New-generation nano-engineered biosensors, enabling nanotechnologies and nanomaterials
The Authors
Vinod Kumar Khanna, MEMS & Microsensors, Solid-state Devices Division, Central Electronics Engineering Research Institute, Pilani, India
Abstract
Purpose – This paper aims to discover the novelties in biosensor fabrication brought about by breakthroughs in nanomaterials and process techniques, the resulting enhancement in biosensor functionalities, new applications and future possibilities.
Design/methodology/approach – The impact of nanotechnology on biosensor advancement has been examined. Different directions of biosensor research in the nano era have been highlighted. These include the efforts made through nanotechnology to improve the performance parameters of the existing biosensors, and for implementation of innovative biosensor concepts.
Findings – Nanotechnology is a key technology in biosensor development. It has permeated into the biosensor field and brought in its wake far-reaching changes.
Practical implications – Biosensor science and engineering are central to virtually all aspects of life including medical diagnostics, environmental monitoring and biotechnological process control. Therefore, the progress in biosensors brought about by nanotechnology influences one's everyday life.
Originality/value – The study helps in understanding the applications of nanotechnology in fabricating a new generation of biosensors with improved characteristics. It provides information of value to those involved in biosensor research.
Article Type:
Technical paper
Keyword(s):
Sensors; Particle size measurement.
Journal:
Sensor Review
Volume:
28
Number:
1
Year:
2008
pp:
39-45
Copyright ©
Emerald Group Publishing Limited
ISSN:
0260-2288
1 Introduction
Sensors were downsized from the conventional macro- to micro-scale for compatibility with integrated circuit signal processing circuits (Stetter et al., 2006). Retrospectively, the commercial success of the semiconductor industry and its downscaling roadmap motivated emulation efforts in sensor industry. Mass manufacturing capability, along with reduction in size, weight, power consumption, etc. were the major advantages derived. But the present age belongs to nanotechnology, nanoelectronics and nanosensors (Bogue, 2004). Nanotechnology involves development of materials (and even complete systems) at the atomic, molecular, or macromolecular levels. The dimensional range of interest is approximately 1-500 nm.
In combination with nanotechnologies, novel functional nanomaterials, nano-biostructures and nano-biotechnologies, sensors are exerting a profound influence on medical treatment (Staufer et al., 2007). In particular, the immense applications of biosensors have made them contemporary areas of vigorous research and development all over the world. Biosensor technology is on the threshold of a new revolution which strikes at the root of device operation through the different physico-chemical phenomena taking place at nanodimensions.
This paper enquires about the effects of “nanoengineering” on biosensor performance. This research has tended to pursue different directions. The paper describes how the use of nanotechnology has resulted in biosensors with improved characteristics and also, how new biosensor ideas in the nano-dimensional range have been realized. It surveys how far nanotechnology has progressed in controlling the properties of biosensors and assesses the changes its presence has made to biosensors. Future research directions are indicated.
2 Glucose biosensor: the perennially studied device
The history of amperometric biosensors is closely related with that of blood glucose monitoring. The enzyme glucose oxidase (GOx) immobilized behind a dialysis membrane on the surface of a platinum electrode, selectively oxidizes glucose by reduction of O2 to H2O2. Either the consumption of O2, or the formation of H2O2, is measured at a platinum electrode.
The vital parameters of a sensor are its sensitivity and detection ranges. Many sensors operate through the variation of a surface parameter like surface conductivity with analyte concentration. Hence, the effective surface area of the device, i.e. the area actually interacting with the analyte, determines the sensitivity. For increasing the surface area, nanotechnology provides an easy answer. The high surface-to-volume ratio of nanoparticles has been exploited for improving the performance of sensors.
In the field of biosensors, carbon nanotubes, the quintessential element of nanotechnology, have received considerable attention because of their inert properties, conducting behaviour, and high-surface area. Particularly, their promotional ability for electron-transfer reactions with enzymes and other biomolecules has made carbon nanotubes the ideal supporting material for heterogeneous catalysts. A carbon nanotube (CNT) is a tubular form of graphite sheet in nano dimensions. A single-wall carbon nanotube (SWCNT), ranging in diameter from 0.4 to >3 nm, can be visualized as formed by the rolling of a layer of graphite, called a graphene layer (Figure 1(a)) into a seamless cylinder (Figure 1(b)). Similarly, a multiwall carbon nanotube (MWCNT), ranging in diameter from 1.4 to >100 nm, can be treated as a coaxial assembly of cylinders of SWCNTs (Figure1(c)).
On the other hand, noble metal nanoparticles have also engaged interest for amperometric biosensors. The reasons are their exceptional catalytic and electrochemical activities for many chemical reactions.
An enzyme-based mediated glucose biosensor prepared by Xie et al. (2007), using platinum-decorated multi-walled CNTs, is shown in Figure 2. They deposited platinum nanoparticles (1-5 nm in diameter) on functionalized multi-walled carbon nanotubes (MWNTs) using a decoration technique, resulting in an enzymatic Pt/MWNTs (multi-walled nanotubes) paste-based mediated sensor. Enclosed in a teflon casing, a circular platinum pad was employed as the conductor. A platinum wire electrode extruded from the top of the casing. They filled as-prepared Pt/MWNTs paste into the cavity at the bottom followed by pressing. The measured sensitivity (∼52.7 μA mM−1 cm−2) of the Pt/MWNT-based electrode is reported to be among the best results cited in the literature.
In the above sensor, platinum decoration produced a larger electroactive surface area, which was beneficial for glucose sensing. Owing to its large surface area, a Pt-decorated multi-walled CNT served as a GOx reservoir serving in uniform immobilization and high loading of enzyme. Furthermore, the large surface area led to higher selectivity for glucose because it supported a kinetically-controlled reaction over the competitive reaction from diffusion-controlled interfering electroactive species. Such species include ascorbic acid, uric acid and pacetamedophenol.
Lim et al. (2005) reported a glucose biosensor based on electrochemical co-deposition of Pd and GOx onto a Nafion-solubilized CNT film. The co-deposited Pd−GOx−Nafion CNT bioelectrode retained its biocatalytic activity and offered efficient oxidation and reduction of the enzymatically liberated H2O2. Thus, it permitted fast and sensitive glucose quantification. Xian et al. (2006) immobilized a composite mixture of Au nanoparticles (NPs) – conductive polyaniline (PANI) nanofibres with GOx and Nafion on the surface of a nanocomposite to realize a sensitive and selective biosensor for glucose. The high conductivity, large specific surface area and outstanding electroactivity of the Au NPs – conductive PANI nanocomposite made it an extraordinary matrix for enzyme immobilization and electrocatalysis.
For potentiometric biosensors, Luo et al. (2004) described an ion-sensitive field-effect transistor-based biosensor using manganese dioxide MnO2 nanoparticles. MnO2 nanoparticles acted as an oxidizing agent to react with H2O2 instead of being a catalyst accelerating the decomposition of H2O2 (as with bulk MnO2 particles). This produced an increase in pH in the sensitive membrane of the enzyme FET (ENFET). Consequently, the range of the sensor was enlarged, as shown in Table I. This table makes a comparison of the aforesaid approaches for construction of nanoparticle-based glucose biosensors.
3 Optochemical nanobiosensors
In this class of nanosensors (Buck et al., 2004), the probe (20-600 nm diameter) is prepared from up to seven ingredients (Figure 3). It is optimized for selective and reversible analyte detection, sensor stability and reproducibility. The fluorescent dye is encapsulated inside an inert matrix. After encapsulation by biologically localized embedding (hence called probe encapsulated by biologically localized embedding or PEBBLE), the probe is delivered into a cell using minimally invasive techniques, e.g. a pico-injector, a gene gun, liposomal incorporation and natural ingestion. This matrix protects the dye from interferences in the sample such as protein binding. The main categories of PEBBLE nanosensors are supported on matrices of cross-linked polyacrylamide, cross-linked poly (decylmethacrylate) and sol-gel silica. These remote nano-optodes have been used for various ions such as H+, Ca2 + , K+, Na+, Mg2 + , Zn2 + , Cu2 + , O2−and Cl− ions (Clark et al., 1998). PEBBLE sensors have mainly utilized the measurement of single fluorescence peak intensity.
In practical applications, however, these sensors have shown a complex behaviour because of signal fluctuations that did not originate from the concentration of the analyte but were attributed to scattering of light or were caused by fluctuations in the excitation source, i.e. a higher excitation power produced a greater intensity of fluorescence. To surmount this problem, ratiometric PEBBLE sensors (Gao et al., 2007), have been used. In these sensors, a fluorescent indicator dye and a fluorescent reference dye are encapsulated inside the inert matrix. The response of these sensors is based on ratios of intensities between the indicator and reference dyes. They provide more accurate information because fluorescence fluctuations that are not directly related to analyte concentration influence the indicator and reference dyes similarly, and therefore their interfering effects are nullified.
Gold nanoparticles (NPs) have been utilized as a new class of universal fluorescence quenchers to fabricate an optical biosensor for recognition and detection of specific DNA sequences (Jianrong et al., 2004). Oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other end were attached to Au nanoparticles. This hybrid bio/inorganic structure spontaneously assembled into a constrained arch-like conformation on the nanoparticle surface. Binding of target molecules led to a conformation change causing restoration of the fluorescence of the quenched fluorophore. The developed biosensor could detect single-base mutations in a homogeneous format.
4 Semiconductor quantum dots
The foremost application of quantum dots (QDs) as sensors is based on the Forster resonance energy transfer effect (FRET). Owing to this effect, the fluorescence emanating from QDs changes from an ON state to an OFF state (Smith and Nie, 2004). FRET takes place when the electronic excitation energy of a donor fluorophore is relocated to a neighbouring acceptor molecule without exchanging light between the donor and the acceptor.
Goldman et al. (2004), used QDs functionalized with antibodies to perform multiplexed fluoroimmunoassays for simultaneous detection of various toxins. This type of sensor could be used for environmental purposes for concurrently recognizing pathogens like cholera toxin or ricin in water. The FRET principle was also applied to a maltose biosensor. The sensing mechanism was the application of semiconductor QDs conjugated to a maltose binding protein covalently bound to a FRET acceptor dye. In absence of maltose, the dye occupied the protein binding sites. Energy transference from the QDs to the dyes quenched the QD fluorescence. When maltose was present, it replaced the dye leading to recovery of the fluorescence.
5 Minimally-invasive biosensors for probing cellular activity
These are fabricated by pulling a larger silica optical fibre using a micropipette puller that has been optimized for optical fibres, yielding fibres with submicron diameters (Vo-Dinh et al., 2001). These fibres typically have distal end diameters in the range between 20 and 80 nm, depending on the pulling parameters used. After pulling of the fibre, approximately 200 nm of Ag, Al, or Au was deposited on the side of the tapered fibre by a vacuum evaporation unit to prevent leakage of light. In the ensuing step of biosensor fabrication, covalent immobilization of receptors onto the fibre tip was done (Figure 4). The small size of the probe allowed easy manipulation of the nanosensor at specific locations within the cells (Figure 5).
Several microscopy techniques require incubation of cells with fluorescent dyes. Other techniques involve “fixing” the sample before viewing. This often destroys the cellular viability. Furthermore, only with optical nanosensors can excitation light be delivered to specific locations inside cells.
6 Localized surface plasmon resonance (LSPR)-based biosensors
The LSPR spectrum shows dependency on the properties of the nananoparticle (NP), i.e. its size, material and shape, and also on the external properties of the nanoparticle environment, thus making noble metal nanoparticles extremely important from the sensing point-of-view (Riu et al., 2006). LSPR spectra are highly sensitive to changes in the local refractive index that occur upon binding of molecules to the metal nanoparticles. The consequent shift in the LSPR spectrum enables the detection of molecules attached to the noble metal NPs.
Localized surface plasmon resonance (LSPR)-based sensors have been utilized for biosensing applications. Streptavidin was quantitatively detected with a subpicomolar limit of detection. For this study, triangular silver NPs with biotinylated self-assembled monolayers were used (Haes and Duyne, 2002, 2003). Triangular silver nanoparticles fabricated by nanosphere lithography (NSL) act as extremely sensitive and selective nanoscale affinity biosensors. These nanoscale biosensors based on LSPR spectroscopy function in a manner similar to their SPR counterparts. They convert small variations in refractive index near the noble metal surface into measurable wavelength shift responses (Figure 6). Biotin-streptavidin (SA) system with its extremely high-binding affinity (K a∼1013 M−1) was selected to demonstrate the features of these LSPR-based nanoscale affinity biosensors (Haes and Duyne, 2003).
The arrays of triangular silver NPs fabricated using NSL Biotynilated SAMs have also been exploited in immunoassays to detect the antibody, anti-biotin. The limit of detection was estimated to be <700 pM (Riboh et al., 2003).
7 Nanomechanical biosensors
Nanomechanical biosensors (Carrascosa et al., 2006; Gould, 2007) represent an important class of nanomechanical sensors. The microcantilevers (Figure 7) translate the molecular recognition of biomolecules into nanomechanical motion (ranging from a few nm to hundreds of nm). This motion is coupled to an optical or piezo-resistive readout system. Microcantilevers are typically constructed from silicon, silicon dioxide, silicon nitride or polymer materials. Their dimensions vary from tens to hundreds of microns in length, some tens of microns in width and hundreds of nanometers in thickness. Si, Si3N4 and SiO2 cantilevers are available commercially in different shapes and sizes, analogous to AFM cantilevers, with lengths of 10-500 μm and ultra-thin cantilevers up to 12 nm thick. When fabricated at the nanoscale (hence called nanocantilevers), the anticipated limits of detection (LODs) range from femtomole (fmol) to attomole (amol) offering the possibility of detection at the single-molecule level in real time. Nanocantilevers display large spring constants and are unsuitable for detecting adsorption-induced cantilever bending. Nonetheless, they can be employed for highly sensitive, dynamic measurements. They are expected to provide ultra-high-sensitivity mass detection.
8 Fabrication of existing biosensors by bottom-up approach
The channel region and gate dielectric of an ISFET has been fabricated by layer-by-layer self-assembly technique, Figure 8 (Liu and Cui, 2007). The advantages of this approach are technological simplicity and the ability to produce low-cost sensors.
9 Analysis and discussions
With diminishing dimensions of a particle, special properties are noticed which are clearly distinguishable from those of bulk matter. The morphology and size of CNTs make them the perfect supporting material for heterogeneous catalysts. Decoration of CNTs is interesting for modification of the properties of pristine CNTs for flexible use. Nanotubes attached with noble metal nanoparticles constitute a type of new nanotube/nanoparticle hybrid material for heterogeneous catalysis. The efficient attachment of platinum nanoparticles to the nanotube surface, as done in the glucose biosensor, enhanced the electron transfer and offered a high-surface area for enzyme loading.
The optical nanobiosensors include multicomponent nanospheres with radii as small as 10 nm, encapsulated by biologically localized embedding and enabling subcellular, real-time, chemical imaging of primary biochemical and physiological processes; functionalized silver, gold and magnetic nanoparticles. Finally, there is an important category of biosensors comprising the non-invasive nanobiosensors for cellular activity studies. Non-invasive biosensors are very useful for continuous health surveillance without any undesirable effects on the individual. An optical fibre pulled down to tips with diameters of 30-50 nm is covalently bound with antibodies for providing in vivo bioanalysis of the single cell. This fibre of a few nanometers diameter can pierce the cell wall and peep into the living cell to view the activities taking place inside. The tip of the fibre is coated with biomolecules, which interact with particular molecules of interest to impart specificity to this sensing. Good cell viability after testing with nanosensors has been obtained, which opens the possibility of non-harmful continuous monitoring in real time.
Figure 9 shows the advantages of nanotechniques for biosensors.
10 Conclusions
This paper has presented a comprehensive exposition of the nanotechnogy-impacted progress made along the different directions of biosensor research, brought out the role of nanotechnology in each case, and explored the underlying physico-chemical phenomena occurring in these sensors. One of the areas where nanotechnology has played a definitive role is in enhancing the performance of sensors. Improvement has been achieved in sensitivity, detection range and response time of sensors. Further, innovative sensor concepts based on nanotechnology have been implemented. But even more profound impact of nanotechnology has been felt in opening opportunities that were hitherto unimaginable, e.g. in realization of sensors for exploring non-invasively and in real time the functioning of a living cell. This has been possible due to the extremely small dimensions of the nanoscale sensors. Another approach involves the use of functionalized nanoparticles themselves as biosensors. Attempts have also been made to fabricate conventional biosensors using bottom-up methods.
Optical nanobiosensors are good examples of the united progress of nano- and biotechnologies. Micro- and nanotechnology, novel materials, and smaller, smarter, and more effective electronic systems will play an increasingly important role in the future. Tiny, wireless, fast, super-sensitive and non-invasive biosensors and micro-instruments can be fitted with chemical, electronic or optical detectors for science missions, particularly for use in on-the-spot analysis and robotic operations. On the whole, the advent of nanotechnology has forced researchers to look at biosensor development from a new perspective, i.e. the nano point-of-view or nano angle.
Figure 1(a) Graphene sheet; (b) SWCNT; and (c) MWCNT
Figure 2CNT-based working electrode for glucometry
Figure 3Schematic representation of a PEBBLE sensor for K+, Na+ and Cl−
Figure 4Nanoprobe fabrication steps
Figure 5Pictorial representation of a nanoprobe penetrating a cell
Figure 6Streptavidin binding to an Ag nanobiosensor fabricated by NSL on a glass substrate
Figure 7Cantilever bending caused by biomolecular interaction occurring only by specific molecular recognition
Figure 8Schematic of ISFET: silicon dioxide-indium oxide structure comprises the following layers: SiO2 nanoparticles/PDDA/PSS/In2O3 nanoparticles/PSS/PDDA
Figure 9Advantages of biosensor development in the nanotechnology age
Table INanoparticle/nanotube-based glucose biosensors
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