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					BIOLOGICAL SENSORS
ABSTRACT
******************* Aligned multi-wall carbon nanotubes (MWNT) grown on platinum substrate are used for the development of an amperometric biosensor. The opening and functionalization by oxidation of the nanotube array allows for the efficient immobilization of the model enzyme, glucose oxidase. The carboxylated open-ends of nanotubes are used for the immobilization of the enzymes, while the platinum substrate provides the direct transduction platform for signal monitoring. Glucose in blood encounters the enzyme quickly reacts to modulate the electronic structure and optical characteristics of the nanotube, ―The more glucose that is present, the brighter the nanotube will fluoresce‖. It is also shown that carbon nanotubes can play a dual role, both as immobilization matrices and as mediators, allowing for the development of a third generation of biosensor systems, with good overall analytical characteristics.

Keywords Carbon nanotube · Biosensor · Array · Glucose oxidase

INTRODUCTION : ****************
Nanotechnology as a force that will change our world. But to become reality, this science of the small will require an affordable, ready source of its primary raw material, carbon. Petroleum is the obvious choice, but as its price goes up, corn-based ethanol could turn out to be the preferred source of raw materials for our nano future. It is estimated that a nanotechnology-based industry will be worth more than $1 trillion by 2015. Experts forecast that nanomaterials could soon give us tires that never go flat, drill bits that never get dull and thousands of other useful products that will benefit from super-strong, yet affordable materials. The basic building blocks of nanotechnology, called carbon nanotubes, can yield ultrastrong fibers that are 5,000 times smaller than a human hair. Carbon nanotubes are of special interest due to their unique electronic, metallic, and structural characteristics.These nano-materials are very promising for the development of novel technological applications, such as batteries ,tips for scanning probe microscopy , electrochemical actuators , sensors etc. Among the anticipated applications of carbon nanotubes is their use as components in biological devices. It has been shown that small proteins can be entrapped into the inner channel of opened nanotubes by simple adsorption. Attachment of small proteins on the outer surface of carbon nanotubes has also been achieved,either by hydrophobic and electrostatic interactions, via covalent bonding or by functionalization of the nanotube sides by polymer coating . However, application of these practices has been limited to the development of microelectrodes to promote bio-electrochemical reactions . One of the key issues in biosensor design is the establishment of a fast electron-transfer between the enzyme active site and the electrochemical transducer. Small surface area leads to constraints concerning the minimum enzyme loading, that can be repeatably immobilized for analytical applications . The structure-dependent metallic character of carbon nanotubes should allow them to promote electron-transfer reactions at low overpotentials. This characteristic, along with their high surface area, provides the ground for unique biochemical sensing systems. The latest advances in production of well-controlled aligned carbon nanotube arrays have shown the way for incorporation of nanotechnology in BIOSENSOR TECHNOLOGY .

Materials and methods :
The nanotube array used was a multi-wall carbon nanotube array,with closed nanotubes 15–20 microns long and 150 nm internal diameter .Multi-wall nanotube arrays, grown by the CVD method on platinum substrate. Cyclic voltammetry was used to monitor the electrochemical characteristics of the nanotubes, proving the metallic character of the array and the chemical stability upon treatment at high oxidative potentials. The original nanotube array was opened using two previously reported etching procedures , namely acid oxidation (sensor 1) and air oxidation (sensor 2) with small modifications. In short, the chemical etching was achieved using a mixture of concentrated H2SO4 and HNO3 acids (3:1, 98% and 65% respectively) for 8 h at 40 °C. Alternatively, air oxidation was achieved at 600°C for 5 min under air flow. Upon completion of the etching procedure the array was washed with deionized water and dried at 100°C overnight. Scanning electron microscopy was used to determine the effect of these oxidation procedures on the arrays. SEM images were obtained with a JEOL-JSM 840 scanning microscope at 10 KV. Subsequently, enzyme immobilization was achieved by incubation for 3 h of both freshly oxidized sensors in an enzymatic solution (1500 to 2500 U/mL).

Results and discussion :
Figure 1 shows the schematic diagram of the carbon nanotube array biosensor presented in this work. The immobilization of the enzyme into the nanotubes would allow for the mediated direct electron transfer to the platinum transducer. FIGURE 1

The two array systems used in this work were either acid treated, or air treated. Acid treatment purified the array by removing any amorphous carbon material or other impurities that occurred during the production procedure. It also reduced the length of the nanotubes by approximately 50% (Fig. 2B), compared with the original array (Fig. 2A). On the contrary, air-oxidation caused the peeling of the outer graphitic layers from the nanotubes producing thinner nanotubes (Fig. 2C). FIGURE 2

After immobilization of the enzyme, the calibration curves to glucose were recorded. Sensor 1, which was constructed using the acid oxidized array (Fig. 3A), had a linear range of response from 0.25 to 2.5 mM glucose (r=0.9986) and sensitivity of 93.9±0.4 A mM–1 cm–2. The limit of detection, based on a signalto-noise ratio of 3, was 0.19 mM. These values are among the best reported for glucose biosensors and make the application of the system in micro-analysis feasible. One very important feature was that even after incubation of the sensor in buffer solution for 24 h (at 4 °C) there still was a significant remaining sensitivity (4.53±0.01 A mM–1 cm–2) (Fig. 3A, inset). On the other hand, sensor 2, which was based on the air oxidized array (Fig. 3B), showed completely different analytical characteristics. The linear range of response of the air-treated sensor was from 0.05 to 0.5 mM glucose (r=0.9707) and the sensitivity was 15.6± 0.5 A mM–1 cm–2, a value much lower than the one obtained from the acid-treated sensor 1, but yet high if compared note that sensor 2 lost all its activity after the first day of operation, suggesting a weaker enzyme immobilization,

or lower enzyme stabilization. In order to exclude any possible adsorption of the enzyme on the platinum substrate, a blank experiment was undertaken.

Fig. 3A,B Initial calibration curves in phosphate buffer (10 mM, pH 7.5) at +800 mV to glucose of: A sensor 1 on the first and the second day (inset), and B sensor 2 in the presence and absence inset) of oxygen

A platinum foil was immersed in an enzyme solution for 20 h and the possible response to the substrate was examined. No signal was recorded, thus the enzyme must have been indeed immobilized on the nanotubes. Based on the abovementioned results, the procedure of chemical etching is more efficient in opening carbon nanotubes and allowing the entrance of the enzyme at the inner channel. What seems most probable is that upon oxidation of the array, the introduced carboxylic groups at the open-ends, provide a stabilizing hydrophilic environment

that allows for the adsorption and insertion of the enzyme into the cavity of the nanotubes, while preserving its functionality. Even if blocking of the entry-ports could occur by the introduced functional groups , this is not significant in this case due to the large internal diameter of the nanotubes (150 nm) compared to the enzyme’s hydrated diameter (approximately 7 nm). The possible electrostatic interaction of the enzyme with the outer wall of the nanotubes is weak , and cannot account for the observed extended lifetime of the sensor. The remaining sensitivity of sensor 1 on the second day shows that a significant amount of the adsorbed enzyme could be in the inner walls of the nanotubes. Fig.2A–C SEM images of the Pt-aligned carbon nanotube array A in original state, B after being chemically etched with a mixture of concentrated H2SO4 and HNO3 acids (3:1, 98% and 65% respectively) for 8 h at 40 °C, washed with deionized water and dried at 100 °C overnight, and C after air oxidation at 600 °C for 5 min under air flow. SEM images were obtained with a JEOL-JSM 840 scanning microscope at 10KV. Bar is 1 m Since carbon nanoparticles have been successfully used as mediators in enzyme biosensors, sensor 2 was additionally evaluated under anaerobic conditions (argon flow) to examine the mediation capabilities of the nanomaterial (Fig. 3B, inset). It has been reported that air oxidation does not lead to significantly increased work functions of carbon nanotubes, as opposed to acid oxidation . Higher work functions would impede reversible electron transfer between the co-factor of the enzyme and the believed mediator. For this reason, only the mediation capabilities of the air oxidized nanotubes (sensor 2) were evaluated. In this case, the observed sensitivity to glucose was 2.003±0.001 A mM–1 cm–2, indicating that the absence of oxygen did not obstruct the enzyme’s catalytic reaction or the operation of the sensor. This observation further enhances the unique character and usefulness of these nano-materials.

Nanotech fibers from ethanol:
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PRODUCTION :
Tiny twisted ropes made by winding freshly made carbon nanotubes onto spinning rods as they came out of a furnace. Mixing ethanol — the carbon source — with a catalyst called ferrocene and another chemical called thiophene that helps the threads to assemble. The mixture was squirted into a hot furnace in a jet of hydrogen gas. Nanotubes formed into a tangled, cotton candy-

like mass and were then wound onto a spindle to form strands.So far, the fibers aren't any stronger than typical textile fibers. But there’s still plenty of room for improving the process to make stronger fibers by finding ways to make the nanotubes line up better. In Kevlar, for example, it’s the good alignment of molecules that generates the high strength. The nano fiber’s strength should be boosted so that it rivals that of standard carbon fibers. And the process is relatively cheap, because it uses an ethanol feedstock that can be made from renewable resources.

IMPLEMENTATION:
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Selective coatings create biological sensors from carbon nanotubes :
Protein-encapsulated single-walled carbon nanotubes that alter their fluorescence in the presence of specific biomolecules could generate many new types of implantable biological sensors . The researchers showed the viability of their technique by creating a near-infrared nanoscale sensor that detects glucose. The sensor could be inserted into tissue, excited with a laser pointer, and provide real-time, continuous monitoring of blood glucose level. Carbon nanotubes naturally fluoresce in the near-infrared region of the spectrum where human tissue and biological fluids are particularly transparent. The researchers have developed molecular sheaths around the nanotube that respond to a particular chemical and modulate the nanotube's optical properties.

BIOLOGICAL SENSORS:
To make biological sensors, researchers associated assembling a monolayer of the enzyme glucose oxidase on the surface of nanotubes suspended in water. The enzyme not only prevents the nanotubes from sticking together into useless clumps, it also acts as a selective site where glucose will bind and generate hydrogen peroxide. Next, the researchers functionalize the surface with ferricyanide, an ion that is sensitive to hydrogen peroxide. The ion attaches to the

surface through the porous monolayer. When present, hydrogen peroxide will form a complex with the ion, which changes the electron density of the nanotube and consequently its optical properties.

GLUCOSE DETECTION :
Researchers working to make sensors that indicate a given chemical or biological agent after sensing only a few or even a single molecule of that substance are turning to the minuscule tools of nanotechnology. Researchers are using carbon nanotubes to sense single molecules, and are tapping the way carbon nanotubes give off near-infrared light in order to read what the sensors have detected.

Miniscule monitor: Nanotubes that act as chemical sensors have been developed that when loaded into tiny glass tubes, shown here on a fingertip, can track blood sugar and other biological changes in the body. Credit: Michael S. Strano.

When glucose encounters the enzyme, hydrogen peroxide is produced, which quickly reacts with the ferricyanide to modulate the electronic structure and optical characteristics of the nanotube. "The more glucose that is present, the brighter the nanotube will fluoresce."

ANALYSIS:
To prove the practicality of the technique, researchers loaded some of the sensors into a porous capillary that confined the nanotubes but allowed glucose to enter. When inserted into human tissue, the fluorescent emission of the sensor corresponded to the local glucose concentration.

The enzyme glucose oxidase, like most other enzymes, acts as an insulator when binding to a conductive surface such as an electrode. However, when the gold nanoparticle is implanted into the enzyme, the gold acts as an electrical "plug," facilitating the flow of electrons to the attached electrode and the oxidation of the glucose by the enzyme. The magnitude of the current reveals the extent of glucose in the system.

ADVANTAGE:
The advantage of the near-infrared signaling to and from such a capillary device is its potential for implantation into thick tissue or whole blood media, where the signal may penetrate up to several centimeters.And, because nanotubes won't degrade like organic molecules that fluoresce, these nanoparticle optical sensors would be suitable for long-term monitoring applications. One important aspect of the new surface chemistry, is that no bonds are broken on the nanotube. "This allows us to shuttle electrons in and out without damaging the nanotube itself." Another important aspect is that the technique can be extended to many other chemical systems. ―We have shown that it is possible to tailor the surface to make it selective to a particular analyte‖.There are whole classes of analytes that can be detected in this manner.

CONCLUSION: **************
In conclusion, we report here the use of carbon nanotubes as immobilization matrix for the development of an amperometric biosensor. The opening and functionalization of large nanotube arrays allowed for the efficient immobilization of the enzyme, while the platinum substrate served as the direct transduction platform for signal monitoring. The carbon nanotube-based biosensor showed good overall analytical characteristics. It was also shown that carbon nanotubes can play a dual role,both as immobilization matrices and as mediators, allowing for the development of a third generation of biosensor systems. The researchers proof-of-concept system detected glucose levels in a sample of blood. The method could be used practically in 5 researchers.

to 10 years, according to the

See no evil?
Debates about nanotechnology’s (and our) possibly apocalyptic future are redundant if we fail to ask and answer the most basic questions about the impact of nanomaterials on the environment today. As was the case with biotechnology, the products of a new technology have been rushed to market without proper foresight and assessment and with little public discussion. The provocative questions raised by a handful of researchers working in the field highlight the need for governments and civil society to come to grips with this powerful new technology. One thing is certain: size matters.

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RESEARCH BY: Mr.STRANO, POSTDOCTORAL RESEARCH ASSOCIATE, UNIVERSITY OF LLLINOIS AT URBANA-CHAMPAIGN, SPACE DAILY .

AUTHORS: K.R.VIJAYA V.B.SRIDEVI C0LLEGE: C.ABDUL HAKEEM COLLEGE OF ENGINEERING AND TECHNOLOGY, MELVISHARAM, VELLORE DISTRICT, 632509. PHONE : 04172-267387 EMAIL ID:vb_sridevi@yahoo.co.in


				
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