ABSTRACT

The emerging nanotechnology has opened novel opportunities to explore analytical applications of the fabricated nano-sized materials. Recent advances in nano-biotechnology have made it possible to realize a variety of enzyme electrodes suitable for sensing application. In coating miniaturized electrodes with biocatalysts, undoubtedly the most of the potential deposition processes suffer from the difficulty in depositing process and reproducible coatings of the active enzyme on the miniature transducer element. The promising prospects can concern to the obtaining of thin protein layers by using, i.e. electrochemical deposition, electrophoretic deposition as well as monolayer methods (Langmuir-Blodgett procedure, Layer-by-Layer—LbL). Many aspects dealing with deposition of enzyme by techniques employing electric field are considered, including surface charge of enzyme, and its migration under applied electric filed. The using of nanoscale materials (i.e. nanoparticles, nanowires, nanorods) for electrochemical biosensing has seen also explosive increase in recent years following the discovery of nanotubes. These structures offer a promise in the development of biosensing, facilitating the great improvement of the selectivity and sensitivity of the current methods. Finally, the perspectives in the further exploration of nanoscaled sensors are discussed.

Keywords:Enzymes, Immobilization, Nanobiosensors, Nanoparticles

1. Introduction

To date, there is an increasing necessity for mighty analytical tools with high sensitivity, fast response, selectivity, accuracy and low cost of production/operation. Notably, biosensors have found comprehensive adoption in the area of environmental control as well as pharmaceutics and medical diagnostics. Consequently, the main objective in biosensors design is the sufficient development of a biosurface, firmly sensitive and selective for a respective analyte, which may be able to generate measurable signals coupled to an adequate transducer.

Thus, many groups of researchers tend also to combine nanoparticles into the materials used for biosensors in order to improve the sensitivity of the system in potential sensing applications. Most recent studies show that biosensors composed with nanoparticles do take on rapid, simple, and accurate measurements, which offers exciting new opportunities for the development of biosensor capabilities. Owing to the emerging roles that nanoparticles are playing in the improvement of biosensors in recent years, it is necessary and meaningful for us to investigate the researches of nanoparticle-based biosensors from the point of view of management of technology. As a vital part of management of technology, grasping the latest development of technology and identifying the emerging characters can help us get competitive advantages in the future.

Several kinds of nanoparticles, including metal nanoparticles, oxide nanoparticles, semiconductor nanoparticles, and even nanodimensional conducting polymers have been used in biosensing systems. Owing to these unique properties, different kinds of nanoparticles always play different roles in different sensing systems. Generally, metal nanoparticles are always used as elements of “electronic wires”. Oxide nanoparticles are often applied to immobilize biomolecules, while semiconductor nanoparticles are often used as labels or tracers [1] .

The development of high performance and reliable miniaturized enzyme electrodes is a crucial objective worth pursuing in the nanosized biotechnology area. The miniaturization bids numerous advantages [2] . Miniaturized enzyme electrodes are useful in analysis of small sample volumes, hence practical if only small amounts of biological fluids are provided or, waste saving if larger quantities are available. These small systems can be also integrated into new technologies like microarray sensor or microfluidic systems. When used in vivo, miniaturized systems create less damage of the tissues and hence quick healing.

Certainly, there are several methods (i.e. Langmuir-Blodgett techniques, LbL) with which enzymes can be deposited including but not limited to entrapment and crosslinking [3] . But an important problem in the application of enzymatic proteins for the development of miniaturized electrodes is the difficulty in depositing uniform and reproducible layer coatings of enzymes on the transducer [4] .

Electrochemical and electrophoretic deposition are offered as techniques, which can employ electric field to produce apparently uniform and reproducible layer coatings of biocatalysts over very small areas. Electrochemical deposition is known for several decades, but by the contrast electrophoretic deposition is rather recent technique [5] . However, further development work needs to be done according to optimize the parameters for a broader use of especially in the fabrication of miniaturized enzyme systems [4] .

2. Immobilization of Enzymes in Miniaturized Systems

2.1. Electrochemical Deposition

A study of literature indicates that electrochemical deposition is one of the most techniques employed for enzyme immobilization, because of simple, cost effective apparatus. Electrochemical deposition can be employed to drive enzymes alone to deposit on the support, as well as with other components including i.e. collagen [6] , noble metal salts (Pt, Pd) [7] , monomers such as pyrrole, 1, 3-diaminobenzene [8] , some redox mediators (i.e. Prussian blue) or redox centers [9] , nanomaterials like carbon nanotubes and metal nanoparticles [10] . The final goal of all these efforts is to fabricate enzyme electrodes with appropriate characteristics in terms of preserved activity, enhanced kinetics and stability to fit with the specific application.

Generally, the activity of enzyme electrode prepared by electrochemical deposition depends primarily on thickness of the enzyme layers [11] .

The latter yields deposition of thin enzyme coatings because only enzymes present nearby vicinity of the electrode surface precipitate. Matsumoto et al. [12] observed that only few tens of nanometer are produced. The thickness of the enzyme coating can be increased to reach much thicker layers if i.e. a surfactant is added to enzyme solution prior deposition.

According to kinetics, it was observed, that enzymes deposited under electrochemical deposition may or may not keep similar kinetics as nativeproteins [13] [14] . This fact depends on the environment of the deposited enzyme coating and presence of other components. In case of stability, enzyme electrodes produced by electrochemical technique have usually moderate stabilities [4] .

2.2. Electrophoretic Deposition

Electrophoretic deposition is carried on from low conductivity aqueous solutions/suspensions (Figure 1). The technique requires high strength electric fields, which can reach several hundreds of volts to move the charged biocatalysts from bulk of the solution to the electrode. Both parameters of elevated zeta potential and high strength electric field yield significant migration of enzymatic proteins under electrophoretic deposition [4] . As a result, more enzymes reach the surface of the electrode and precipitate to form thick-deposited coatings [15] . It is weighty, that when high strength electric fields are employed in electrophoretic deposition, continuous direct current can no longer be utilized because it can induce heat and extensive water electrolysis [4] . This situation causes changes in temperature and pH in the environment of electrodes which maylowered the activity of the protein [16] . To avoid the problem it is possible to apply pulsed direct current and alternating current [4] .

Alternating current is no fixed anode or cathode but the polarization of each electrode changes continuously between the positive and negative signs. Due to the fact, that a negatively charged enzyme is subjected to asymmetrical alternating current signal, it should only oscillate in one location because the migration achieved during the first half cycle when one of electrodes is positively charged should be neutralized during the second half process when the other electrode becomes positively charged [4] . According to that, the migration of charged enzyme under symmetrical signals is zero. In results, only thin enzyme coatings can be deposited [5] [17] . Whereas, under unsymmetrical alternating current field is applied to negatively charged protein, large amount of enzymes accumulate nearby the surface of electrode and the enzymes precipitate to yield thick deposited layers. Furthermore, because alternating current fields generate the minimum of water electrolysis as well as heat, the maximum enzymes activity could be preserved after deposition [5] [17] . There is found a several enzymes (i.e. glucose oxidase, peroxidase), which have been successfully immobilized by this method [4] [5] [17] .

2.3. Thin Layer Methods

Adsorption of organic molecules on solid conducting supports to produce thin nanostructured films are one of the most employed architectures and represents an important approach in the field of nano-manipulation. Langmuir-Blodgett (LB) technique promotes a high control of the physical and chemical properties of nanostructured organic films and plays an important role in the production of miniaturized devices applicable as platforms for enzyme immobilization [18] .

Other pathways to prepare platforms based on nanomaterials, aiming the fabrication of electrochemical biosensors are dispersion in solvents, adsorption (e.g. LbL), formation of covalent bonding.

As well, the utilization of hybrid organic/inorganic thin films can, in a simple manner, be employed in solid conductor electrodes. The possibility to incorporate hybrids containing nanostructured materials for enhance electrochemical properties makes these techniques much attractive in the field of bionanoelectrochemistry [19] [20] .

The aim is to preserve the native enzyme molecular conformation and to arrange it in a suitable position for the molecular recognizing of an external molecule of a solution put on contact with the Langmuir-Blodgett device. As defended in a review by Girard-Egrot [21] , the successful incorporation of enzymes on a preformed Langmuir monolayer depends strongly on the methodology employed. The most one commonly used is the adsorption of the enzyme from the subphase, avoiding direct adsorption of the macromolecule present at the water surface. This strategy was used to produce electrochemical sensors containing i.e. phytic acid [22] , horseradish peroxidase [23] , hemoblogin [24] , and urease [25] , tyrosinase [26] , to detect a diversity of substances such as including phytic acid, hydrogen peroxide, glucose, choline, urea, and phenols.

In these types of sensors the sensor sensitization can be achieved by i.e. an amphiphilic heterocyclic semiconducting structures admixed into the film [27] or other, more sophisticated architectures have been developed in order to enhance the performance of LB-based enzyme electrochemical sensors. For instance, Sun et al. [28] used pyriduylthio-modified carbon nanotubes as Langmuir-Blodgett films to support hydrogenase added in a subsequent adsorption from solution.

Alternatively, a modern concept of self-assembly was introduced by Decher and co-workers [29] [30] at 90 decade as a low-cost and simple method to obtain nanostructured thin films under controlled conditions (pH, temperature, polyelectrolyte concentration, ionic strength, etc.). For this purpose, a large variety of materials for electrochemical sensing and biosensing can be obtained [31] -[33] . Basically, the processes of film fabrication by LbL technique (Figure 2) is governed by the adsorption of organic polyelectrolytes with opposite charges present on their molecular structure, in such a way that film roughness, thickness, porosity and morphology can be controlled at molecular level [34] . Important advantage in the use of LbL technique to construct biosensors is the possibility to incorporate organic/inorganic composite materials that contributes for the maximization of the biodevices electrochemical signal [35] . Also, it is important to emphasize that most hybrids based on nanomaterials has been utilized to detect electrochemical signal from biochemical reactions.

Proteins have also been used in LbL method to construct alternate multilayer of ceramic nanotubes (halloysite), spherical particles leading to an array of new ordered nanoparticles-tubules, which were applied to load co-enzymes (NAD) for the development of enzymatic nanoreactors. Decher and co-workers [36] also reported the use of protein/polyelectrolyte hybrid films via specific recognition. One of the main challenges is to maintain the integrity of the native protein structure to promote their utilization for technological applications.

Ram and co-workers [37] reported the utilization of LbL technique to produce nanostructured films of poly(ethylene imine) and poly(sodium polystyrene sulfonate), cholesterol oxidase and cholesterol esterase. The strong stability of multilayer films was also evaluated and contributes directly for the electrochemical properties of the film warranting the glucose oxidase immobilized on solid conductor supports remained active on the electrode surface.

3. Summary and Future Trends

Electrochemical nanobiosensors offer without doubts an important step toward development of selective, down to few target molecules sensitive biorecognition device for medical and security applications. In their case, very high amplification of signal could be reached, i.e. using high diameter carbon nanotubes filled with nanoparticles and their following electrochemical stripping.

The utilization of nano-manipulation techniques has also become an interesting approach to fabricate electrochemical devices with high specificity and molecular order. Moreover, the sensitivity and overall behavior of biosensors has grown rapidly as an outcome of incorporating different nanomaterials in their construction.

Electrochemical nanobiosensors consisting from single carbon nanotube are clear examples of future path of biosensor development. These strategies waits for exploration. There is high expectation that such devices will develop toward reliable point-of-care diagnostics of cancer and other diseases, and as tools for intra-operation pathological testing, proteomics and systems biology.

Acknowledgements

Authors are gratefully acknowledged for financial support of NCN-Grant no. 2012/05/B/ST5/00749 and Wrocław University of Technology.

References

NOTES

^*Corresponding author.