. (2))

Different types of nanomaterials used for different biosensing applications.

Carbon Nanotubes (CNTs)

CNTs are made up of hexagonal pattern-organized carbon atoms that form six-member carbon rings. These rings interlink to produce a graphene sheet, which subsequently generates CNTs, which become uniform cylindrical tubes and have a length of micrometres and a diameter of roughly 100 nm [10]. CNTs are classified into two types depending on the layered architecture of graphene sheets: single-walled carbon nanotubes or SWCNTs (diameters ranging from 0.4 to 2 nm), which include just one layer of the graphene sheet, and multi-walled carbon nanotubes or MWCNTs (diameter ranges from 0 to 3 nm.), which comprise several stacked graphene layers [11, 12]. Their additional benefit over other NMs is a unique blend of electromagnetic, optical, mechanical, and electrochemical attributes that hold promising potential for a variety of implementations, particularly biosensing [13]. These biosensors are classified as electromechanical transducers, electrochemical-based CNT biosensors, immunosensors, and optical-based CNT sensors based on their substrate identification and processing mechanisms. CNTs have outstanding physicochemical qualities, including excellent mechanical properties, ultra-lightweight, unique electronic frameworks, and great thermochemical persistence. Additionally, their simplicity of use and well-studied organic modifications adds novel features to the nanostructured working electrodes, such as specified docking sites for macromolecules or redox regulation of bio-electrochemical events. CNTs also have the capacity to easily penetrate biological membranes allowing them to be used in vivo with minimum intrusiveness, and they could also be used for photoacoustic cell imaging. Importantly, CNTs have quite a high specific surface area (SSA) that allows for the immobilization of a significant number of multifunctional entities at the CNTs surface, including receptor molecules for biosensing purposes. CNTs also have intrinsic optical features such as powerful resonance Raman scattering as well as near-infrared photoluminescence properties [14].CNTs may be constructed using three different fabrication methodologies: chemical vapour deposition, electric arc deposition, as well as laser deposition [15].They also have semi- and metallic conducting qualities, making them good materials for disease diagnosis, food hygiene, and environmental pollution monitoring. One of the biggest challenges with CNTs for biological implementations is their innate complexity in handling. CNTs prefer to accumulate into bundles due to their strong, attractive associations that are hard to break. The addition of reactive groups to the surface of CNTs, therefore, aids in their solubilization and enables their analysis [16]. The specific electric characteristics of carbon nanotubes (CNTs) have been used in field-effect transistor (FET) biosensing systems, within which variations in the conductivity of the CNT medium or alternation of the Schottky barrier after the specific analyte recognition event contributed to high specificity and low detection thresholds down to single compounds [17]. For example, a FET-CNT immunosensor has been designed to measure osteopontin protein (OPN), a biomarker of prostate tumor, by anchoring a genetically-engineered single-chain dynamic segment protein with a strong affinity for OPN, and is being used to track this molecular marker in the presence of a high concentration of bovine serum albumin (BSA) [18]. MWCNTs have great prospects in biosensors due to their convenience of immobilization while retaining protein inherent activity [19]. Beden et al. (2015) constructed electroactive adducts to create an electrochemical sensor device for sub-nanomolar sensing of dopamine. For enhanced quantitative metrics, the biosensor was upgraded using MWCNTs and AuNPs. When the electrodes were changed using nano-hybrids, the sensing device responded better. The sensor demonstrated an excellent and broad linear range, as well as a reduced sensing threshold [20].

Graphene

Graphene, a two-dimensional (2D) carbon substance with a one-atom-thick, has emerged as a popular study issue in the realm of biosensing. Graphene, like carbon nanotubes, is tightly packed in a honeycomb lattice arrangement. Graphene and its variants, such as graphene oxide (GO), reduced graphene oxide (rGO) and so on, have various admirable qualities due to its peculiar architecture, notably high heat conductance, remarkable electrical properties, enhanced optical features, high flat surface, excellent flexibility, high degree of freedom, and good tensile stability [21, 22]. GO is a functionalized graphene generated via oxidative extraction of graphite, which has a morphology comparable to graphene. Chen et al. constructed a fluorescent biosensing device based on GO to detect and quantify dopamine in biological sample specimens. This monitoring methodology relied on dopamine assembly onto the surface of GO via various non-covalent contacts, and considerable fluorescence quenching revealed its effectiveness as a tag-free fluorescent biosensor for dopamine sensing method with a limit of detection (LOD) of 94 nM [23]. Nanobiosensing (NBS) devices have been developed based on graphene-based materials that can be used in various detection methods, including optical, electrochemical, and electrical. These remarkable features allow graphene to be an appealing contender for the construction of a new line of NBS devices with several benefits, such as excellent performance, specificity, affordability, scalability, flexibility, and stability. Graphene and graphene-related NMs are currently in production using a variety of physicochemical methods, including physical exfoliation of graphite, chemical vapour deposition or CVD of hydrocarbons on metallic surfaces, and thermo-chemical or liquid-phase exfoliation of graphite oxide layer [24]. Graphene-based glucose biosensors are often constructed by immobilizing glucose oxidase (GOx) enzyme onto the surface of graphene sheets, as in the graphene-FET described by Huang et al. [25]. Among the most critical concerns for graphene's biological uses would be its short and long-term toxicity. Reduced graphene oxide (rGO) is formed out of GO that has been reduced chemically or physically. Due to their diverse capabilities, reduced GO may be effectively employed to construct extremely efficient electrochemical and biochemical sensors that can be tailored to be very susceptible to minor changes in the biochemical environment [26]. In recent times, efficient multifunctional biosensors with multiple detection outputs on a single system have been proposed [27]. Ouyang et al. demonstrated a unique dual-spectroscopic all-in-one approach for quantifying aristolochic acids in complicated biological specimen matrices. After extraction, aristololactam (AAT), a bioproduct of aristolochic acid I (AAI), was identified instantly by fluorescence spectrometer, whereas AAI was identified by Surface-enhanced Raman scattering or SERS using a graphene-enhanced absorption and magnetically recovery method [28]. Despite several valuable findings on graphene's in vitro cytotoxic activity effects, there is currently no comprehensive knowledge of the associated processes of graphene's cellular toxicity in the previous research; hence, this problem needs to be thoroughly investigated. After extraction, aristololactam (AAT), a biological product of aristolochic acid I (AAI), was identified instantly by fluorescence spectrometer, whereas AAI was identified by Surface-enhanced Raman scattering or SERS using a graphene-enhanced absorption and magnetically recovery method.

Nanodiamonds

Nanodiamonds (NDs) have already been emphasized as a novel group of carbon-based nanostructured materials due to their distinctive qualities, such as non-toxicity, sustained fluorescence, accessible functionalization, inherent biocompatibility, and many other basic traits of pure diamonds [29-31]. Since the first NDs production in the 1960s [48], a substantial number of ND research have been published over the last several years. NDs of various architectures and sizes have been synthesized using various processing techniques and are extensively used in various applications, including drug administration, biomedical imaging, biosensor, power storage systems, and catalysis [32-34]. Prominent methods for producing NDs of various sizes and architectures include detonation methods, high pressure and temperature (HPHT), ball milling, laser ablation, and chemical vapour deposition (CVD) [35-37]. NDs appear to be a much more biocompatible and non-toxic variant in their family among carbon-based NMs. Other studies found that ND had no effect on cell survival, cell membrane oxidative stress, or intracellular oxidative stress, and that their biological effects were less than those of other nanocarbons [38, 39]. As a result, biological characteristics, including aggregate formation, metabolism, internalization, and toxic effects, may be regulated, providing a superior risk-benefit ratio for medicinal and biomedical imaging approaches. NDs are categorized into many classes according to their core size and biosynthesis mechanism. Detonation nanodiamonds (DNDs), also called ultra-dispersed diamonds, comprise diamond NPs sizes ranging from 3 to 10 nm produced by carbon-containing explosives detonated under circumstances of negative oxygen equilibrium [40]. FNDs (fluorescent nanodiamonds), which have a wider size distribution than DNDs, are primarily formed from costly high-pressure and temperature (HPHT) diamonds and mechanical procedures, which give them very stable flaws [41, 42]. Chemical vapour deposition or pulsed laser ablation in liquids can also produce nano-sized diamonds, although these methods are less frequent and more expensive [41, 43]. NDs are processed to remove impurities (non-diamond carbons, metals, oxides, and many other impure particles) and manage their surface morphology and chemistry before being used in bio application formulations. Different solutions regulated by physical principles or chemical changes can be used to solve the restricted aggregation effect of bare NDs, which is a commonly debated subject. NDs have adapted to work with a variety of medications, displaying favourable input and less adverse effects. For example, tests of materials containing doxorubicin (DOX) revealed remarkable tumour growth inhibition, delayed cytoplasmic release, and enhanced drug uptake in the nucleus of cancer cells [44]. NDs have outstanding luminescent features, such as high quantum yield and stable emission from colour centres, such as Nitrogen-Vacancy centres glowing in the far-red/near-infrared, making them ideal for biological labelling [45]. NDs have been shown in previous investigations to have nitrogen-vacancy centres with intrinsic fluorescence characteristics, making them useful imaging and diagnostic tools [46]. Furthermore, because NDs have a higher refractive index (RI) than cytoplasm, they usually generate a powerful light scattering response, allowing optical microscopy to easily discern them in a cell [47]. The surface of NDs has a high affinity for proteins. Li et al. investigated the receptor-mediated endocytosis of fluorescent NDs connected to transferrin because of this high affinity for proteins. Their findings revealed that crosslinking nanoparticles with proteins improves cellular absorption stability and efficiency [48]. Biocompatibility has been demonstrated for NDs in a variety of biosensing and biomedical applications. Despite its unique properties, the idea of improving its compatibility with various solvents and polymers has not been fully investigated. Furthermore, it has been difficult to disperse NDs in an aqueous solution for successful usage in the biomedical field until now, thus future research should look into particle systems for this reason.

Organic Nanoparticles As A Biosensor

Organic compounds may be encountered in numerous sectors, ranging from medicines to consumer goods and services, including dyes, ink solvents, flavouring agents and household cleaning products. Chemical modifications are used to improve the quality and performance of many of these organic molecules. Many chemicals necessary for formulation are insoluble in nature, which limits their activity and application. Organic substances are essentially and eventually miscible in aqueous or aquatic conditions, but at a considerably slower speed than their inorganic equivalents, hence organic nanoparticles (NPs) will not be discovered in the environment for an extended period of time, considering them environmentally advantageous. These organic NPs are more environmentally benign, less expensive, and better suited to a variety of biological applications [49]. Organic nanoparticles or polymers include liposomes, dendrimers, micelles, and ferritin. Certain organic NPs, such as micelles and liposomes, also known as nano-capsules, are susceptible to temperature and electromagnetic radiation like heat and light, as well as some organic NPs, are biocompatible, biodegradable, and non-toxic in behaviour.

These NPs have distinctive attributes that render them suitable for therapeutic medicine administration as well as a wide range of biological functions. Aside from the conventional qualities like size, structure, chemical composition, surface form, and delivery methods, the drug-carrying potential and resilience, whether an entrapped drug or immobilized drug system, affect their range of applicability and performance. Since they are effective and can be administered into the body system, organic NPs are most typically used in medical applications, including targeted drug delivery applications. The term targeted drug delivery system refers to a system that delivers drugs to specified regions of the body [50].

Dendrimers

Dendrimers are symmetrically nano-sized molecules containing a small atom surrounded by uniform branches called dendrons. Dendrimers are branching molecules with features similar to polymers and tiny entities. Dendrimers can have a structure of up to 4 nm, but most are in the 1-2 nm range [51]. Unlike other NPs such as lipid nanoparticles and micelles, these nanostructures do not have a completely central cavity and would instead be comprised of a polymeric matrix of repeating units that expands from the inside out. The NM's framework is built in the manner of an onion, with the shells representing repeating units that have been joined to the next inner cell, incrementally shrinking at the core. Thus, every generation begins with the core, and the beginning of the shell can be thought of as a focal point where the new repeating units begin.

A dendrimer is sometimes a tree-like structure since it has a confined branch-like structure. The dendrimer's structure is made up of three structural components [52]. Dendrimers' cores are shielded from the environment, providing a unique microclimate. The outer shell consumes a well-defined micro-environment immediately beneath the surface. A large number of possible active sites can be found on the multivalent surface. Dendrimers' most appealing applications are in the pharmaceutical and medical fields. Dendrimer is used as a contrast