D:\Docstoc\Working\pdf\eb1ca84d-f53a-49d9-b6c7-ced4364350b6.doc 12/02/10 17.32
Activities of the Laboratory of Analytical Chemistry at the
Institute of Chemical Sciences of the University of Bologna
The laboratory of Analytical Chemistry at the Institute of Chemical Sciences of the
University of Bologna offers technical expertise in
developing analytical bioluminescent and chemiluminescent methods for complex
utilising diverse analytical techniques for the analysis of food, environmental and
The main research topics carried out in the laboratory of Analytical Chemistry at the
Institute of Chemical Sciences of the University of Bologna regards various aspects of the
application of bio and chemiluminescent (BL and CL) dosages.
ANTI-OXIDANT ACTIVITY: A microplate chemiluminescent method based on the
inhibition of the peroxidase-catalysed reaction of luminol was developed. The method
was applied to the analysis of sewage, allowing to verify the state of activity of
wastewater plants and the excessive presence in the output stream of substances
used for sterilisation (peracetic acid).
ERWINIA AMYLOVORA: Erwinia amylovora (Ea) is the causal agent of fire blight of
rosaceous plants, the most destructive infection of cultivated fruit trees like pears and
apples. A molecular diagnostic technique (polymerase chain reaction enzyme-linked
immunosorbent assay [PCR-ELISA]) with chemiluminescent detection was developed
for the detection of Ea. As few as 4 x 10 2 CFU/g of artificially infested pear wings were
detected. This specific, sensitive and reliable method was applied to the control of the
Emilia-Romagna territory, the main European producer of pears. Bees pollen was
analysed in order to discover the presence of eventual infections. The assay was able
to detect the presence of Ea in zones in which the symptoms of the infection had not
still been identified, demonstrating the efficacy of this system, the first one utilising
bees to detect the presence of bacteria in the environment.
BIOLUMINESCENT BACTERIA: In vivo luminescence is a sensitive indicator of
xenobiotic toxicity: if noxious substances are present, the luminescence decreases
proportionally to their concentration. A bioassay for monitoring of toxic compounds was
developed based on bioluminescent bacteria. The assay was performed at room
temperature and proved to be rapid and versatile, even if unspecific. Bioluminescent
bacteria were employed to detect the presence of several chemicals at various
concentrations: heavy metals (mercury and lead), BTEX (benzene, toluene,
ethylbenzene, xylene), and antibiotics. Bacteria could be easily stored at –20°C and
transported after lyophilisation. The use of 96 wells microplates allowed reducing the
working volumes and therefore the cost per assay. Mussels grown in cadmium polluted
water were also analysed by the luminescent bacteria toxicity test. After
homogenisation, mussels were directly tested. Polluted samples were clearly
distinguished from the blank. Mussels in association with bioluminescent bacteria
therefore represent a possible bioindicator to monitor coastal pollution.
River water was also analysed to test the presence of industrial wastewater.
Luminescent bacteria allowed to differentiate samples collected from rivers containing
and non-containing industrial emissions.
The luminescent bacteria toxicity test, compared with other bioassays, proved that its
average sensitivity is well within the same order of magnitude as other tests.
ATP BIOLUMINESCENT ASSAY: the bioluminescent ATP assay was employed to
develop a method for the determination of the bacteria content in sludge from
purification plants. The same principle was employed for the optimisation of a method
for the control of surfaces cleanliness in rooms characterised by the presence of
several people, as for example lecture halls. The method was then employed for
monitoring sanitation efficacy in the lecture halls of the Faculty of Pharmacy of the
University of Bologna.
PESTICIDES: In the framework of an European Copernicus projects,
chemiluminescent ELISA and flow immunoassays were developed for the
determination of DDT and endosulfan in food and environmental samples.
Chemiluminescent detection displayed a lower consumption of reagents and a slightly
better sensitivity than traditional colorimetric one. The I 50 obtained with the
chemiluminescent ELISA was in the range 3-9 nM and 13-22 nM for DDT and
endosulfan respectively. Repeatability and reproducibility were found to be in the range
10-30 %. The lowest limit of detection (LOD), 1 nM p,p-DDT, was obtained with a
membrane-based flow immunoassay with HRP-labelled specific antibody.
A multiresidual method for the gas chromatographic analysis of organophosphorus
and carbamates pesticide residues in honeybees was also developed. The bee is a
good direct indicator of insecticides because it responds with an intense and extensive
mortality to this type of treatment. The method is currently applied to the monitoring of
pesticides and radionuclides in the surroundings of Bologna, on behalf of several
municipalities. Every monitoring station covers an area of approximately 6 km 2 and it
costs about 3000 Euro (, plus vat) for 30 weeks of activity, with a starting initial cost
(only one time) of about 700 Euro. The information supplied concern the utilised
pesticides, the type of crops, and the presence of -emitting radionuclides. The
monitoring, funded from local agencies, is co-ordinated from the environmental cultural
co-operative “La Carlina” and is taken advantages of the collaboration of
entomologists, agronomists, bee-experts, bee-keepers and environmental
ANTI-OXIDANT ACTIVITY: the chemiluminescente method, in cuvette or microplate
format, based on the inhibition of the peroxidase-catalysed reaction of luminol was
applied to the analysis of various complex matrices: wine, beer, oil.
PHOSPHOLIPASE D ACTIVITY AND CHOLINE: A chemiluminescent flow sensing
device for the determination of phospholipase D activity and/or choline in biological
samples using choline oxidase and horseradish peroxidase immobilised on Eupergit C
was developed. The flow biosensor was used for the analysis of samples of different
origin (salad, tomatoes), including rape seeds during maturation.
ALKALINE PHOSPHATASE (ALP): A chemiluminescent assay for ALP based on
dioxetane hydrolysis was developed. Measuring enzymatic activities in milk is useful to
establish the effectiveness of milk hygiene. The presence of ALP indicates inadequate
pasteurisation, mixture with raw milk or bacterial contamination. The assay, able to
detect up to 0.5 nkat/L proved to be simple, effective, rapid and easily performed.
XANTHINE OXIDASE (XOD): A sensitive and rapid chemiluminescent assay for the
determination of the activity of XOD was developed and applied to milk analysis. XOD
content in pasteurised milk depends on the fat content and disappears in UHT milk
owing to the heat treatment.
D- AND L-LACTIC ACID: A bioluminescent flow sensor was applied to the analysis of
the D- and L-lactic acid in beer for the unwanted presence of lactic bacteria.
BACTERIAL COUNT: Several fast and sensitive chemiluminescent methods were
developed for the analysis of milk. The bioluminescent ATP assay was optimised for
the application to bacterial count in milk samples. After the elimination of somatic ATP ,
the measured bacterial content by means of the bioluminescent test proved to be in
good agreement with the classic cultural method and with the results obtained with the
instrument Bactoscan 8000.
Chemiluminescence already had a considerable impact on biomedical analysis. In this
context various chemiluminescent assays were developed for clinical applications,
including the determination of superoxide dismutase activity in erythrocytes, the detection
and quantitation of Parvovirus B19 DNA using a chemiluminescent competitive PCR, the
determination of the antioxidant capacity of serum.
He received his degree in Chemistry at the Faculty of Sciences of the University of
Bologna in 1975. Since 1981 he is researcher at the Faculty of Pharmacy of Bologna
University and since 1987 he is professor of Analytical Chemistry and Clinical Analytical
Chemistry at the University of Bologna. He was in Scientific committees of national and
international Symposium. He made some stages and collaborative works abroad (UK, E,
P) and he was member of PhD Commissions (Cz, E). Several foreign researchers and
students visited his laboratory, also in the frame of Tempus and Copernicus UE Projects.
He is referee for the Journals of Analytical Chemistry. He published more than 130 papers
including reviews and monographs (11), book chapters (11), scientific articles published in
international (82) and national (29) journals. Patents: co-author of the Italian Patent
BO94A000527 for the bioluminescent determination of bacteria in air. He co-ordinated or
participated to several Italian (60%, 40%, National Interest, CNR, Agricultural Ministry and
Industrial grants) and International (EU Copernicus, Tempus, Collaborative Actions IT-E e
Severino Ghini is researcher at the University of Bologna. His main research topics are in
the field of analytical chemistry. In particular he is an expert of environmental biomarkers
(honey bees), of gas chromatographic analysis of pesticides, and of enzyme
immobilisation on various supports, as nylon membranes, methacrylate and controlled
pore glass beads.
Elida Nora Ferri
Elida Nora Ferri is researcher at the University of Bologna since 1996. She carried out
various stages in foreigner countries. Her main interests of research are in the field of
biochemistry and analytical chemistry. In particular she concentrates on the development
of bio and chemiluminescent biosensors for the determination of metabolites, enzymes,
chemicals and bacterial polluting agents in food and in the air. She also works in the field
of electrochemical and luminescent studies on the production of free radicals, and of
radioisotopic marking of drugs and biologically active molecules. She is co-author of more
than 80 scientific papers published on international and national journals, of four licences
and of a book..
After the degree “with honour” in Pharmaceutical Chemistry and Technology at the
University of Bologna (march 1996), she obtained a Ph.D. in Pharmaceutical Sciences at
the University of Bologna, that ended in March 2001. The main fields of interest are the
development and application of immunoenzymatic assays and of flow assays with
chemiluminescent detection in environmental and food analysis. F. Fini is co-author of 18
papers and book chapters published in international and national scientific journals.
After the degree in Pharmacy (July 1998) with an experimental
thesis about "Bioluminescent analyses of micro-organisms and
antibiotics in milk", he has continued to carry out research
activity at the Institute of Chemical Sciences of the University
of Bologna with a fellowship of the project of National Interest
“Endocrine modulators in water and biological systems: new
analytical methods and toxicological impact”. In particular he
develops analytical methods for the control of toxic substances
(heavy metals, BTEX, drugs) by means of bioluminescent bacteria.