Question

Answer ALL parts of this question.

(a)       Describe the design and operation of glucose biosensors.

(8 marks)

(b)       Discuss TWO of the following techniques.

            (i)            Inductively Coupled Plasma – Mass Spectrometry                                (ICP-MS).

            (ii)           Cold Vapour Generation Atomic Absorption                                           Spectroscopy (CVAAS).

            (iii)          Laser Ablation ICP-MS

(2 x 4 marks)

(c)       The calibration data for the determination of lead levels in salmon by a new atomic absorption spectroscopy method are shown below.

[Pb] / mgL-1	0	5	10	20	40
Absorption	0.000	0.109	0.240	0.507	0.981

When these data were plotted using linear regression a correlation coefficient, R², of 0.9993 and a line of best fit were obtained;

y = 0.0248 x - 0.0042

Use this information to determine the lead levels in the unknown sample, which had been diluted by a factor of 25, from the following replicate measurements; 0.675, 0.681, 0.686, 0.679 and 0.684.

Answer 1

(a)

Glucose oxidase has become an important tool in several different industries, its uses ranging from a glucose biosensor for the control of diabetes, to a food preservative and colour stabiliser.  Some of its current applications in industry are described below.

Glucose biosensor for diabetes monitoring

People with diabetes mellitus need to constantly monitor their blood glucose levels in order to detect fluctuations in glucose level that could lead to hyperglycaemia (high blood glucose levels) and hypoglycaemia (low blood glucose levels) so as to control the disease.  Currently, such monitoring is done using finger-prick blood samples and a portable meter several times a day.

Biosensors are also being developed to measure blood glucose levels.  Glucose oxidase is one of the possible enzymes that a biosensor can use.  Biosensors work by keeping track of the number of electrons that pass through the enzyme by connecting it to an electrode and measuring the resultant charge. Alternatively, some biosensors use sensitive fluorescence measurements, monitoring changes in the intrinsic FAD fluorescence of glucose oxidase.

(b) (I)

ICP-MS (inductively coupled plasma-mass-spectrometry) is a technique to determine low-concentrations (range: ppb = parts per billion = µg/l) and ultra-low-concentrations of elements (range: ptt = parts per trillion = ng/l). Atomic elements are lead through a plasma source where they become ionized. Then, these ions are sorted on account of their mass. The advantages of the ICP-MS technique above AAS (Atomic Absorption Spectroscopy) or ICP-OES (inductively coupled plasma optical emission spectrometry) are:

• Extremely low detection limits
• A large linear range
• Possibilities to detect isotope composition of elements

The ICP-MS technique has a multi-element character and a high sample throughput, like ICP-OES, but it allows one to perform more sensitive measurements. Disadvantages and weaknesses of the ICP-MS detection are the occurrence of spectral and non-spectral interferences and the high costs.

(ii)

Atomic Absorption Spectrometry (AAS) determines the quantities by “measuring the absorbed radiation by the chemical element of interest. This is done by reading the spectra produced when the sample is excited by radiation.”[i] CVAAS was born when Hatch and Ott used an attachment for a flame atomic absorption spectrophotometer that enabled the reduction of Hg2+ in a solution to ground state atoms (Hg0). The ground-state mercury atoms were then transported to an optical cell and detector for measurement. Shortly after Hatch and Ott introduced the technique to the market, the United States EPA adopted CVAAS for the determination of mercury in water, soil and fish.

The early CVAAS systems provided:

• Detection limits in the single-digit part per trillion range (ng/L)
• A linear dynamic range of 3 to 4 orders of magnitude
• An abundance of analytical methods designed to determine the mercury concentration in almost any sample matrix

Today’s CVAAS systems are more sensitive, automated, smaller, faster and less expensive than the early generic flame spectrometers with the cold vapor attachments. Modern models provide trace to ultra-trace detection limits of ng/L and can analyze samples in a minute. The systems also do not require a lot of operator interaction or bench space.

Most CVAAS instruments have a peristaltic pump that transports sample and stannous chloride into a Gas Liquid Separator (GLS) where a stream of pure, dry gas (typically argon) is introduced to the liquid mixture to release mercury vapor. Gas carries the mercury in the vapor phase through a dryer and into an atomic absorption optical cell. Once in the absorption cell, the elemental mercury will absorb light at 253.7 nm in logarithmic proportion to its actual concentration in the sample. Using this principle the detector, in combination with the software, is able to determine the quantity of mercury present in the sample.

CVAAS can use several commercially available approaches to convey the reduced mercury in solution to the gas stream and then onward to the spectrometer, including:

• Bubbling gas through the sample
• Thin film interface
• In-Situ Reduction

(iii)

LA-ICP-MS (Laser Ablation Inductively Coupled Plasma Mass Spectrometry) is a powerful analytical technology that enables highly sensitive elemental and isotopic analysis to be performed directly on solid samples.

LA-ICP-MS begins with a laser beam focused on the sample surface to generate fine particles – a process known as Laser Ablation. The ablated particles are then transported to the secondary excitation source of the ICP-MS instrument for digestion and ionization of the sampled mass. The excited ions in the plasma torch are subsequently introduced to a mass spectrometer detector for both elemental and isotopic analysis.

Benefits of LA-ICP-MS

LA-ICP-MS is one of the most exciting analytical technologies available because it can perform ultra-highly sensitive chemical analysis down to ppb (parts per billion) level — without any sample preparation.

Samples can be both conducting or non-conducting, and the analysis can be performed in the air without the need for a complex vacuum system. Results are available within seconds; therefore LA-ICP-MS delivers that fastest analysis speed of all analytical techniques with the limit of detection approaching ppb level.

The sample mass size required for LA-ICP-MS analysis is sub-microscale — picograms to femtograms. Traditional liquid nebulization approaches for ICP-MS require the removal of milligrams of sample mass in order to be effective.

When applied with optimized laser ablation conditions and ICP-MS data acquisition protocols, LA-ICP-MS allows versatile solid sampling schemes that include:

• Bulk analysis
• Local inclusion and defect analysis
• Depth profiling
• Elemental/isotope mapping

(c) Average absorption of unknown sample = (0.675 + 0.681 + 0.68 + 0.679 + 0.684)/ 5 = 0.681

Equation

Abs = 0.0248 × Concentration -0.0042

Now we have absorption = 0.681

So concentration = (0.681+ 0.0042)/0.0248 = 27.63 mg/L

Dilution factor is 25

Initial concentration of Unknow sample = 25 × 27.63

= 690.72 mg/L