Liquid Gated Biosensor - Research Paper Example

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Liquid gated biosensor using reduced graphene oxide and silicon dioxide substrate as a detection platform for cytochrome c (protein) using the liquid gated method Name: Name: Tutor: Date: Objectives 2 The main objective of the study include; 3 Characteristics required in a biosensor 4 Biosensors are used in food analysis 4 Carbon based biosensor 5 Electrochemical biosensors 6 Surface Plasmon resonance biosensor 7 Surface acoustic wave-based sensors 9 Graphene 9 Reduced grapheme oxides 10 Cytochrome c 10 Binding mechanism for reduced graphene oxide and cytochrome c applied 12 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide 12 Structures and stages of EDC 12 Liquid gate modulated nano-sensors exhibit optimal sensitivity 13 Highly Reduced Graphene Oxide, Graphene Oxide and Graphene 14 Atomic Structure in Reduced Graphene Oxide 15 Optimization of the CNTs Biosensor Response 16 Conductometric Biosensor mechanism 16 Infrared Spectroscopy mechanism 17 A quartz crystal microbalance (QCM) mechanism 17 Fig. 6 18 Conductometric biosensor 19 Surface acoustic wave-based sensors 20 Bibliography 20 Farhan A.(2008 ).Nanobiosensors .American Society of Civil Engineers.Methods Mol Biol. Pp.115-26. 24 Further reading 24 Palchetti I, Laschi S, Mascini M. (2010). Electrochemical biosensor technology: application to pesticide detection. Universita degli Studi di Firenze. Italy. 25 Background/introduction Nanotechnology advances are opening development for the biosensors on micron size features that are suitable on detection of molecules in biological sciences. Based on the size micro is added onto the biosensor to indicate the scale. Development of biological sensors for water industry is experiencing growth on the two decades past (Lim et al 2005). The progress on the developments in nanotechnology provides the nano-materials (Vaseashta 2005). Nanoscale materials show extraordinary electrical and optical as well as mechanical properties in comparison to their counterpart. Sensors using these materials interact with the cells such as bacteria, protozoa and virus as well as proteins, antibodies. They also interact with chemical species to produce a signal. These signals are converted to property with a measurable response such as current or light intensity etc. which may be amplified or processed and even stored for the analysis (Erickson et al. 2008). Objectives The main objective of the study include; -To use reduced graphene oxide to detect cytochrome c. - To describe why reduced graphene oxide is used as a detection platform for cytochrome c (protein) using the liquid gated method Literature review The main applications for the biosensors are focused on the water safety for detecting waterborne pathogenic organisms. Diseases from pathogens that are water borne give a great challenge in developing and developed countries. According WHO (2005), World Health Organization estimated deaths in year 2005 of about 1.6 million on consequences diseases that are water borne. These risks have not been completely in elimination with best practices of water treatment in this industry. The predominant techniques identified waterborne disease causing pathogens and rely on culture based practices that are selective and also time consuming. A study conducted by Farhan (2008) reveals that, Biosensors meet most requirements hence leading to highly sensitive and specific as well as a rapid platform in detecting the waterborne pathogens. Studies have reported on biological molecules techniques and also transduction techniques on various pathogens (Farhan 2008) .At present very few assays meeting the requirements for assay time and also detection limit. The high percentage applications of biosensors in food industry are on development of the portable detection platform (Arora et al 2006). In Consideration of the diverse and complex environmental samples and wide range on environmental applications and also economical benefits that are low. Presently more need is of portable biosensors that have rapid detection in waterborne pathogen due to increasing waterborne disease epidemics (Vockenroth & Atanasova 2005). These portable devices should address the large water volumes so that the biosensors are useful. Diagnostic market represents large and also established markets that are continually expanded in current climate for the prevention and not remedy for detection in an increasingly lower limits and many diverse areas. Estimates for the market size as well as projections are difficult and inaccurate. Applications for the biosensors are many and include; i. Biodefense ii. Food industry iii. Clinical diagnostics iv. Detecting micro organism that are pathogenic either in water or other environmental matrices. Characteristics required in a biosensor i. Relevance in output signal for measuring environment ii. Accuracy iii. Repeatability iv. Sensitivity and resolution v. Dynamic range vi. Speed of response vii. Temperature compensation viii. Insensitive to other interference(environmental and electrical) ix. Easy to test and calibrate x. Reliability xi. Physical robustness xii. Service requirements xiii. Capital cost xiv. Running costs and life xv. Acceptability by user xvi. Product safety Biosensors advantage includes; i) High sensitivity because of size of the sensor element in comparison to target species and also high surface area in comparison to volume ratio. ii) Low materials cost and reagents. iii) Short assay time. iv) portability v) use as a point of care devices Biosensors are used in food analysis Biosensors are used in food analysis. Optic coated that have antibodies are used in detecting pathogens and toxins that are in food. The system of light in the biosensors is fluorescence hence this optical measurement greatly amplifies the required signal. Immuno-binding and ligand binding assays detection and measurement in molecules such as vitamins are water-soluble. They are also contaminants (drug residues) like Beta-agonists which have been made for use on sensor systems. Types of Biosensors Carbon based biosensor Carbon based biosensor use Carbon nano-tubes, CNTs, that exhibit a very unique combination of mechanical and electrical as well as electrochemical properties. These carbon nanotubes have stimulated interest (increasing) in application components for the biosensors or sensors at large. There are various design methods for Carbon nano-tube based biosensors and use on detection of biomolecules. In recent developments the fields of Carbon nano -tube chem-resistors and also chemically sensitive transistors have been presented (Krishnamurthy et al 2010). Carbon nanotubes consist of graphene sheets that are cylindrical a with nanometer diameters presenting various unique characteristics e.g. It has large ratio of surface area to mass, electrical conductivity as well as remarkable strength mechanically. CNTs may be single-walled or multiwall structures. Single walled CNTs comprise of cylindrical sheets of graphite with nanoscale diameter being capped by the hemispherical ends. Multi walled CNTs comprise of several incommensurate cylinders that are concentric and have graphitic shells that have a layer spacing of about 0.3 to 0.4?nm. The target analyte get involved for the reaction which takes place on active electrode surface and this reaction cause either transfer of electron across a double layer which produce a current or contribute to double layer potential that produce a voltage. We can measure the current or rate of flow for the electrons which is proportional to analyte concentration and at a potential which is fixed or measured at zero point current. This gives logarithmic response. The potential of working or the active electrode is sensitive space charge and is often used. The label-free as well as the direct electrical detection for peptides or proteins is possible due to their intrinsic charges and by using bio-functionalized and ion sensitive field effect transistor. Carbon based biosensor extensive applications are found in physical and chemical or even material science fields. The advantages of CNTs are; i. High surface area, ii. Has favorable electronic properties iii. Good electro catalytic effect iv. Used in making of electrochemical biosensors. These Electrochemical biosensors such as enzyme electrodes benefited from the CNTs based transducers on promoting the electron transfer in an enzymatic reactions Electrochemical biosensors Electrochemical biosensors are usually on enzymatic catalysis for the reaction which produces and consumes electrons that are called redox enzymes. The substrate of the sensor usually contains electrodes; 1. Reference electrode, 2. Working electrode 3. Counter electrode. The potentiometric biosensor produces potential at zero current and gives a logarithmic response at a high dynamic range. These biosensors usually made of screen printing of the electrode patterns to plastic substrate and coated with conducting polymer and some protein such as enzyme or antibody are attached. They have two electrodes and may be extremely sensitive and highly robust. They usually enable detection of analytes previously on level and are achievable. All biosensors involve sample preparation for biological sensing and are highly selective for analyte concerned. These signals get produced by the electrochemical changes in the polymer layer on changes occurring to the surface of a sensor. These changes are attributed to the ionic strength and pH as well as hydration and the redox reactions. In recent years the electrochemical sensors and the biosensors are becoming accepted part of the analytical chemistry since they satisfy an expanding need of rapid and reliable measurements. An area that electrochemical biosensors show the diversity and potential in development involves measurement of environmental parameters. The increasing pollutants in the environment has urge for fast and cost-effective as well as analytical requirements. In this context biosensors may appear as the alternative or the complementary analytical tools. Electrochemistry is a very powerful tool on real-time detection in comparison with fluorescence and spectrophotometry that involves expensive detection systems. A combination of reactions that are enzymatic with electrochemical method in monitoring electro-active products and allows the development of the enzyme based electrochemical biosensors on sensitive and rapid determining the important environmental pollutants (Krishnamurthy et al 2010). Surface Plasmon resonance biosensor The excitation in surface plasmons is by use of light. This is denoted as surface plasmon resonance (SPR) for the planar surfaces for metallic structures in nanometer size. The phenomenon is the standard tools for adsorption measurement of material of planar metal usually gold and silver surfaces. It is fundamental to many color biosensor applications and different sensors. Surface Plasmon’s are electromagnetic waves propagating in direction parallel to metal. Since the wave acts at the boundary of metal and medium oscillations are sensitive to change of boundary such as adsorption of molecules in metal surface. To describe properties for surface Plasmon you can choose various models such as quantum theory or Drude model.The way to approach a problem is treating material as homogeneous continuum that is described by frequency between the medium and the metal surface. Localized surface polaritons (LSPRs) are electron charge causing oscillations in nano-particles which are excited by the light. Light intensity is important aspect and localization means that LSPR has high spatial resolution and limited by size of nano-particles. Because of field amplitude effects depends on the amplitude that include magneto optical effect that are enhanced by LSPRs. Fig. 5 A conductometric biosensor A conductometric biosensor,is usually based on an immobilized whole cell (Chlorella vulgaris microalgae) which acts as a bio-receptor and interdigitated conduct metric electrodes which act as a transducer. It was developed and also tested for the alkaline phosphatase activity analysis. These sensors were used for detecting of the toxic compounds such as cadmium ions and in aquatic habitats. Algae immobilized in the bovine serum albumin membranes which were cross linked by glutaraldehyde vapors. The detecting of local conductivity variances which were caused by enzymatic reactions of the algae was achieved. The inhibition of vulgaris microalgae alkaline activities and with cadmium ions presence was measured. Results were compared to measurements of bioassays. It appeared conductometric biosensors that used algae were more sensitive than that of bioassays in detecting low levels cadmium ions; the limit for the first experiments detection was 1 ppb (Krishnamurthy et al. 2010). The main advantages to alkaline phosphates biosensors is that it consist of high specificity with regards to toxic compounds. They enable detection on they have high stability due to they are contrary to biosensors that are enzymatic. They use the algae cells in whole. Surface acoustic wave-based sensors The QCM in a wider class of instruments for sensing is on acoustic waves at the surfaces. Instruments may share similar principles in operation with horizontalsurface acoustic wave devices, Love-wave devices and also torsional resonators. Surface acoustic wave devices make use of reflectivity of acoustic wave by the crystal surface which depends on the impedance of the medium. Acoustic sensors of temperature and pressure use the fact that speed of sound in a crystal depends with temperature and pressure and also bending. This sensor does not use surface effects (Krishnamurthy et al. 2010). Graphene Graphene this is a rising star of carbon and has attracted interest from theoretical and experimental, scientific communities and is discovered and isolated from graphite which is bulk few years ago. Graphene is one atom thick and planar sheet of bonded carbon atoms that are arranged ( honey comb like) crystal lattice and the difference in structure of graphite’s is that Graphene is wrapped up to zero dimensional fullerenes and also rolled to one dimensional nano-tubes or is stacked to three dimensional graphite. Hence, a two dimensional carbon graphene, is taken as the building blocks of the graphitic materials with all the available dimensionalities. These owes to the extraordinary electron transportation which is very fast and have high surface area that is uniquely graphitized on basal plane structure and manufacturing low cost numerous applications on graphene nanosheets was investigated .The diagram below shows synthesis of grapheme on the silicon dioxide substrate (Krishnamurthy et al 2010). Fig. 1 Reduced grapheme oxides Reduced graphene oxides are attracting considerable interest due to their applications on electronic devices and circuits. However, little is brought to attention regarding the chemical reduction induced method on graphene oxide in gas and solution phases and exception of hydrazine as reducing agent although essential in vapour for patterning the hydrophilic Graphene oxides on the pre-patterned substrates and reduction to the hydrophobic Reduced Graphene Oxides. Reducing agent system allows an efficient reduction of the solution of Reduced Graphene Oxide powder and vapor of Reduced Graphene Oxide paper and in thin film. This reducing agent system provides highly qualified Reduced Graphene Oxides and by mass production. Results is highly conducting Reduced Graphene Oxide and its paper or thin films prepared within low temperatures of about 40 °C and these are found to be applicable in flexible devices. The one-pot method used is expected in advancing research for graphene platelets of high conductance. Cytochrome c Cytochrome c is a small and water soluble protein associated to the inner membrane of mitochondrion. It a very essential link to the electron transport that through which cells perform their controlled burning glucose and also capture that released energy and store it in ATP. The cell’s energy molecule for distribution is ATP. Every cytochrome c carries an electron from one to the electron transport complexes that are embedded in inner membrane of the mitocondrion. In this, cytochrome c undergoes in repitition either oxidation or reduction and does not bind the oxygen. Cytochrome c is particularly studied because due to its tiny size of about 100 amino acids and the level of water solubility that permit researchers in isolating it from the other mitochondrial proteins that tend to be larger than cytochrome c and also soluble in fat and get embedded in membrane. The factors involved combined may have led researchers in determining the amino acid patterns for the cytochrome c which occur in various organisms such as human and yeast Cytochrome c is usually found in aerobic organisms and its comparison to amino acid structures of the molecule of diverse species indicates a deal of similarity amongst the animals and plants. Such similarities usually suggest common ancestor must have used this protein before basic divergences on plants and animals. Cytochromes are membrane hemoproteins and have heme groups that transport electron. A heme is a prosthetic group which is the non protein component of protein molecular complex that comprises iron atom in the center of heterocyclic organic molecule (porphyrin. Hemoproteins). That is part of the large class in metalloproteinase that includes complexes. Porphyry group contains different metal atom at its center not only iron. Cytochromes are found as monomeric proteins and enzymatic complexes which catalyze redox reactions. They are found in mitochondrial membrane and endoplasmic reticulum of the eukaryotes in chloroplasts and in bacteria. The heme group electrons are mobile and surrounds iron ion that readily converts from its primary states of oxidation. The iron converts from the Fe2+, reduced to Fe3+, oxidized Cytochromes are therefore capable of performing oxidation and also reduction. Because cytochromes are held in membranes in organized way and redox reactions are done in the proper sequence of maximum efficiency. Cytochromes c is electron transporting proteins having several heme groups bound in the surrounding structure. The fifth iron ligand is provided by a histidine. Cytochromes c have many properties and functions in different redox processes (Pettigrew and Moore 1987). Cytochrome c's structure comprises 100 amino acids. R. P. Ambler (1991) classified four classes of cytochrome c: Class 1; the low­spin cytochrome c in mitochondria and bacteria. Class 2 the high­spin cytochrome c'. Class 3 comprises low redox potential cytochromes. Class 4 holds complex proteins that have prosthetic groups. Binding mechanism for reduced graphene oxide and cytochrome c applied 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide); this is water soluble carbodiimide obtained as the hydrochloride. It is employed at 4.0-6.0 pH range. It is used as carboxyl agent on coupling of amines to give amide bonds. EDC can activate the phosphate groups. Common uses include; i. Peptide synthesis, ii. Protein cross linking to the nucleic acids iii. Preparation of immuno-conjugates. EDC is a combination of N-hydroxysuccinimide (NHS) to increase efficiency of coupling. Structures and stages of EDC O N N NH NH N=C=O H2N N -H2O C N N Fig. 3 Liquid gate modulated nano-sensors exhibit optimal sensitivity Biosensors that use the nanowires (NWs) are highly employed in this case, as the conducting channels usually have the potential in providing high sensitive that can be detected. This is good for features of a high surface to volume ratios and sizes that are comparable with biomolecules. However, the fluctuations based on electrical characteristics should not be neglected if feature sizes for the electronic devices are brought down to nanoscale. It is most likely NW biosensors that are made using same fabrication process still exhibit variations on their performances even by applying same operational bias in conditions. The good thing is that the uncertainty is fixed by the liquid-gating to the electrical modulation on the sensitivities of the biosensors to the limit. Researchers from National Nano- device Laboratories and also National Chiao Tung University in Taiwan found that the NW biosensors exhibited a more optimal sensitivity when applied a to a specific liquid gate voltage and corresponded to the maximum value of the trans-conductance and conductance of the NW biosensor. The impact of electrical performance and its fluctuations may be minimized and optimal sensitivity determined. This method may be applied practically in the field of the nanoscale biosensors. There has been developed extensive range for chemical and biological sensors in complementing the Rapid Scan Reader.  These sensors may be based on electrochemical sensing principles that range from selective membranes ions to immobilized enzymes and also a peptide as well as DNA molecular probes.  Fig. 2 Advantages of these sensors: i) Requires only a drop of the sample ii) High detection limits iii) Fast results iv) Provide quantitative readings v) Simple and also easy to use vi) portable design and mobile vii) No additional reagents that are required viii) Non-colorimetric and they are not affected by original sample’s color These sensors are useful for organizations and individuals in: a. Quality control b. Point of care diagnostic c. Field monitoring and enforcement d. Scientific field studies Areas of applications include: Environmental monitoring Production Quality Control Biomedical diagnostics Food safety monitoring Highly Reduced Graphene Oxide, Graphene Oxide and Graphene Isolated grapheme is a two dimensional analog of the fullerenes and the carbon nanotubes that recently brought the great excitement in our scientific community due to its excellent mechanical and also electronic properties. Particularly it is attractive its availability of bulk in quantities of graphene as colloidal dispersions as well as powders that enables the fabrication of carbon based materials. Large amounts of the graphene are easily produced through reducing graphene oxide: oxygenated grapheme that are sheets covered epoxy or hydroxyl as well as carboxyl groups and offers tremendous opportunities on access to functionalized graphene based materials. Both the graphene oxide as well as grapheme may be processed to a wide variety of materials and has distinctly differing morphological features where the carbonaceous nanosheets serve as the sole component in papers or thin films as well as the fillers in polymer and also inorganic nanocomposites. This summarizes techniques of preparing such materials that are advanced through graphene oxide which is stable and also highly reduced graphene oxide and graphene dispersions that are in aqueous and organic medium. The mechanical and electronic properties to the resulting materials may be highlighted and with a forward outlook to the applications. Atomic Structure in Reduced Graphene Oxide Fig. 4 Optimization of the CNTs Biosensor Response In the current the voltammetric characteristics on acetylthiocholine substrate and thiocholine which is an enzymatically generated product on carbon nano-tube electrode was investigated by square voltammetry wave of phosphate buffer. Typical square voltammograms wave are shown as two oxidation processes of thiocholine. For a glassy carbon electrode that was modified with carbon nanotube film. The optimal potential of biosensor operation and also current time responses are obtained in thiocholine in a carbon nano-tube electrode. In order for optimizing the CNTs the biosensor’s performance the following an experimental variables was investigated and were substrate concentration and also the incubation time. The influence of the substrate concentration to the response of the biosensor that was studied and the purpose was to increase the signal which was obtained for the enzymatic reaction. Therefore the effect of substrate concentration on CNTs biosensor’s responses was being investigated. Conductometric Biosensor mechanism Illustratively, Food borne illnesses may be widespread and pose growing health problem. The problems are with conventional pathogens which are detected using methods that have speed and sensitivity such as Conductometric biosensor. Conventional culture methods consume and laboriously and usually take 2-7 days in order for it to yield results. Conductometric biosensors are may be powerful tools to incorporate biological elements on selective detection of a wide range in chemical substances. Biosensor technology is quite exciting since it shows a great potential to providing cost effective and highly accurate as well as specific detection to pathogens and in real time. Researchers investigated types of biosensors and the conductometric biosensor on detection of Salmonella which is a common food pathogen. Based on research the student investigated and designed as well as built a conductometric biosensor right from scratch with available materials. Infrared Spectroscopy mechanism This mechanism (Infrared spectrum) reveals a partial structure in a substance. In some cases this process is like using and reading an X-ray. This discloses the structures and their deformities or arrangement and alignment. The interaction of the infrared radiation and molecules requires a similar reading as well as treatment. A quartz crystal microbalance (QCM) mechanism In this mechanism, the quartz crystal microbalance measures mass per unit area. It measures the change of frequency in a quartz crystal resonator. The resonance is disturbed and additional or removal of small masses due to oxidation growth or decay or even film deposition from surface of acoustic resonator. The QCM maybe used under vacuum or gas phase and also in liquid environments. It is very useful in monitoring rate of deposition of film deposition systems while in vacuum. In liquid usually is highly effective to determine the affinity to molecules i.e. Proteins to surfaces with recognition sites (Harthorn, 2009).. Larger things such as viruses are investigated in QCM. QCM is also used to investigate interactions on biomolecules. Frequency measurements are easily made on high precision hence easy in measuring mass densities to the level below 1 ?g/cm2. In measuring the frequency the dissipation often is measured for helping in analysis. The dissipation parameter quantifies the damping in system and is usually related to the viscoelastic properties of the sample. The photograph is of typical quartz crystal resonators Fig. 6 Table 1. Advantages and disadvantages of various biosensors Biosensors Advantages Disadvantages Carbon based biosensor High surface area, Has favorable electronic properties Good electro catalytic effect Used in making of electrochemical biosensors. Potential is measured at fixed or zero point current A quartz crystal microbalance (QCM) High precision Larger things such as viruses are investigated In liquid it is highly effective Frequency measurements are easily done Expensive. Resonance is disturbed hence accuracy may be affected. Additional or removal of small masses due to oxidation growth Conductometric biosensor They are powerful in incorporating biological elements e.g. detecting pathogens on food. Time saving compaired with conventional methods Specificity with regards to toxic compounds High stability Incorporate selective detection of a wide range in chemical substances Surface plasmon resonance biosensor High spatial resolution Limited by size of nano-particles Because of field amplitude effects depends on the amplitude Surface acoustic wave-based sensors This sensor does not use surface effects but reflectivity Hence less disturbance to the substance. Depend on pressure and temperature hence reduced precision Infrared Spectroscopy Fast Efficient High detection limits Radioactive used may alter the substance due to its effects. Electrochemical biosensors May be extremely sensitive Highly robust. 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Selected Papers on Nanotechnology—Theory & Modeling (Milestone Volume 182). SPIE Press, Bellingham, WA, USA. ISBN 0-8194-6354-X. Fritz Allhoff and Patrick Lin (eds.), Nanotechnology & Society: Current and Emerging Ethical Issues (Dordrecht: Springer, 2008). Fritz Allhoff, Patrick Lin, James Moor, and John Weckert (2007) "Nanoethics: The Ethical and Societal Implications of Nanotechnology". Hoboken: John Wiley & Sons.. Further reading Genetic Engineering & Biotechnology News (January, 2008), Getting a Handle on Nanobiotech Products Regulators and Companies Are Laying the Groundwork for a Predicted Bright Future Hari Singh Nalwa (2004), Encyclopedia of Nanoscience and Nanotechnology (10-Volume Set), American Scientific Publishers. ISBN 1-58883-001-2 Hunt, G & Mehta, M (eds)(2008) Nanotechnology: Risk, Ethics & Law, Earthscan, London. 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