Detecting water contaminates using a CNT-based biosensor

Transcription

1 Nanotechnology and Nanosensors Final Project Detecting water contaminates using a CNT-based biosensor 1 Table of Contents Abstract... 1 Introduction Carbon Nano-Tubes Literature Review Project's Goal... 3 Method of Fabrication Characterization and Applications Conclusions... 9 References Abstract Enzyme-Coated Coliform Sensitive Carbon Nano-tube Biosensor is proposed as alternative cheap, on site and fast technique for water monitoring. The highly selective sensor based on interconnected array of carbon nano tubes coated with coliform-sensitive bio-engineered enzyme. This paper includes literature review, application and fabrication of the proposed bio-sensor. 3 Introduction In the last decade contamination of water reservoirs has become an environmental hazard threatening public health. The main concerns are microbiological contaminants such as bacteria viruses, protozoa and parasites that can reach water reservoir and subsequently, hundreds of thousands households connected to the contaminated water source. The high 1

2 risk and the biological hazard of contamination require a constant monitoring of drinking water; the inspection must provide fast accurate and reliable results [1]. Most disease-causing organisms (Pathogens) that can contribute to water contamination originated in the faces of warm-blooded animals and humans. There are many different types of pathogens, each requires an individual inspection. Most of which are time consuming, expansive and it is almost impossible to inspect the whole verity. Instead of looking for pathogens, a single coliform bacteria examination can be made, its presence in drinking water indicates the presence of pathogens, which means that a further investigation is required [2]. Until recently, the most common way of testing water contaminants was taking routine water samples and testing them in a lab for pathogens. As mentioned, this technique is: slow ineffective, expensive and most important, cannot provide a real time results, thus doesn t always prevent biological hazard [3]. 3.1 The sense of Taste Taste is one of the five traditional senses. Humans perceive taste through sensory organs called taste buds which concentrated on the top of the tongue. Each taste bud contains 50 to 100 taste receptor cells. The taste sensation produced when a substance in the mouth reacts chemically with receptors of taste buds. Taste buds are able to differentiate among different tastes through detecting interaction with different molecules or ions [4]. Analogous to the taste sense (taste buds), the bio-sensor proposed in this work based on an array of carbon nano-tubes coated with coliform-sensitive bio-engineered enzyme that detect coliform bacteria by a selective chemical reaction. 4 Carbon Nano-Tubes Literature Review Carbon nano-tubes are composed of carbon atoms linked in 3D shapes, with each carbon atom covalently bonded to three other carbon atoms. CNTs have diameters as small as 1 nm and lengths up to several centimeters. Though CNTs are strong, they are not brittle. They can be bent, and when released, they will spring back to their original shape. There are two main types of CNT: (1) The single-walled CNTs (SWCNTs) are sp 2 hybridized carbon in a hexagonal honeycomb structure that is rolled into hollow tube morphology and (2) A multi- 2

3 walled CNT (MWCNTs), a multiple concentric tubes encircling one another. The physical and catalytic properties make CNTs ideal for use in sensors. Most notably, CNTs display high electrical conductivity, chemical stability, and mechanical strength. CNTs offer unique advantages including enhanced electronic properties, a large edge plane/basal plane ratio, and rapid electrode kinetics. Therefore, CNT-based sensors generally have higher sensitivities, lower limits of detection, and faster electron transfer kinetics than traditional carbon electrodes. CNTs are commonly incorporated onto electrodes by directly growing CNTs on the electrode surface, adsorbing them on existing electrodes, imbedding them in polymer coatings, or combining CNTs and a binder to make a paste electrode. The common electrochemical methods used with CNT sensors include voltammetry, amperometry, electrical impedance spectroscopy, and potentiometry [5], [6]. 4.1 Enzyme Based CNT Sensors Enzymes are often incorporated into biosensors to selectively detect an analyte and create an electroactive product after the enzymatic reaction. The reaction can be followed by either direct measurement the significant electrical resistance change that occurs when biomolecules are absorbed on its surface, or by indirect concentrations measuring of enzyme- analyte reaction byproducts. Most of the research on enzyme electrodes has focused on glucose because of the rising number of diabetes patients worldwide. Glucose oxidase (GOx) is the most common enzyme used in enzyme electrodes. It catalyzes the oxidation of glucose according to the following reaction: The reaction can be followed directly, by measuring electrons transferred to GOx or indirectly by measuring hydrogen peroxide concentrations [5]. 5 Project's Goal The work will describe an innovative, efficient and cheap new method of drinking water monitoring that can be easily implemented and can provide reliable, real-time data on the amount (concentration) of coliforms within it. 3

4 6 Method of Fabrication [7, 8] Biosensing can be performed using a broad spectrum of techniques. Methods like mass spectrometry and biolabeled fluorescence usually require several steps before the biomolecule is detected. Electronic and electrochemical detection techniques, in comparison to classical methods, are advantageous in this aspect. Carbon nanotubes (CNTs) have high electrochemical and electrical properties, which make them ideal for use as both electrodes and transducer components in biosensors. Single wall Carbon nanotubes (SWCNTs) are known to be extremely sensitive to their surrounding environment. Chemiresistors and chemically sensitive field-effect transistors (FETs) based upon pristine or functionalized CNTs have been shown to be capable of detecting biomolecules. In the following, the fabrication of a biosensor based on CNT attached enzyme will be described. 6.1 CNT electrodes Common electrochemical biosensors are based on either glassy carbon electrodes (GCE) or metal electrodes (e.g. Au, Pt or Cu) for amperometric or voltammetric analyte detection. These electrodes are known to have poor sensitivity and stability, low reproducibility, large response times and a high overpotential for electron transfer reactions. The use of carbon nanotubes as nanoelectrodes in sensor applications can overcome most of these disadvantages due to the CNT ability to undergo fast electron transfer and the resistance of CNT-modified electrodes to surface fouling. 6.2 CNT electrodes enhanced by immobilized enzymes Enhancing the selectivity and sensitivity of CNT-modified electrodes further can be achieved through the immobilization of enzymes. In these electrodes, CNTs act as transducers, communicating the signal from the enzymes to the substrate. Various techniques based on noncovalent or covalent bonding of the enzyme have been developed. The noncovalent approach has very little effect on the activity of the enzyme and can be categorized into adsorption, entrapment and encapsulation techniques. In adsorption, the enzyme is attached onto the CNT with the help of a binder or an ion-exchange resin. Alternatively, the enzyme can be immobilized using surface groups of self-assembled monolayers or Langmuir Blodgett (LB) films. In the entrapment method, a polymerization reaction is carried out during which the enzyme is incorporated into the resulting polymer matrix. The encapsulation method makes use of hydrogels or sol-gels to immobilize the enzyme. 4

5 6.3 Enzyme adsorption In Enzyme adsorption, the CNT electrode is prepared by evaporating or casting the CNTs onto a GCE, and then dripping the desired enzyme on top of the electrode using a Nafion solution and allowing it to evaporate. For example through adsorption, a tyrosinase-based amperometric sensor was developed for the detection of phenolic compounds. The immobilization of enzymes via adsorption has several disadvantages, such as the low quantity of adsorbed enzyme, leaching of the enzyme, etc. Some of these limitations can be overcome by adsorbing enzymes onto CNT-modified GCEs decorated with metallic nanoparticles; this solution has been demonstrated for a Pt-NP/CNT/GC electrode modified with glucose oxidase (GOx). A different method for enzyme adsorption involves a layer-by-layer technique in which alternately charged layers of polyelectrolyte and enzyme are deposited on the electrode surface. By using the adsorption method a random distribution of the enzymes on the electrode surface is normally received. Direct attachment of the enzymes to the CNT framework can be obtained through the covalent immobilization approach. This can also enable direct electron transfer to the active enzyme. Covalent attachment has been used to immobilize GOx onto an array of SWCNTs grown on a gold substrate, which lead to direct electron transfer between the enzyme molecules and the SWCNTs. First a covalent bond between the molecule flavin adenine dinucleotide (FAD) and the SWCNT is made and then the GOx enzyme is connected to the immobilized FAD. In Figure 1 a fabrication scheme of such an electrode can be seen. Figure 1 Schematic fabrication scheme of a SWCNT-based GOx electrode 6.4 Nanoscale CNT electrode Utilizing the miniature size of a SWCNT can be done by connecting the SWCNT to a field- 5

6 effect transistor (FET) configuration. This further improves the properties of the electrode used. Usually an array of SWCNT is used and through chemical functionalization of the SWCNT sensitivity and selectivity towards a specific analyte is achieved. Once more the example of Glucose sensors is shown in Figure 2. The sensor is achieved by immobilizing the GOx enzyme onto the SWCNTs with the aid of a linker molecule, which binds at one end to the SWCNT through van der Waals interactions. Figure 2 Schematic depiction of a semiconducting SWCNT with GOx immobilized on its surface. The enzyme is attached with the help of a linker molecule 7 Characterization and Applications [7, 8] The suggested biosensor is made of interconnected CNT array with coliform-sensitive bioengineered enzyme, as was described earlier. The system is based upon the enzyme, which determines the application of the nano-sensor since it leads to change in signal in the system, and the CNT are the electrodes. The type of enzyme should be determined according to usage and application of the purposed nanosensor. 7.1 Electrochemical properties and detection of biomolecules The most important property for the enzyme is its reaction efficiency to the specific bacteria. The structure of this nano-sensor, where the enzyme is immobilized to the electrode, makes the device very sensitive and selective to a specific molecule, thus increasing its efficiency. CNTs were chosen to act as the electrodes, thus communicating the changes from the enzyme reaction to the substrate. The use of CNTs is very convenient to that purpose since it allow surface modification of the CNTs endings via an attachment of almost any desired molecule. CNT exhibits additional extraordinary properties which paved a way to new and improved sensing devices. 6

7 Electrochemical sensors for the detection of biomolecules contaminates in a solution are highly attractive due to their simplicity and the relative ease of calibration. These sensors can be based on potentiometry, amperometry, voltammetry, coulometry, AC conductivity or capacitance measurements. Most CNT-based electrochemical biosensors perform the detection of biomolecules amperometrically. 7.2 Wide range of sensitivity When analyzing a biosensor, a number of aspects are to be considered. The first important sensor parameter is the sensor sensitivity range. Due to the high sensitivity of carbon nanotubes, it can detect low concentrations, as well as high concentrations of the wanted molecule. For example, a glucose sensor used to detect diabetes needs to be sensitive enough in order to detect glucose in the range of a few μmol/l to 15 mmol/l. This is due to the fact that a level of less than 6 mmol/l of glucose in blood is considered to be normal, while a level of 7 mmol/l or higher suggests diabetes. In environmental studies, sensors for the detection of organophosphorus compounds must detect the compounds in the ppb range. 7.3 Electrical sensitivity The need for lower sensitivities would require enhanced electronics in order to detect the low currents, which would result in an increase to the cost of the sensor. One of the significant properties of the CNT is that the CNT resistance can change significantly when molecules adsorbs on it, making it a very high sensitivity device. 7.4 Biological properties It has been shown that pathogens can be grown on carbon-based materials. This lie in contradiction to the cytotoxic properties of pristine CNT which prohibits the growth of pathogens on its surface and could potentially contribute to self-cleaning properties of CNT surfaces. Pristine CNTs exhibited antimicrobial characteristics over a wide range of microorganisms including: (a) bacteria e.g. Mi- crococcus lysodeikticus, Streptococcus mutans, E. coli, Salmonella and bacteria endo- spores (Krishna et al., 2005). Figure 3 show SEM images of E. coli bacteria species incubated with single nano-tubes: 7

8 Figure 3 (a) E. coli cells exposed to SWNT's and (b) Salmonella cells exposed to SWNT COOH. Detecting total coliforms contaminates in water in a quick fashion can be achieved by using a CNT based sensor. The sensor incorporates an array of CNTs with an immobilized enzyme to bind the coliforms. Each and any bacteria that are trapped will cause a change to the substrate electrical resistance. By mapping and calibrating the resistance change caused by each of the Bactria belonging to the total coliform group, a feedback system connected to the sensor could count the number of sites occupied and calculate the relative ratio between the occupant bacteria according to the change in resistance to the CNT. Figure 4 show a schematic representation of the proposed biosensor system. A CNT attached to a substrate is terminated by an immobilized enzyme, which catalyze the coliform reaction thus binding it to the CNT. The biosensor would be composed of an array of CNTs. The array would have a known number of reaction sites, making the calculation of the ratio between the coliforms possible. Figure 4 - a schematic representation of the proposed biosensor system 8

9 Storage costs are to be considered also, since many enzyme-based biosensors contain biodegradable components, they need to be stored under special conditions to avoid degradation of the active material. Wrong storage can degrade the sensor rapidly thus rendering it useless. 8 Conclusions In this paper a new innovative approach regarding water monitoring was presented using an array of carbon nano-tubes with immobilized enzyme. The high efficiency of this method is mainly due to the selective and unique properties of CNT, making it a real-time, sensitive and accurate sensing device. This work is focused on water contamination but there is no doubt this method can be extended to other detection types like blood sugar level or cholesterol tests and many more. 9 References Jacobs, Christopher B.; Peairs, M.Jennifer; Venton,B.Jill "Review: Carbon nanotube based electrochemical sensors for biomolecules" Analytica Chimica Acta 2010, Vol. 662(2), Wang J. "Carbon-Nanotube Based Electrochemical Biosensors: A Review", Department of Chemistry and Biochemistry, New Mexico State University Las Cruces, NM 88003, USA 7. Balasubramanian K, Burghard M. "Biosensors based on carbon nanotubes", Anal Bioanal Chem. 2006, Vol. 385(3), Upadhyayula VK, Deng S, Mitchell MC, Smith GB. "Application of carbon nanotube technology for removal of contaminants in drinking water: a review" Sci Total Environ. 2009, Vol. 408(1),