BIOLUMINESCENT WHOLE-CELL

BIOSENSORS
E. Michelini, L. Cevenini, M. Calabretta, A. Roda
Department of Chemistry G. Ciamician, University of Bologna, Italy
INBB, Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy

Why whole-cells for biosensing?

provide information about bioavailable concentration
provide more comprehensive and complex functional information
(e.g., mechanisms of toxicant, apoptotic or necrotic cell death) than
nucleic acid and immunochemical methods
can be grown in two-dimensional (2D) or three-dimensional (3D)
cell-culture systems that can mimic in vivo responses of animal
tissues
can be incorporated in a sensor for multianalyte detection in an
array format
the analysis time and cost are contained

and

why bioluminescent reporter genes

Bioluminescence is the production and emission of light by a
living organism, that involves enzymes or photoproteins.

high quantum yield emission

high specific signal (low background)
no excitation light source is required

BL reporter proteins and photoproteins

BL reporter protein

Organism

Reporter
gene

Mw (kDa)

Substrate

Bacterial luciferase

Vibrio,
Photobacterium,
Xenorhabdus
genera)

lux

80 (dimer)

Aliphatic
aldehyde

Firefly luciferase

Photinus pyralis

Fluc

61 (monomer) Luciferin

Luciola italica

Lit

61 (monomer) Luciferin

Hotaria
unmunsana

Hluc

61 (monomer) Luciferin

Renilla luciferase

Renilla reniformis

Rluc

35 (monomer) Coelenterazine

Railroad-worm
luciferase

Phrixotrix hirtus

PxLuc

60 (monomer) Luciferin

Gaussia luciferase

Gaussia princeps

Gluc

20 (monomer) Coelenterazine

Aequorin

Aequora victoria

Aeq

22 (monomer) Coelenterazine

Obelin

Obelia longissima

Obe

22 (monomer) Coelenterazine

Whole-cell bioluminescent 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
receptor or regulatory protein

mRNA
Reporter
O/P 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.

Whole-cell biosensors bioanalytical applications

Water analysis
(Roda et al., Anal
Bioanal Chem 2011)

Drug screening (Michelini et al,

Anal Chem 2008)

Detection of androgenic
compounds in clinical samples
(Michelini et al., Clin Chem 2005;
Michelini et al., Nat Protoc 2008)

Analyte
Vitality
control

Receptor

ARE P

Evaluation of water
treatment plant
efficiency
(Michelini et al, Biosens
Bioelectron 2005)

Detection of pathogens
effect on cells
(Michelini et al., 2013)

PpyRed

PpyGreen
Specific BL
signal

Assessment of hormonal activity in

moisturizing lotions (Leskinen et al.,
Chemosphere, 2005)

Detection of dioxin-like
compounds
(Michelini et al Anal Lett
2006)

An example: multiplexed BL assay with a new luc from

Luciola italica for of cholesterol-lowering drugs
(In collaboration with Prof. Bruce Branchini, Connecticut College, USA)

A new reporter gene from

Bologna hills

Lit WT

Ppy WT

PCT/US2007/003546

An example: multiplexed BL assay with a new luc from

Luciola italica for of cholesterol-lowering drugs

Coelenterazine

Analyte

Vitality
control

MetLuc
(secreted)

FXR

CMV FXR

CMV Met Luc

Cyp7A1

cyp7a1 RED Luc

RedLuc

D-Luc
cyp27a1 GREEN Luc
GreenLuc

HepG2

Cyp27A1

Turning whole-cell sensing systems into biosensors:

unsolved issues and directions
Long term preservation of cells (e.g., immobilized or liophylized) to
obtain living reagents that can be easily stored and transported)
Multiplexing should be increased (spatial, spectral and temporal)
Robustness
Response time (avoid transcription and reporter protein production)
and possibly re-usability
Containment of genetically modified organisms to avoid
unintentional GMO spreading into the environment

An example: BL biosensor for androgens with internal

viability control
Internal viability control based on constitutive expression of the
P. pyralis red-emitting mutant thermostable luciferase (max =
618 nm)

S. cerevisiae cells were

genetically engineered
to express the human
androgen receptor
(hAR) which drives the
expression of P. pyralis
wild-type luciferase
(PpyWT, max = 557 nm)
through the regulation
of the androgen
responsive element
(ARE) in presence of
hAR agonists

Analyte

hAR

ARE P

PpyRE8
Viability
BL signal

PpyGreen
Androgen
induced
BL signal

Roda et al., Biosens Bioelectron 26 (2011) 364736532

Analytical procedure
Method
Growth of bioreporter yeast cells in
selective medium until specific OD600
A volume of 90L of cell suspension is
transferred to a sterile 96-well microtiter
plate with 10L of sample
Incubation for 2h at 30C
Automatic addition of BL substrate (1mM
D-luciferin, pH 5).
Luminescence measurements: 1 s
integration with 530-570 nm and 610-650
nm band pass filters

530-570 nm
bandpass filter

610-650 nm
bandpass filter

Dose-response curve for testosterone

Non corrected doseCorrected BL signal
BL signal (RLU)

response curve

Viability control signal

Competition studies
IC50
5 mol/L bisphenol A
10 m/L vinclozolin
0.5 nmol/L hydroxyflutamide

The presence of a second luciferase,

requiring the same substrate but
emitting at different wavelength,
allows to correct the aspecific
samples matrix effects with respect to
cell growth and metabolism leading to
increased robustness and dynamic
range.
LOD=0.05nM
Dynamic range 0.5nM-1M
Intra-assay variability: CV%=10%
Inter-assay variability: CV%=14% *

*The intra- and interassay CVs

using separate control strain were
13% and 22%

Portable instrumentation setting

From this...

to that

Cell immobilization
whole-cell viability and light emission kinetics
Yeast cells were immobilized onto a
polymeric matrix comprising natural
and synthetic polymers.
The polymeric matrix was able to
keep cells alive in ready-to-use
cartridges for up to 35 days at 4C.

Calcium alginate immobilization

CV < 18%, 10 replicates

Polymeric matrix immobilization

CV < 10% ,10 replicates

Yeast
Bacteria

Multi-biosensing platform with contact imaging detection

Different whole-cell biosensors
specific for different analytes can be
immobilized in different positions
Entrapped
living cells

3x4 wells array

CCD sensor

Fiber optic
taper

CONTACT IMAGING

Sony MZ-2PRO CCD cooled camera;

sensor: 10,2x8,3mm; 1,4 MP

high light collection efficiency

(up to 50%)
very simple and compact
devices

Integration into a portable device

Components of
the device

Sample analysis procedure

Cartridge selection
(stored at +4C)

Addition of the sample

(5l);
incubation (2h at 30C)

Substrate addition (30 l

D-luciferin 1mM, pH 5)

Bioluminescent signal
acquisition (1min)

PCT/IB2010/050625

Analytical problem

Our BL androgen-responsive biosensor

can be used as a rapid cost-effective antidoping screening tool?
Would our assay be able to detect doping
cheats when conventional tests fail?

BL biosensors for antidoping

(In collaboration with Prof. Rane, Karolinska Institutet)
We analyzed serum and urine samples of 55 men who were injected
testosterone.
17 of the men tested negative with the standard urine test, never reaching
the cut-off value of 4 due to a genetic variant.
The result is that they may be able to take testosterone with impunity.

The androgenic activity

measured with our
biosensor could be
used to reveal false
negative samples?

Average urinary T/E ratios for 15 d in

the different genotype groups

Analysis of urine samples

Urine samples of these individuals were analyzed with the wholecell biosensor
The total androgenic activity was
evaluated as testosterone
equivalents on day 2, 4 and 15
after testosterone administration
with respect to the basal activity
at day 0.
AR activity was normalized for
creatinine level in order to
correlate for variances in urinary
dilution.
The mean androgenic activity
increased 4-5 fold on days 2 and
4 after testosterone intake.

Basal AR activity

del/del

ins/del

ins/ins

The increase in AR-activity

was independent of the
genetic variants
Ekstrm L et al., Eur J Clin Invest. 2013 Mar;43(3):248-55.

Analysis of synthetic AAS

Dose-response curves for synthetic AAS
obtained with the biosensor

(Dianabol)

(Nandrolone)

(Androstanolone)

Analyte

EC50 [nM]

LOD [nM]

Testosterone

15 3

0.57 0.04

Androstanolone

7 2

0.33 0.05

Dianabol

18 4

0.78 0.05

Boldenone

22 4

1.2 0.2

Nandrolone

35 6

1.5 0.6

Corrected BL signal

(Boldenone)

Analyte [M]

The yeast bioreporter was able to detect androgenic activity

of exogenuos synthetic anabolic androgenic steroids
commonly used in doping practice

Androgenic activity of drugs seized by State Police

(In collaboration with Dr. M. DElia,
Scientific Police)

AR activity expressed as percentage of

maximal DHT induction.

Sample No. 14
Counterfeit Norma
Hellas nandrolone
decanoate
(testosterone
propionate)

Sample No. 15
Pink five-sided pill with
a twisted-snake
symbol containing 5
mg/tablet
methandrostenolone

Anti-androgenic activity of drugs seized by State Police

Normalized AR activity
(% of induction by DHT)

BLIND ANALYSIS OF SAMPLES

100

50

H
T

10
n
bl M
an
k
1
2
3
4
5
6
7
8
9
10
11
12
13
16
17

Sample

Strong anti-androgenic activity of

samples containing 9-THC,
cannabinol and cannabidiol

Sample No

Identified compounds

9% morphine, thebaine, codeine, papaverine

Diacetylmorphine

Methamphetamine

Gamma butyrolactone

3,4-methylenedioxymethamphetamine (MDMA,
ecstasy)

Methadone

1-(3-trifluoromethylphenyl)piperazine (TFMPP)

4-methylmethcathinone (mephedrone, 4MMC)

7% (9)-tetrahydrocannabinol (9-THC),
cannabidiol (CBD), cannabinol (CBN)

10

3% 9-THC, cannabidiol (CBD), cannabinol (CBN)

11

4% 9-THC

12

23% diacetylmorphine, caffeine,

dextromethorphan, mannitol

13

30% Cocaine, 30% paracetamol, levamisole

16

13% 9-THC

17

88% Cocaine

Cevenini et al., Anal Bioanal Chem, 2013, Jan;405(2-3):1035-45

Field deployable bioluminescent whole-cell

biosensors: so near and yet so far
Despite the obvious potential of genetically engineered BL whole-cell
biosensors for field-deployable applications, their actual exploitation is very
limited.
Some technical limitations, mostly related to their scarce robustness and
short shelf-life of cells, together with current regulatory issues explain this
lack of commercial exploitation (applications outside laboratories until now
are restricted to naturally occurring luminescent bacteria)
Hybrid devices

New approaches to keep cells

in a dormant state

Acknowledgements
Group of Analytical and Bioanalytical Chemistry headed by Prof Aldo Roda
(University of Bologna)
Jenny Schulze, Lena Ekstrom, Anders Rane (Karolinska Institutet)

Marcello dElia, Gabinetto Regionale di Polizia Scientifica per l'Emilia

Romagna

Looking for
collaborations to develop
high-tech low-cost
biosensors

Detection: optics-based vs. contact imaging

Conventional, optics-based imaging

Contact" imaging

CCD

Imaging device (imaging

sensor plus optics)

Light collection angle

Imaging lensless sensor

q
CCD

Measurement
cell

Pros:
high resolution
low cross-talk
Cons:
low light collection efficiency (only a
small fraction - usually less than a few
percent of the emitted light is
collected)

Pros:
high light collection efficiency (up to
50%)
very simple and compact devices

Cons:
the absence of optics reduces the
resolution and increases cross-talk

polymeric matrix

Kinetic measurements with immobilized

cells showed a temporal window of about
5min (ranging from 4 to 9min), in which
the intensity of the BL signal was
maintained at an approximately constant
level

calcium alginate
liquid

Cell immobilization
The matrix thickness was critical for BL
substrate diffusion and/or oxygen
limitation in cell-matrix layers.
In a 3mm cell-matrix layer, 30 s after
substrate addition, 60% of the BL signal
arose from the uppermost 1mm-layer it
took 5min for the BL signal to be almost
uniformly distributed across the whole
cell-matrix layer.

Calcium alginate immobilization

CV < 18%, 10 replicates

Polymeric matrix immobilization

CV < 10% ,10 replicates

30 s

5min