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II PROGRESS REPORT
Contract n.° EVK4-CT2000-00028
PART B: DETAILED REPORT OF THE CONTRACTOR
Participant 2
Contractor: URTV.DSTC
Principal Investigator:
Prof. Giuseppe Palleschi
Scientific staff:
Prof. Danila Moscone
Prof. Aziz Amine
Dr. Silvia Piermarini PhDS
Dr. Josefina Calvo Quintana PhDS
Address:
University of Rome “Tor Vergata”
Department of Chemical Sciences and Technologies,
Via della Ricerca Scientifica, 00133 Rome, Italy
Telephone
Fax
E-mail
+39 06 7259 4423/4421
+39 06 7259 4328
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
I. OBJECTIVES FOR THE REPORTING PERIOD:
The contribution of this group was to participate in workpackage 4 “Damage quantification by
microsensors’’ tasks “3, 4, 5”. The investigations were planned concerning the application of
potentiometric microelectrodes for the measurements of calcium, potassium, ammonium and nitrate,
the development of amperometric microsensors to measure phosphate, the assessment of the
feasibility of a multiparametric device for the different sensors.
II. OBJECTIVES FOR THE NEXT PERIOD:
The objectives foreseen for WP4 in the third year project include the measurements of potassium,
calcium, ammonium, nitrate and phosphate on biofilms and on unaffected and colonised rocks.
Moreover, the application of portable sensor monitor together different microsensors developed
from Participant 9.
III. MAIN RESULTS OBTAINED (METHODOLOGY, RESULTS AND DISCUSSION):
WP 4
Task 3: Development and application of potentiometric microsensors for the measurement of
potassium, calcium, ammonium and nitrate.
Potentiometric microelectrodes for the measurement of potassium, calcium, ammonium were
constructed as explained in the first annual report and applied to the measurement of their respective
analytes in cyanobacterial biofilms collected from Roman Catacombs.
Concerning potassium and calcium measurements, a high variability between biofilms was found
but no light-dependent variations could be detected. In fact, measurements performed at increasing
irradiance showed that no significant variation in potassium and calcium concentration took place
during the whole experiment. However, the potassium and calcium mean values measured in the
collected biofilms resulted 20-50 times higher when compared with the concentrations measured in
monospecific biofilms cultured in laboratory (Tab. 1).
Table 1. Averaged mM values and standard errors of potassium and calcium concentrations in three
biofilms.
n = number of measurements.
Biofilm
[K+] mM
Sampling sites Mean value  SE
[Ca++] mM
Mean value  SE
(n=5)
(n=3)
CD 15
7.3  1.8
6.9  0.5
CD 13
3.3  0.5
29.0  0.1
CSC 16
2.2  0.3
12  2
Then we realised ammonium and nitrate microelectrodes in the same way as for calcium and
potassium. The calibration curves of the realised microsensors showed a linear response in the range
10-4 to 10-1 M with a slope of 54.0 ± 0.5 mV NH4NO3 for ammonium microelectrode, and a slope of
–55.6 ± 0.6 mV in the range 10-4 to 10-1 M KNO3 for nitrate microsensors.
The ammonium measurements, however, are highly influenced by the presence of potassium, which
represent its main interferent ion. For this reason, ammonium calibration curves in presence of
different potassium concentrations were carried out, in order to quantify this interference by
obtaining the selectivity coefficient.
Next figure shows the calibration curves obtained in absence of potassium (black line) and in
presence of 10 mM of K+ (red line). The selectivity coefficient KNH4+/potassium obtained by two
different methods was equal to 0.1.
Figure 1. Calibration of a NH4+-selective
microelectrode showed the effect of K+ on
the calibration curves (in absence () and
in presence () of 10 mM of potassium).
0
-20
-40
E mV
-60
-80
-100
-120
NH4NO3
-140
NH4NO3 + KCl 10 mM
-160
-180
0.1
1
10
100
+
[NH4 ] mM
Table 2. Average mM values
of potassium and ammonium
concentrations biofilms (n.d.=
not detectable).
Moreover, measurements of ammonium concentration on different biofilms collected in the
Domitilla and S. Callistus catacombs have been carried out, measuring at the same time and on the
same sample the K+ concentration by a potassium microelectrode, and taking in account its amount
in the calculation of the NH4+ concentration. Results are reported in table 2, showing an averaged
concentration below the millimolar range.
The measurements of nitrate concentration of cyanobacterial biofilms are in progress.
Task 4: Development and validation of amperometric microelectrodes for determination of
orthophosphate
Electrochemical microelectrodes to determine orthophosphate in cyanobacterial biofilms have been
developed. The method is based on the reaction of ammonium molybdate under acidic conditions
with orthophosphate to form a heteropolyacid, the molybdophosphoric acid. This latter can be
electrochemically reduced, and the resulting current is proportional to phosphate concentration.
Different materials have been tested as transducer for the reaction, i.e. Pt, Glassy Carbon and
Carbon Paste. From the results, CP has been selected for its best performances and for its suitability
to be assembled as microelectrode. Pastes obtained by thoroughly hand-mixing of 80% of graphite
powder (pre-treated with aqua regia) and 20% of mineral oil were used for microelectrode
assembling. The carbon paste was packed into 20 µm tip of glass capillary tubes. The electrical
contact was established via a tungsten wire (Ø=100 µm) embedded into the carbon paste. As
reference electrode, a silver wire was oxidised at constant potential for 20 - 30 min in a saturated
potassium chloride solution, then introduced into another capillary tube. Both the prepared carbon
paste microelectrode and the Ag/AgCl (0.1 mol/l KCl) reference electrode were placed into a metal
tube acting as counter electrode.
In order to choose the best potential for amperometric determination of phosphate, the
hydrodynamic voltammogram was recorded in the potential range 0.0 V-0.4 V (Fig.2). The
reduction peak at 0.10 V was large but difficult to reproduce due to an apparent absorption
phenomenon. Moreover, we observed high noise of molybdate reduction at this potential. A
potential between 0.25 and 0.30 V (0.28V) was then selected as the best compromise between signal
intensity, baseline noise and rejection of silicate interference.
6
phosphate
Figure
2.
Histograms
represent the phosphate and
the
interference
current
intensity at different applied
potentials.
[phosphate] =
50 µM and [silicate] = 1 mM.
5
Current (nA)
silicate
4
3
2
1
0
0
0.1
0.15
0.2
0.25
0.3
0.4
Potential (V)
The pH effect on the electrochemical behaviour of PMo12O403- shows that protonation accompanies
the reduction. Molybdate concentrations lower than 1mM resulted inadequate to obtain
phosphomolybdate complex, while concentrations higher than 1 mM seem to inhibit the electrode
response. For further experiments 0.2 M nitric acid and 1 mM molybdate were employed at a fixed
concentration of [PO43-] 10-4 M.
The calibration curve (Fig. 3) of CP microelectrode showed a linear response between 0.5 - 20 µmol
l-1 but could be utilised until 100 µmol l-1.
10
2
Analytical parameters
I (nA)
8
6
1
Linearity range
Detection limit
Sensitivity
RSD% (n=3)
r2
0
I (nA)
0
5
[PO4
10
3-
15
20
] (µM)
4
0.5 - 20 µmol l-1
0.5 µmol l-1
0.104 nA µmol l-1
4
0.990
2
0
0
20
40
60
80
100
120
3-
[PO4 ] (µM)
Figure 3. Plot of steady state current at +0.28 V (vs. Ag/AgCl) vs. concentration of phosphate added to 1
mM Mo7O24(NH4)6 in 0.2 mol/L HNO3.
This method was applied to different cyanobacterial biofilms collected from roman catacombs. Four
samples were analysed and results were compared with values obtained using the
spectrophotometric reference method (Tab. 3). The concentration of phosphate found varied
between 5 and 20 g/g.
Table 3. Determination of phosphorus in cyanobacterial biofilms with microelectrode (electrochemical
method) and with spectrophotometric method. Error% = (g/g electr. - g/g spectr.) / g/g spectr. * 100.
The principle of the construction and the use of an amperometric carbon paste microsensors has
been successfully demonstrated. This method showed a good selectivity, a low detection limit and
great advantages for measurements on cyanobacterial biofilms.
Task 5: Construction of a commercial portable device holding together the different
microsensors will be planned and designed.
All the electrical parameters characterising the realised amperometric and potentiometric
microsensors have been communicated to Participant 9 for the assessment of the feasibility of the
multiparametric device. Based on these characteristics, a first prototype of the device has been
prepared.
Deviation from description of work and reasons:
As highlighted in the 1st report, oxalate microsensors were not realised (task 4) given the absence of
this compound in the hypogean sites in study.
Participation to national and international meetings:
Calvo Quintana J., Piermarini S., Moscone D., Palleschi G., Albertano P. – ‘Potentiometric
microsensors for monitoring of cyanobacterial biofilms in hypogean monuments.’ – 7° National
Conference on sensors and Microsystems, Bologna, February 4 – 6, 2002.
Piermarini S., Calvo Quintana J., Albertano P., Palleschi G., Moscone D. – ‘Microsensori: studi
degli effetti della fotosintesi sulle variazioni di pH, potassio e calcio in cianobatteri di ambiente
ipogeo.’ – Convegno in memoria del Prof. Arnaldo Liberti, Roma, 20 – 22 Febbraio 2002.
Calvo Quintana J., Amine A., Piermarini S., Palleschi G., Moscone D. – ‘Studio preliminare per la
determinazione elettrochimica selettiva di ortofosfato in cianobatteri.’ – XVII Congresso
Nazionale di Chimica Analitica, Viareggio, 24 – 28 Giugno 2002.
Publications:
Calvo Quintana J., Piermarini S., Albertano P., Palleschi G., Moscone D. – ‘Potentiometric
microsensors for monitoring of cyanobacterial biofilms in hypogean monuments.’ –
Proceedings of the 7th Italian Conference on Sensors and Microsystems, Bologna, February 4 –
6, 2002. In Di Natale et al. (eds).