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  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
       matrices.
      utilising diverse analytical techniques for the analysis of food, environmental and
       pharmaceutical samples.


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.


ENVIRONMENTAL APPLICATIONS
 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




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  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.




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 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
   associations.


FOOD APPLICATIONS
 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




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   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.


CLINICAL APPLICATIONS
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.




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STAFF
Stefano Girotti
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
IT-PT) projects.


Severino Ghini
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



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than 80 scientific papers published on international and national journals, of four licences
and of a book..


Fabiana Fini
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.


Bolelli Luca
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.




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