Prof. Aldo Roda
Laboratory of Analytical and Bioanalytical
Chemistry
Dept. of Pharmaceutical Sciences
Alma Mater Studiorum-University of Bologna
Layout of the group activities in the field of biosensors
Bioluminescent whole-cell
biosensors
Analyte
Bio-chemiluminescent
miniaturized biosensors for PointOf-Care (POC) applications
Microfluidic devices
based on
chemiluminescence
“contact” imaging
detection
Electronic nose applications
Signal
1) Bioluminescent whole-cell biosensors:
reporter gene technology at a glance
Bacteria, yeast or mammalian cells
A cell is genetically modified with the
introduction of a reporter gene, whose
expression is regulated by a a receptor
or regulatory protein
mRNA
O/P
Reporter
gene
Receptor or
binding protein
Signal
Reporter
protein
When the analyte enters the cell, it binds the receptor
and interacts with the O/P.
This activates the expression of the reporter gene,
with synthesis of mRNA
Analyte
and consequently of reporter protein.
The measurement of the reporter protein provides the
analytical signal.
Biosensors with internal vitality control
Spectrally resolved luciferases
requiring the same BL substrate
Recombinant S.cerevisiae cells for androgenic
compounds with internal vitality control:
- GREEN firefly Luc is androgen dependent
Analyte
Vitality
control
Receptor
PpyRed
Vitality
control
Corrected BL signal
- The RED thermostable mutant is constitutively
expressed
8
6
4
2
0
10 - 1 2 10 - 1 1 10 - 1 0 10 - 9 10 - 8 10 - 7 10 - 6 10 - 5 10 - 4
Testosterone [M]
ARE P
PpyGreen
Specific BL
signal
Corrected BL signal
4
3
2
1
0
10 - 1 2 10 - 1 1 10 - 1 0 10 - 9 10 - 8 10 - 7 10 - 6 10 - 5 10 - 4
Testosterone [M]
Increased robustness and dynamic range
Immobilization of living cells by entrapment in polymeric
networks
Ca2+ alginate
Ca2+ alginate
The viability of the immobilized yeast and
bacterial cells is maintained for up to 35
days at 4 °C.
polymeric matrix
The BL cell-based biosensor comprises two main parts: a CCD sensor and a multiwell
cartridge with immobilized cell. These are in direct contact via a fiber optic taper
2) Microfluidic devices based on chemiluminescence
“contact” imaging detection for Point-of-Care
applications
Point-Of-Care Testing (POCT): Analytical testing performed outside the physical
facilities of clinical chemistry laboratories using kits, devices and instruments suitable for
on-field use.
POCT analytical methods should offer the possibility to:
•Perform sensitive and quantitative assays
•Detect panels of analytes (e.g., sets of biomarkers for a given disease)
•Perform all the analytical steps in a single analytical device (lab-on-a-chip devices)
One of the most promising approaches for POCT development is the combination of:
Biospecific molecular recognition reactions (e.g., antigen-antibody
reactions, nucleic acid hybridization reactions), offering high selectivity and
high affinity/binding constants
Chemiluminescence detection, one of the most sensitive detection
principles, also in small volumes
Microfluidic devices based on chemiluminescence “contact”
imaging detection for Point-of-Care applications
CL substrate
Our final goal is the
realization of a
multiplexed miniaturized
analytical device, where
different types of
chemiluminescence
bioassays (e.g.,
immunoassays and gene
hybridization assays) are
combined to detect, with
high sensitivity, a
complete panel of
biomarkers of a given
pathology.
CL substrate
Enzyme
Enzyme
Immobilized
capture
antibody
Immobilized
oligonucleotide
probe
Our POCT prototype consists in three different components
1_A plastic or glass transparent surface is
functionalized with multiple spots of biospecific
probes (e.g., DNA probes), able to capture the
analytes (e.g., target DNA sequences) present in
the sample.
2_The surface is coupled with a
microfluidic module to deliver samples
and reagents on the functionalized
spots.
3_The surface is placed in direct contact
with an ultrasensitive imaging sensor (e.g.
a cooled CCD sensor) to acquire the
chemiluminescence emission from each
spot. The whole system is included in a
dark box to prevent interference from
ambient light.
Chemiluminescence “contact” imaging detection
Contact imaging, in which the analytical surface is placed
directly in contact with the sensor without intervening optical
systems, was employed in order to exploit its characteristic high
light collection efficiency (due to the large light collection angle)
and to obtain a compact device.
θ
Biofunctionalized surface
Efficient photons
transfer was
obtained employing
a fiber optic taper
Highly
sensitive
cooled CCD
sensor
(Magzero
MZ-2PRO)
CCD sensor
Application: detection of Parvovirus B19 DNA and genotyping
The method provides quantitative
information for B19 DNA, with low LOD.
The method can distinguish between
the three B19 genotype DNA
sequences.
R/S
S/N40
~40
S/N
R/S~8
8
Genotype 1
CL signal (RLU)
10 8
Genotype 2
10 7
LOD= 50fmol/mL
10 6
10 - 3
10 - 2
10 - 1
10 0
10 1
10 2
pmol/mL
(calculated as blank + 3 standard deviations)
Genotype 3
3) Identification of viable pathogenic bacteria by an
olfactory MOS-based sensor array coupled with FieldFlow Fractionation
Our aim: detecting viable pathogenic bacteria through the analysis of their characteristic
olfactory fingerprint by means of a MOS-based electronic nose.
Biomarker VOCs
in headspace
The electronic nose proved
able to discriminate
between E. coli O157 and Y.
enterocolitica when present
in pure bacterial cultures.
Olfactory fingerprint
Y. enterocolitica
However
E. coli
It did not work
efficiently when
bacteria were
present in a
mixture.
Thus, the electronic nose sensing system was coupled with the Gravitational
Field-Flow Fractionation (GrFFF) technique
GrFFF: separation, which takes place within an empty capillary channel, is due to the
combined action of a laminar flow of mobile phase with parabolic profile and of the Earth
gravitational field that acts perpendicularly to the flow. Cells are separated on the basis of
their size and morphological characteristics.
Earth gravitational field
thickness
80-250 µm
Sample in
F1
Flow with parabolic profile
F2
Bacteria populations
elute at different times
GrFFF is a “soft” separation technique, thus after fractionation bacteria retain their viability
and metabolic activity.
Analytical set-up
pump
Gravitational field
600
0.
7
5
0
.
7
5
Injection
port
YERS.
12
3
4.
5
6
GrFFF channel
UV/vis
detector
E.COLI
1 Bacteria mixtures are injected into the
GrFFF system to obtain bacteria
fractionation.
3 Data are elaborated by multivariate
statistical analysis.
2 Collected fractions are subjected to the
electronic nose analysis.
PCA analysis results
Y. Enterocolitica (n=10) E. coli (n=10)
F2 (n=10) F1 (n=10) MIX (n=2)
F1
F1 (Y. Enterocolitica)
F2 (E. coli)
mix E.coli + yersinia
E.coli
yersinia
0,10
0,08
AU
0,06
F2
0,04
0,02
0,00
0
2
4
6
8
10
12
14
16
18
20
tempo di ritenzione (min)
22
24
26
LDA analysis results
Canonical variable2
Graphical representation of the distribution of objects
in terms of the first two canonical variables
Canonical variable 1
E. coli
Y. enterocolitica
TOT
Ability of correct
classification
89%
91%
90.0%
Ability of correct
prediction
90%
92%
91.0%
Selected bibliography
Roda A, Roda B, Cevenini L, Michelini E, Mezzanotte L, Reschiglian P, Hakkila K, Virta M. Analytical strategies for improving the
robustness and reproducibility of bioluminescent microbial bioreporters. Anal Bioanal Chem. 2011, 401:201-11.
Roda A, Mirasoli M, Dolci LS, Buragina A, Bonvicini F, Simoni P, Guardigli M. Portable device based on chemiluminescence lensless
imaging for personalized diagnostics through multiplex bioanalysis. Anal Chem. 2011, 83:3178-85.
Roda A, Cevenini L, Michelini E, Branchini BR. A portable bioluminescence engineered cell-based biosensor for on-site applications.
Biosens Bioelectron. 2011, 26:3647-53.
Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A. Cell-based assays: fuelling drug discovery. Anal Bioanal Chem. 2010, 398:22738.
Michelini E, Cevenini L, Mezzanotte L, Roda A. Luminescent probes and visualization of bioluminescence. Methods Mol Biol. 2009,
574:1-13.
Michelini E, Cevenini L, Mezzanotte L, Leskinen P, Virta M, Karp M, Roda A. A sensitive recombinant cell-based bioluminescent assay
for detection of androgen-like compounds. Nat Protoc. 2008, 3:1895-902.
Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini BR, Roda A. Combining intracellular and secreted
bioluminescent reporter proteins for multicolor cell-based assays. Photochem Photobiol Sci. 2008, 7:212-7.
Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini B, Roda A. Spectral-resolved gene technology for
multiplexed bioluminescence and high-content screening. Anal Chem. 2008, 80:260-7.
Roda A, Mirasoli M, Michelini E, Magliulo M, Simoni P, Guardigli M, Curini R, Sergi M, Marino A. Analytical approach for monitoring
endocrine-disrupting compounds in urban waste water treatment plants. Anal Bioanal Chem. 2006, 385:742-52.
Michelini E, Magliulo M, Leskinen P, Virta M, Karp M, Roda A. Recombinant cell-based bioluminescence assay for androgen bioactivity
determination in clinical samples. Clin Chem. 2005, 51:1995-8.