20 Nanotechnology and Biosensors

Dr Hardeep Kaur

Nanotechnology and Biosesnors

25.1. Introduction

25.2. Nanomaterials used in Biosensors

25.2.1. Gold nanoparticles

25.2.2. Carbon Nanotubes

25.2.3. Graphene

25.3 Nanofabrications in Biosensors

25.3.1 Microcantilever based biosensors

25.3.2 Integrated microfluidics based biosensors

25.1 Introduction

Nanotechnology is the term associated with nanosize materials, their creation and employment in functional materials, devices and systems. It exploits the novel phenomena and properties arising from the nano meter length scale. One nanometer is one billionth (10-9) of a meter, very close to the atomic level. To provide a practical perspective, a typical human hair is about 10,000 nm in diameter. Although nanotechnology is a relatively recent phenomenon, the development of its central concepts was initiated over a long period of time. In the 1990s, the term “nanotechnology” gained serious attention by the scientific community. Materials at the nanometer scale exhibit unique size dependent properties that are different from their bulk state. Nanotechnology then involves manipulating these size-dependency to create new and novel products and systems in various sectors, including biotechnology, health care, food safety, water quality, chemicals, electronics, computers, tools, equipment, systems and diagnostic devices. Nanotechnology is increasingly playing a major role in the development of biosensors to achieve high sensitivity, specificity, robustness, reproducibility and long-shelf life that are critical to diagnostic assays. Nanomaterials are used to introduce new signal transduction mechanisms in biosensing modalities. They are also used to allow rapid and simple analysis, improve portability and increase utility at the point of care and point of use.Nanomaterials (NM), or matrices with at least one of their dimensions ranging in scale from 1 to 100 nm, display unique physical and chemical features because of effects such as the quantum size effect, mini size effect, surface effect and macro-quantum tunnel effect. Because of their small size, nanosensors, nanosystems and nanoprobes are transforming the fields of chemical and biological analysis, to enable rapid analysis of multiple substances in vivo. This chapter focuses on the major aspects of the nanotechnology-based biosensors.

25.2. Nanomaterials used in Biosensors

The nanotechnology products can be classified into three categories based on the number of dimensions “pushed” to the nanometer scale:

Thin films, such as coatings of implants for biocompatible purposes, anticoagulant coatings of stents, and coatings of pills and other therapeutic agents, have only one dimension pushed to the scale of few tens or hundreds of nanometers, while the other two dimensions can still extend up to millimeters; NMs, such as carbon nanotubes (CNTs), silicon nanowires, nanorods, and fibers, have two dimensions pushed to the nanometer scale; and NMs, such as quantum dots, gold, magnetic and polymeric nanoparticles, and liposomes, have all the three dimensions pushed to the nanometer scale. Various kinds of nanomaterials, such as gold nanoparticles, carbon nanotubes (CNTs), magnetic nanoparticles and quantum dots, are being gradually applied to biosensors because of their unique physical, chemical, mechanical, magnetic and optical properties, and markedly enhance the sensitivity and specificity of detection (figure 1).

During the last decade, NMs have been widely used in the fields of in vitro diagnostics, imaging, and therapeutics. They have enabled the simultaneous multiplex detection of many disease biomarkers and the diagnosis of diseases at a very early stage. They have also opened the possibility to explore the detection of ultra-trace concentrations of target analytes and have led to ultra-sensitive, rapid, and cost-effective assays requiring mini-mum sample volume. The NMs are being seen as the most promising candidates for the development of high-throughput protein arrays. The size, shape, composition, structure, and other physical/chemical properties of NMs can be tailored in order to produce the desired materials with specific absorptive, emissive, and light-scattering properties. The bioconjugated nanoparticles (NPs) have also been employed for signal amplification in assays and other biomolecular recognition events. However, the most promising application of nanotechnology will be in the field of point-of-care diagnostics, which will enable the primary-care physician and patients to perform assays at their respective settings. The long term stability of NPs in addition to their brightness and sharp bandwidth will be of tremendous significance to devise new methods for ultra-sensitive biomarker discovery, validation, and clinical use. The gold NPs (GNPs) tagged with short segments of DNA can be employed to detect the genetic sequence in a sample, while the use of nanostructures (nanopores, nanowires, nanopillars, and nanogaps)-based devices can further provide the single-molecule detection capability. NMs such as QDs and NPs are good imaging agents due to their enhanced performance and functionality. They can be targeted to the specific disease sites in the body by conjugating them to biomarker-specific vectors.). The most widely used NMs in biosensor development are described below.

25.2.1 Gold nanoparticles (AuNP)

Surface chemistry plays a pivotal role in biosensing especially in electrochemical investigations, and it need to be managed to produce desired signal outcome. Certain inorganic components have been explored to functionalize the electrode surface that facilitate rapid electron transfer and improve recognition molecule adsorption on the electrode surface. The prerequisite for surface modifiers are that these should be biocompatible, thermally stable, easily functionalizable and provide antifouling effect. AuNPs or gold nano clusters are among the recognized nanomaterials famed for their highly conductive nature and enlarged surface area. Such materials not only facilitate biomolecule immobilization on transient platforms but also expand electrochemical properties such as low background current, high signal to noise ratio and amplified signals. These properties help to attain surface characteristics that is ought to deliver sensitive output signals. Among all the nano- materials, AuNPs are laced with all attributes required for electrochemical biosensing. Non-toxic, simple and rapid synthesis, convenient functionalization, large surface area to volume ratio and high electrical conductivity has raised their preference for clinical diagnosis and biosensing of environmental pollutants. Size of nanoparticles is an important parameter considered for their easy functionalization which is necessary for stable adsorption of DNA probes, aptamers or antibodies. Also, electrodeposition or direct adsorption of AuNPs on gold platform decreases interfacial resistance offered by biomolecule adsorption on electrode surface. Association of AuNPs with some other nanocomposits escalate the signal amplification effect. For instance, amalgamating Zinc oxide (ZnO) and Gold – Titanium Oxide (Au-TiO2) nano composites offers fast electron transfer with biocompatible interface for biomolecule immobilization. Not only for electrochemical analysis, AuNPs have also crested their place in optical studies, such as introduction of AuNP to enhance sensitivity of ELISA as well as fluorescent and colorimetric assays. An example of AuNP based detection of lead ions (Pb II) is depicted in figure 2, where a DNA duplex called as DNAzyme is used as the bioreceptor against Pb (II). The DNAzyme contains a substrate strand called 17 DS with a ribonucleotide at the cleavage site and an enzyme strand called 17 E. In the presence of Pb (II) the 17 E strand cleaves 17DS at the cleavage site. In order to develop a Pb (II) biosensor with this DNAzyme the substrate strand was labelled with AuNP at both the ends as illustrated in figure 2c. In the absence of Pb (II), no cleavage happens and the AuNP aggregate at 50 ºC which in turns change the AuNP colour from red to blue (due to aggregation). But, in the presence of Pb (II), substrate stand was cleaved which displaced the AuNP away from each other so the colour remained red. Such colour based biosensors have been developed for various analytes through AuNP.

25.2.2 Carbon NanoTubes (CNTs)

During the past decade, CNTs have been one of the most extensively used NMs in biosensors, diagnostics, tissue engineering, cell tracking, labelling, and delivery of drugs and biomolecules. They are hollow cylindrical tubes composed of one, two, or several concentric graphite layers capped by fullerenic hemispheres, which are referred to as single-, double-, and multi-walled CNTs respectively. They have unique structures, excellent electrical and mechanical properties, high thermal conductivity high chemical stability, remarkable electrocatalytic activity minimal surface fouling, low overvoltage, and high aspect ratio (surface to volume). CNTs-based biosensors and diagnostics have been employed for the highly sensitive detection of analytes in healthcare, industries, environmental monitoring, and food quality analysis. They have been predominantly used in electrochemical sensing, mainly for glucose monitoring but also for the detection of fructose galactose, neurotransmitters, neurochemicals, amino acids immunoglobulin, albumin, streptavidin, insulin, human chorionic gonadotropin, C reactive protein, cancer biomarkers cells, microorganisms, DNA, and other biomolecules

25.2.3 Graphene

Graphene, an atomically thin layer of sp2 -hybridized carbon is another most extensively used NM for diagnostics and biosensors in the last few years due to its interesting and exciting properties, such as high mechanical strength, high thermal conductivity, high elasticity, tunable optical properties, tunable band gap, and very high room temperature electron mobility. It is a transparent material with a very low production cost and low environmental impact. It has been extensively employed in electrochemical, impedance, fluorescence, and electro-chemiluminescence biosensors for the detection of a wide range of analytes such as glucose, cytochrome c, NADH, hemoglobin, cholesterol, ascorbic acid, dopamine, uric acid, hydrogen peroxide, horseradish peroxidase, catechol, DNA, heavy metal ions, and gases. Graphene has also been incorporated with quantum dots for sensitive estimation of environmental pollutants. Further chemically prepared graphene oxide (from graphite) has been modified to carboxylic graphene for immobilization of acetyl cholinesterase and detection of pesticides. Figure 4 represents the SEM images of original and modified forms of graphene with some nanomaterial.

25.2.4 Quantum Dots

Quantum dots (QD) are very small semiconductor particles, only several nanometres in size, so small that their optical and electronic properties differ from those of larger particles. They are a central theme in nanotechnology. Many types of quantum dot will emit light of specific frequencies if electricity or light is applied to them, and these frequencies can be precisely tuned by changing the dots’ size, shape and material, giving rise to many applications. Quantum dots have been subject to intensive investigations because of their unique photoluminescent properties and potential applications. So far, several methods have been developed to synthesize water-soluble quantum dots for use in biologically relevant studies. Typical dots are made of binary compounds such as lead sulfide, lead selenide, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide. Dots may also be made from ternary compounds such as cadmium selenide sulfide. These quantum dots can contain as few as 100 to 100,000 atoms within the quantum dot volume, with a diameter of ~ 10 to 50 atoms. This corresponds to about 2 to 10 nanometers, and at 10 nm in diameter, nearly 3 million quantum dots could be lined up end to end and fit within the width of a human thumb. Quantum dots have been used successfully in cellular imaging, immunoassays, DNA hybridization, biosensor, and optical barcoding. Quantum dots also have been used to study the interaction between protein molecules or detect the dynamic course of signal transduction in live cells by Fluorescence Resonance Energy Transfer (FRET). These synthesized quantum dots have significant advantages over traditional fluorescent dyes, including better stability, stronger fluorescent intensity, and different colors, which are adjusted by controlling the size of the dots. Therefore, quantum dots provide a new functional platform for bioanalytical sciences and biomedical engineering.

In the field of biosensors, QD have established a special niche due to their unique optical properties. A lot luminescence based biosensors have been developed for clinical diagnosis and environmental monitoring using QD. As an example we may discuss the detection of breast cancer cells of cell line MCF-7n through quantum dot based optical biosensor. In this case QDs are labelled with primary antibodies against MCF-7 cell surface proteins and subjected to sample containing MCF-7 cells. Additions of secondary antibody labelled magnetic nanoparticles enable their magnetic separation to obtain fluorescence emission spectra as illustrated in figure 5.

25.3 Nanofabrications in Biosensors

Nanotechnology ply vital role in today’s biosensors as incorporation of nanomaterials greatly enhances the desirable attributes of biosensors. There is another way of nanotechnology application in biosensors, which is through the miniaturization of devices and fabrication of nanosensors. Nanotechnology plays a major role in fabrication of small compact devises that provides the merits automation, small sample volumes and high throughput. In this section we would discuss few examples of such nanofabrication that has enhanced the biosensor efficiency and would lead to commercial devices in future.

25.3.1 Microcantilever based biosensors

Micro cantilevers are the miniaturized flat surfaces anchored to one end to a support and a flexible protruding end for mass stress based study (figure 6a). The main principle of microcantilever based sensor is converting the mechanical stress on the cantilever into measurable signal. Differential stress on the surface of the cantilever causes deflection which can be read out by different methods such as, piezoelectric, piezo resistivity, optical reflection etc. Micro cantilevers have become a versatile tool in biosensing due to its high sensitivity, intrinsic flexibility and low cost of mass production. Reduction of dimensions of microcantilever to nano scales may lead to faster, cheaper and more sensitive response.

To understand the concept figure 6B illustrates the working principle of a microcantilever based biosensor. Here the biocomponent against a specific target such as antibodies to an antigen are immobilized which causes bending of the lever. Further the interaction of appropriate antigen with antibodies causes more deflection and change in resonance frequency which is monitored and related to the mass/concentration of the antigen as per the given equation.

25.3.2 Integrated microfluidics based biosensors

Microfluidics is the science of controlling fluids in multi-micro channels for analytical purposes. The target-biocomponent interaction is done in micro channels and the products are then channelled to sensing platforms. It has found wide application in biosensor technology as small volumes of samples are needed in this technique to produce highly sensitive responses. Microfluidics provides fast turnaround times along with multiplexing which has widened its application in biosensing. This technology has also given rise to lab on a chip concept which deals with the miniaturization of whole sensing platform.

Here we would like to discuss a recent example to detection of food borne pathogen Listeria through microfluidics based device. The device consists of two separate chip chambers, one for separation of analyte-biocomponent complex and other for detection of the formed complex based on impedance phenomenon. The bio complex included magnetic nanoparticles modified with anti-listeria monoclonal antibodies, gold nanoparticles modified with anti-Listeria polyclonal antibodies and urease. All these components were incubated in the separation chamber and separated from the mixture by the magnetic stirring bar. The separated complex was then treated with urea, which was hydrolysed by the urease into carbonate ions and ammonium ions. The formed products increased the ionic strength of the solution which was then channelled in the detection chamber and the change in impedance was measured by the interdigitated microelectrodes as illustrated in figure 7.

Similar devices are under fabrication, which may allow simultaneous detection of multi analytes with high specificity and sensitivity with very low amounts of sample volumes.

Concluding Remarks

Nanotechnology is the most upcoming and multidimensional field that has diverse applications. It has provided the biosensor technology with miniature devices that has allowed high throughput analysis of analytes and will reduce the cost in future. The incorporation of nanomaterials such as gold nanoparticles and carbon based nanomaterials has highly improved the detection sensitivities of amperometric and optical biosensors. Currently, integration of nanotechnology has delivered various point-of-care (POC) devices for clinical diagnosis and environmental monitoring and would play a more critical role in future for their commercialization.