URGENT REVISION: editing a report Essay Example

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Practical Experiment

TestingGlucose Biosensor Fabrication and

ExperimentAims of the

The major aim of this experiment was to fabricate a carbon nanotube (CNT) biosensor electrodes modified using glucose oxidase enzyme. The CNT biosensor is later used to determine the amount of glucose in beverages. The second objective of this experiment is to enable one to understand principles and techniques of nanotechnology and bio-sensor technology.

improvement of the original technique. Continuous glucose sensors have been made using a wide variety of substrates and electrode materials. This study presents a detailed information on the use of printing and other additive processes to fabricate a novel amperometric glucose biosensor, which find important applications in food science and clinical chemistry.
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Abstract

In this paper, fabrication of an enzyme modified
biosensor electrode based on CNTs has been reported. It involves a 3-electrode setup and utilizes
the enzyme glucose oxidase (see figure 1) as a sensitive and selective form of glucose determination. This
Amperometric experiment
is very
stable and
resistant, as well as versatile and relatively inexpensive. A brief explanation of the function of an Amperometric biosensor will come later in the report. This experiment has been used in line and in comparison,
with standard methods of glucose determination. It is based on the increasing applications of nanotechnology and its importance in teaching biochemistry,
biotechnology, and analytical
chemistry, enabling the development of analytical skills among researchers. CNTs have been used in the experiment mainly because
of their electro-catalytic and electrochemical properties. Other advantages of CNTs include faster electron transfer and increased surface.

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FIGURE.1: Ribbon Diagrams of the Enzyme Glucose Oxide.

Introduction

Amperometric biosensors are self-contained integrated devices that operate on the basis of current emerging from oxidation or reduction process to provide a quantitative analytical information. They generally have response times, dynamic ranges and sensitivities. The simplest amperometric biosensors in common usage is made of the Clark oxygen electrode (see figure.2). The biosensor is about 1 cm in span but has been scaled down to 0.25 mm diameter using a Pt wire cathode within a silver plated steel needle anode and utilizing dip-coated membranes.

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(Chaplin, 2016).Simple Amperometric BiosensorFIGURE.2:

expected that this current will be between 5uA and 50uA. This is because of the fact that the amperometric signal was the highest when peptide nanotubes (PNT) and HQ were used together. A platinum wire is ideally used as a counter electrode.hydroquinone (HQ)-mediated electron transfer. It is in the 2O2The set up would be a simple 3-electrode cell utilizing a conventional gold electrode and layer- by-layer electrostatic assembly. The working electrode would be gold modified with glucose oxidase (GOx) and a poly-diallyldimethylammonium (PDDA) coating. Glucose will be oxidized to Gluconic and hydrogen peroxide in the presence of the oxygen. Potentiostat (an electronic instrument), is the device to monitor the redox current produced by Hof gold nanoparticle (AuNP) and carbon naotube (CNT) composites. In this experiment, we want to measure the concentration of glucose in different aqueous substance samples. To achieve this, we should fabricate CNT-glucose oxidase-modified amperometric biosensor electrodes. This design produces a fast and controllable production

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FIGURE.3: Representation of the God Reaction

Experimental Details and Equipment

Immobilisation of the bio-element

To immobilise the bio-element we go through a series of steps. The gold electrodes (GEs) are polished using 1.0μ malumina followed by 0.05μm alumina, and rinsed with deionised water. A small volume of the oxidised multi-walled carbon nanotube (MWNT-COOH) suspension, 10 μL, is pipetted onto the surface of each GE. The GEs are allowed to dry at 100 °C for 10min and are then cooled.

A small volume of the poly-diallyldimethylammonium (PDDA) solution, 10 μL, is then pipetted on the tip of each GE and dried at 100 °C for 10 min. After rinsing by suspension in deionised water, a layer of PDDA remains electrostatically attached to the negatively charged MWNT−COOH surface of the GEs because the PDDA solution carries a net positive charge. The buffered GOx solution, 10 μL, is sonicated for 1 to 2 minutes. It is then pipetted onto the tip of each MWNT-COOH-PDDA electrode and dried at 100°C for 10 min. GOx has an isoelectric point of approximately 4.5. It carries a net negative charge in this solution. Therefore, after rising as described above, a layer of GOx remained electrostatically attached to the positively charged PDDA. Ideally, the drying of the electrode after casting with the enzyme should be done at ambient conditions due to the potential of enzyme denaturation (approximately 2 hours); however, it was found in previous attempts at this process that drying the electrode at 100 °C for 10 min did not result in any noticeable change in the activity of the enzyme.

Finally, 10 μL of the PDDA solution is pipetted again onto the tip of each MWNT−COOH−PDDA−GOx electrode, dried at 100 °C for10min, and rinsed by suspension in deionised water. A layer of positively charged PDDA remains electrostatically attached to the negatively charged GOx layer beneath. The GOx is thus immobilized between two layers of PDDA.

Signal Transducer:

A transducer is an electronic device that converts energy from one form to another. In this experiment, a transducer located in the biological sensor is used in the recognition of a transduction component within the biosensor structure. It is comprised of intimately coupled divisions that include the biological recognition and the physicochemical transducer. Both of these components act together in converting the biochemical signals to electronic or optic signals.

On the other hand, the physicochemical transducer is intimate and in controlled contacts with a recognition layer. The physicochemical change occurs within the bio-recognition layer, which is evaluated using a physicochemical transducer, which produces the proportionate signal to the analyte concentration. The transducer may be optical, gravimetric, piezoelectric, or electronic. Thus, the transducer acts as the detector element within the biosensor system. This implies that the transducer element transforms the signals, which originate from interactions of biological elements into an electrical signal. This enables measurement and quantification of the signal.

Materials and Instruments

The following table contains all materials and equipment that we need to achieve the best results of this empirical experiment.

Materials and InstrumentsTable 1:

Multi-walled carbon nanotubes

Sulfuric acid

———-

Nitric Acid

Deionised water

Available

Dimethyl formamide

———-

Phosphate buffer

0.1M, pH 8.0

Ag/AgCl reference electrode

Platinum wire counter electrode

Alumina polish

Gold electrode

Polishing pad(s)

glass cell

Sonicator

Glucose solution

Phosphate buffer

0.1M, pH 8.0

Glucose Oxidase

Stirrer/hot plate

Fume hood cupboard

for weighing or dealing with the acid, dimethyl form-amide and PDDA

Like the other experiments that involve working with materials and instruments, we must closely observe safety procedures in this experiment to prevent risks and hazards that may occur to the person carrying out the experiment as well as other people around. It is not acceptable if we do not pay attention to the rules. It is compulsory that all the laboratory work must be performed under the fume hood cupboard; a laboratory coat and safety glasses must be worn at all times. Closed footwear (preferably steel-cap boots) must be worn; no open or exposed footwear is permitted into the laboratories. No food or drinks are allowed in the laboratories; ensure safety and full checks before commencing any experimental procedures. It is also necessary to know where policy and procedures are kept, who the WHS representative is and how to perform evacuation plan in the laboratory in case of any problem. The table below shows the data for chemicals to be used.

Table 2: Relative Safety Data for the Chemicals to be used.

Hazard (0=low,5=extreme)

0.5M NaCl

Phosphate Buffer

Flammability

0

0

0

0

Skin Exposure

Reactivity

Chronic Risks

0

0

0

0

0

Electrode Preparation and Fabrication

C for about 12 hours. A gold electrode (GE) would be prepared by polishing with alumina and rinsing with deionised water. 10 μL of the oxidised multi-walled carbon nanotube (MWNT-COOH) would be pipetted onto the surface of each GE and allowed to dry at 100 °C for 10 minutes.oTo prepare the electrode(s), we need to follow the steps some steps. These steps may vary depending on the type of materials used to fabricate different electrodes. MWCNT preparation would require that 5mg/ml of material be sonicated in a 3:1 mix of Sulphuric/Nitric acids for a period of about 4 hours. These CNTs would then need to be rinsed with distilled water to remove the acid and dried at 100

10 μL of the PDDA solution will be pipetted on the tip of each GE and dried at 100 °C for 10 minutes. After rinsing by suspension in deionised water, a layer of PDDA remains electrostatically attached to the negatively charged MWNT−COOH surface of the GEs because the PDDA solution carries a net positive charge. The buffered GOx solution, 10μL, is then sonicated for 1 to 2minutes and pipetted onto the tip of each MWNT-COOH-PDDA electrode and dried at 100 °C for 10 minutes. GOx has an isoelectric point of approximately 4.5. It carries a net negative charge in this solution. Therefore, after being rinsed as above, a layer of GOx remained electrostatically attached to the positively charged PDDA. Finally, 10μLof the PDDA solution is pipetted again onto the tip of each MWNT−COOH−PDDA−GOx electrode, dried at 100°C for10 minutes and rinsed by suspension in deionised water. A layer of positively charged PDDA remains electrostatically attached to the negatively charged GOx layer beneath. The GOx is thus immobilized between two layers of PDDA. These steps produces an electron fabrication as shown in figure 4.

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Schematic of Functionalised Gold ElectrodeFigure 4:

Results and Analysis

URGENT REVISION: editing a report  6The experiment will be conducted in a 25-ml glass cell at room temperature using a standard electrode set-up shown in figure 5 below.

Experimental Set-up for Glucose BiosensorFigure 5:

is produced and more current flows through the cell. Thus, the current produced is proportional to the concentration of glucose, allowing the biosensor to have excellent selectivity. The calibration curve of current vs. glucose concentration in the glucose concentration range of 0-2.5 Mm.2O2The process of catalysis of glucose oxidation by glucose oxidase immobilized on the surface of the electrode produces hydrogen peroxide which increases the amount of current. As more glucose is added to the electrochemical cell, more HAmperometric response current versus various glucose concentrations would be measured by immersing the electrode in phosphate buffer and raising the glucose levels in the test-cell by adding 2 mM every 90 seconds for 9 minutes drop wise. These responses would be compared to responses using standard solutions of glucose in water (0mmol, 0.5mmol, 1mmol, 2mmol, 4mmol) prepared from a stock solution and the current response would be measured using a potentiostat in micro-amps. Based on the data received during the experiment, the following graphs reveal the trend of this process (see figure 6 to 16).

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: Biosensor Results Figure 7Biosensor Results Figure 6:

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Biosensor Results Figure 9: Biosensor ResultsFigure 8:

As can be seen in the graphs above, the biosensor shows how the amount of current produced within a stipulated period. The concave nature of the graph increases from figure 6 to figure 10 respectively. This indicates a constant increase and then drop in the levels of glucose.

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Biosensor ResultsFIGURE 10:

The following figures (figures 11 to 16) are also obtained from the experiment. Current increases as time increases. The data, which can be extracted from the graphs show a relationship between the amounts of glucose and the currents produced in the experiment.

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Figure 12: Current vs. TimeFigure 11: Current Vs. Time

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Figure 14: Current Vs. TimeFigure 13: Current Vs. Time

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Figure 15: Current Vs. Time Figure 16: Current Vs. Time

As figure 12 reveals, the current had been reduced during the first 80 seconds and remained constant after 100 seconds until the last time. A glucose concentration level of 0.125 mM corresponded to a current of approximately 10nA. An observation made from the graphs is that the trend is not having any significant change from 0.125mM to 0.25mM. In figure 13, we see that the current is still about 10nA.

However, it gradually increases by 0.5 and 1mM. At a glucose level of 2mM, we did not expect to have a constant trend. Between 450s and 550s, the amount of current changes and becomes very unstable. The signal is expected to increase linearly, step by step, in proportion to glucose concentration. These results will be compared with theoretical calculations based on the surface area of the gold electrode to determine performance efficiency. Sensors will be stored under refrigerated conditions to maintain the activity.

The below table shows the approximate results of current output versus the increase of glucose concentration.

Current and Glucose Concentration correspondentTable 3:

Glucose concentration (mM)

0

Current (nA)

0

Using the data from the above table, we can produce the following graph as the typical calibration for total current versus Glucose concentration.

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Typical Calibration for Current vs. Glucose ConcentrationFIGURE 17:

relationship. This difference could be brought about by an experimental error. linearThe correlation coefficient calculated above shows that the experimental results are close enough to the results obtained from the calculations. Based on Pearson product-moment, correlation coefficient measures linear relationships. In this respect, a correlation of zero does not imply zero relationship linking two variables. However, it means zero

Timeline of the Experiment

August 25, 2016

  • Preparation of CNTs: They meant to be dried overnight

August 26, 2016

  • During the time period between 10:00 and 13:00, the solution had been prepared and the first layer of the polymer had been deposited

  • In the afternoon of the same day, from 14:00 to 17:00, the electrode functionalising was performed and the electrodes stored in a fridge.

September 02, 2016

  • During the morning session, between 10:00 and 13:00, group1 repeated the experiment with modifications. Group2 tested the biosensor from previous week.

  • The test and the electrode functionalization had been done between 14:00 and 17:00 and then stored in a fridge

September 09, 2016

  • Electrodes from the previous week were tested from 10:00 to 13:00 in morning session

  • Characterization of the electrode surface was done by AFM in the afternoon session from 14:00 to 17:00

Conclusion

The materials used in this experiment are readily available in a typical biochemistry lab, and the procedures can simply be performed by undergraduate students. The quality of electrode fabrication direct effects the quality of the results obtained. The liner range can go as high as 15 mM, depending on the quality of the electrode. By getting more accurate results and improvements on the procedure, students can be able to use this experiment to understand the technique of nanoscale science and sensor technology.In this experiment, we managed to fabricate carbon electrodes that are modified using CNTs and glucose oxidase by following a layer-by-layer procedure. Amphoteric experiments use a simple 3-electrode cell that is selectively sensitive to the concentration of glucose. Thus, its application in determination of glucose levels in commercial drinks.

References:

  • Web Image, https://cdn.rcsb.org/pdb101/motm/images/77_1gpe-rasmol.gif,August

  • R.D O’Neill, S. Chang, J.P Lowry, C.J Mcneil “Comparisons of platinum, gold, palladium and glassy carbon as electrode materials in the design of biosensors for glutamate”, Biosensors and Bioelectronics, 2004, pp.1521–1528.

  • Herold, K E, and Avraham Rasooly. Biosensors and Molecular Technologies for Cancer

Diagnostics, 2012. Internet resource

  • M. Hobbs, N. Patel, D. Kim, J.Rugutt, A. Wanekaya, “Glucose Determination in Beverages Using Carbon Nanotube Modified Biosensor: An Experiment for theUndergraduate Laboratory”Journalof ChemicalEducation, 2013, pp.1222-25

  • G. Liu, Y.Lin, “Amperometric glucose biosensor based on self-assemblingglucoseoxidase on carbon nanotubes”Electrochemistry Communications, 2006, pp.251-56

  • . Available from: <http://www1.lsbu.ac.uk/water/enztech/amperometric.html> [Accessed 7 Nov. 2016].Amperometric biosensors2016, Chaplin, M,

  • http://whatis.techtarget.com/definition/transducer

  • http://www.chsmith.com.au/Wiki/Transducer.html

  • http://jss.ecsdl.org/content/4/4/P3069.full

  • https://www.ncbi.nlm.nih.gov/pubmed/23601276

  • http://www1.lsbu.ac.uk/water/enztech/amperometric.html

  • https://en.wikipedia.org/wiki/O-Phenylenediamine

  • http://www1.lsbu.ac.uk/water/enztech/amperometric.html