UNESCO Chair in
interdisciplinary Biotechnology

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UVO-ROSTE Contract 875.771.0 (MPC.9)
Report on the activities

the Report on the ctivities
(Word x Windows)

(Josefina Calvo Quintana)

The activity of Josefina Calvo Quintana in the context of the UNESCO Chair in Interdisciplinary Biotechnology will be carried out in the field of Biosensors and will consist in teaching and research.

Teaching activities

o Teaching Activity will be performed with students and doctoral students attending the faculty of Chemistry.
The teaching activity will consist in the preparation of teaching materials and in the organisation of Seminars concerning to:

1. sensor and biosensor applications

The proposals definition for IUPAC of:

"Chemical sensors": they are miniaturized transducers that selectively and reversibly respond to chemical compounds or ions and yield electrical signals which depend on the concentration. Interpreting this strictly, one only has a true sensor if concentration changes in both directions can be detected.

Similarly there is a consensus of opinion in the relevant IUPAC committees that a large analytical instrument (e.g, a mass spectrometer) used with an external probe or a flow injection system (FIA) cannot be regarded as a sensor.

According to these nomenclature proposals biosensors can be regarded as a sub-group of chemical sensors, in cases where biological recognition mechanisms or principles are used as a means of recognizing substances or molecules. Some types of " biomolecules" isolated from living systems, such as enzymes, antibodies, or receptors, are able to recognize chemical compounds with high specificity and can be adapted to generate signals. Were first described in 1962, even if the term "Biosensors" to describe enzyme-modified ion-selective electrodes appeared in the analytical literature in 1977.

Biosensors are nowadays available commercially for many highly relevant substrates such as glucose, lactate, urea, penicillin, are vastly superior to chemical sensors as regards selectivity. They would be the ideal sensors if the lifetime were not severely limited by stability problems.

An important technological application of biosensors is the control of fermentation processes. Here it is desirable to measure continuously as far as possible, to ensure optimisation of the fermentation process and to minimize the consumption of expensive nutrients.

Another area in which biosensors are used, is food technology and food testing. Of interest is the glucose content of wine and fruit drinks, lactate production during milk processing. Most of the sensors developed so far are based on the combination of microorganisms and transducers. Other systems are used in particular to monitor pesticide contents or concentrations of heavy metal ions and the inhibition of the microorganisms is measured by the reduction in CO2 production.
A final example of a field in which biosensors are finding application is medical technology. The most frequently performed analyses are glucose and lactate ones, and it is therefore in this area that the most intensive research is being undertaken.

2. K+, Ca2+ and NH4+ microsensors construction

Sensors and biosensors basic principles are described by three basic components:
Ø the receptor (the recognition system).
Ø the part of the instrument aimed to detect a substance-specific signal, in other words to transform an energy quantity into an electrical signal proportional to the concentration.
Ø the electronic unit (a preamplifier, impedance, converter, analogue-to-digital converter, etc).

The words sensors, microsensors, ultramicro-sensors in analytical system are often used in the literature, even though there is no real consensus on the size limit between macro-, micro- or ultramicro-dimensions. In the field of electro-analytical chemistry, the last two terms are sometimes used to devote dimensions of 10-100 mm and 1-10 mm respectively.

The main advantage of microsensors is the very small size of the sensing tip and disadvantages are that they are much more fragile, noise sensitive and difficult to prepare than macro-sensors, i.e. they are not the instruments of choice for routine.

K+, Ca2+, NH4+ microsensors construction involves capillaries pulling, glass silanization, shielding, filling with electrolyte and membrane liquid, and application of a coating.

The selectivity is partially an intrinsic property of the membrane, often determined by the size and shape of sensor.

3. different rapid silanization techniques of a glass surface: choice of reagent and effect of experimental parameters on hydrophobicity, in order to obtain the best results in the microelectrodes construction.

The quality of the silanization may vary, as it is a poorly controllable process, influenced (in a way that is not fully understood) by local temperature, humidity, and shape of the tip.

Today, the various techniques can be classified into liquid (dip-and-bake) and vapour (exposure-and-bake) methods.

The silanization was carried out with 4 silanes, the effectiveness of the different agents shows that the aminosilanes react better than chlorosilanes.

4. general overview upon characteristic parameters used to describe the above mentioned microsensors.

Calibration measurements of the different developed microelectrodes at different concentrations of investigated analytes.

Sensitivity and selectivity of the microelectrodes obtained in order to get the lower and upper limit of detection, sensor lifetime, signal shift evaluation and the reproducibility of the potential measurements.
Comparison of the above mentioned microelectrodes with the commercially available macroelectrodes.
Application of these microsensors to measure the release of K+ and Ca2+ of cyanobacteria which cause damage to cultural heritage surfaces inhabiting roman hypogea.

5. Biosensor applications in biotechnology .

Electrochemical biosensors for phosphate and oxalate will be assembled using the enzymes immobilised on the electrode surface and H2O2 electrochemical transducers. For phosphate analyses a xantine probe based on xantine oxidase enzyme will be used. The enzyme is active in the presence of phosphate, after a careful work on the optimisation of analytical parameters as the amount of enzyme, immobilization, pH, temperature, buffer. Calibration curves for phosphate analyses will be run and the detection of phosphate in biological media and in the environments will be performed.

Analysis of oxalate will be carried out studying the enzyme oxalate oxidase and by its immobilization on the H2O2 electrode surface. Also in this case all the analytical parameters will be optimised, and this probe will be applied on the detection of oxalate in the environment and in the artistic manufacts to study their degradation.

Research activities

1. Construction of potassium and calcium ion potentiometric microelectrodes.

This activity will be the first one in the field of biosensors during the 2000-2001 chair in Biotechnology. The will be a selection of the appropriate "cocktails" to prepare the membranes for K+ and Ca++, also the best strategy to assemble the microprobes will be studied and discussed.

2. The second part of this initial activity will be the optimisation of the reference electrode as the silver-silver chloride microfilament. This is an important step of the procedure, which will deal with the future high stability of the electrodes. This microreference probe will be compared with the traditional reference electrodes in terms of potential stability and probe reproducibility, to complete the first phase of the work-plan.

The capillary preparation using a sophisticated instrument to pull them to the right dimension will be set up and performed. The prepared capillary will be filled with the internal solution and used for future work.
All this activity will last 6 months.

3. The second six months will be devoted to the analytical optimisation of the microelectrodes. Calibration curves of potassium and calcium ion electrodes will be performed using standard solutions. The sensitivity of the electrodes will be tested to obtain the lower and upper limit of detection; even the selectivity of the probes will be tested and compared which that of the commercially available macroprobes.

An important study to be carried out will be the sensor lifetime and drift. Some sensors will be prepared and tested every day to evaluate the signal shift and the reproducibility of the potential measurements.

4. At the end of the first second three months the evaluation of the probes should be completed and the last second three months will be devoted to the application of these microsensors to measure the release ok K+ and Ca++ of cyanobacteria which cause damage to cultural heritage surfaces-inhabitating roman hypogea.

Scientific activities are co-ordinated by Prof Palleschi
Teaching activities are co-ordinated by Prof Colizzi and Prof. Palleschi