Transcript Optical biosensor for the detection of NF-kB

Optical fibre sensors for biomedical and environmental applications
resp. F. Baldini ([email protected])
absorbance
Carbon dioxide partial pressure (pCO2) is an important parameter to evaluate the tissue oxygenation. In healthy people, the CO2 level in the stomach equals that of the blood, whereas it
increases in the case of reduced oxygen supply in the gastro-intestinal region, which for instance is the case in shock or heavy inflammation. Its monitoring in the stomach has been shown to
be a convenient method in critically-ill patients and seems to be of particular relevance to patient morbidity and mortality. The present method is gastric tonometry, which is based on the
equilibration of the gastric pCO2 with the sample contained in a silicone balloon inserted inside the stomach (Tonocap, Tonometrics, Datex Ohmeda) . This approach prevents the continuous
monitoring of pCO2 and is characterised by long response times (30-40 minutes).
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An optical fibre sensor has been developed, which is based on the utilisation of a CO2-sensitive layer which changes its absorption with the change in
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CO
[hPa]
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the CO2 concentration (Fig. 1). The membrane consists of three layers: the carrier, the indicator (CO2 sensitive) layer and the protective coating
(Fig.2). The CO2-sensitive layer consists basically of a dye/quaternary ammonium ion pair, which is dissolved in a thin layer of ethylcellulose, together
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with a basic agent. Cresol red and tetraoctylammonium are the pH-sensitive dye and the quaternary ammonium ion used in the ion pair, respectively.
This layer is covered and protected by a gas-permeable and reflective white silicone coating, which prevents interferences (by e.g. sample pH or ionic
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strength), leaching of the dye or the quaternary ammonium salt and allows for the
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gastric juice
measurement of the reflected light by means of optical fibres. The probe head consists of
a piece of black plastic that contains the CO2-sensitive membrane (Fig.3). The optical
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protective coating
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probe head is 7 mm in diameter and 9.5 mm in length. The optolectronic unit makes use of
sensitive layer
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LEDs and solid-state photodetectors as optical sources and detectors, respectively;
carrier
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processing of the signal is performed by means of a lap-top connected to the device via an
wavelength [nm]
A/D card. A catheter containing a single fibre (core diameter: 0.6 mm) is connected with
Fig. 1. Absorption spectra of the sensing
Fig. 2. Cross section of the CO2-sensitive
Fig. 3. Photo of the the front panel of the optoelectronic device, and is coupled with the probe head. The
membrane
optical probe head
membrane for different CO2 values
sensor was carefully characterised in laboratory and compared with Tonocap. The optical
fibre catheter was combined with the Tonocap catheter used for gastric tonometry (Fig. 4). The characteristics of the sensor are summarised in the Table.
Both laboratory characterisation (Fig. 5) and clinical tests (Fig. 6) confirmed the superiority of the optical fibre sensor: thanks to its short response time, the developed sensor is able to
detect rapid changes in pCO2, which Tonocap is unable to detect. Clinical validation was carried out on volunteers and on intensive care patients. Besides the pCO2 measured with the optical
fibre sensor and with Tonocap, the end-tidal CO2 (EtCO2) and arterial pCO2 (PaCO2) were also measured.
Optical biosensor for the detection of NF-kB
required
measured
Measurement range
0-145 hPa
0–150 hPa
Resolution at 0 hPa
1.33 hPa
0.2 hPa
Resolution at 150 hPa
1.33 hPa
Response time (t90)
Response time (t99)
Accuracy
0.6 hPa
10 min
< 1 min
10 min
< 2 min
±5% (±2.66 hPa)  2.5 hPa
Measurement period
24 hours
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optical sensor
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time [h]
Fig. 5. Laboratory characterisation:
comparison with Tonocap.
Fig. 6. Clinical test carried out on an
intensive care patient
In collaboration with: Institute of Clinical Physiology – CNR (Pisa, I), Oak Ridge National Laboratory (Oak Ridge, Tennessee, USA)
Nuclear Factor kappa B (NF-kB), a redox-sensitive transcription factor regulating a battery of inflammatory genes, has been indicated to play a role in the
development of numerous pathological states. Acetylsalicylic acid inhibits not selectively the COX, but recognizes as important co-mechanism of action an anti NF-kB
effect, which provides the means to inhibit simultaneously the expression of numerous inflammatory mediators.
Thereafter, when the anti-inflammatory therapy is applied to cardiovascular diseases, for which the detection
of the efficacy of the therapeutic agent is particularly important, the monitoring of NF-kB concentration (for
example in cellular lysate) represents an important target. The so called NF-kB is the nuclear factor that binds
to the k light-chain enhancer of B cells. It is present in the active form in the nucleus of mature B cells and in
some T cell lines. In most of the other cell types, it is present in an inactive form in the cytoplasm where it is
activated in the presence of inflammatory events. Detection of NF-kB in a biological sample is important to
evaluate both the cellular physiophathological processes and the anti-inflammatory agent efficacy. Some
consolidated techniques for quantify NF-kB (for example ELISA) are not able to discriminate the active form
(able to bind DNA) from the inactive one (IkB complexed), and other method (for example band shift assay)
need laborious steps. In order to evaluate the therapeutic agent efficacy, the detection of NF-kB active form
results extremely important. The demand of a simple and direct method to evaluate the amount of active NFkB in a biological sample can be satisfied using a suitable and reusable biosensor.
Scheme of the optical biosensor to be developed. NF-kB
decoy is a double-stranded DNA containing a NF-kBbinding site which binds activated NF-kB.
Labelled
NF-kB;
Inhibited NF-kB;
Activated NF-kB
Fig. 4. The combined probe
48 hours
Tonocap
pCO2 [hPa]
Continuous monitoring of gastric carbon dioxide
European project coordinated by IFAC. Partners: Joanneum Research (Graz,A), Siemens (Stockholm,S), Prodotec (Firenze,I), Karl Franzens
Universitat, Department of Internal Medicine (Graz, A), Università degli studi di Firenze,Faculty of Medicine and Surgery (Firenze, I)
Our aim is to develop an optical biosensor capable of detecting active NF-kB
concentration, based on the competition between the fluorescent labelled
protein and the protein contained in a biological sample.
Planned tasks
 Synthesis and purification of NF-kB decoy
 NF-kB decoy immobilization on capillary tubing
• NF-kB labelling
• NF-kB detection
• Biosensor regeneration
•
•
•
•
cytokines (IL-1, TNF)
hypoxia/anoxia, hyperoxia
protein kinase C activators
mitogen-activated
protein
kinase (MAPK) activators
• phorbol 12-tetradecanoate 13acetate(TPA);phorbol
12myristate 13-acetate (PMA).
• bacterial products (LPS)
• viral products (dsRNA or Tax
protein)
• UV-radiation
• cytokines (IL-1, TNF, INF-, IL-6)
• iNOS, COX-2
• Adhesion molecules(ICAM-1, VCAM-1)
• immunoreceptors(MHC class I, II)
• Acute-phase proteins
NF-kB specifically recognizes kB DNA
elements with a consensus sequence:
5’ - G G G R N Y Y Y C C - 3’
R = unspecified purine
Y = unspecified pyrimidine
N = any nucleotide
Optical biosensor for the detection of photosynthetic herbicides
With a few exceptions, release of NF-kB is mediated by the degradation of IkB. The inducible
degradation of IkB occurs through consecutive steps of phosphorylation, ubiquitination and
proteasomal degradation.
In collaboration with: Institute of Clinical Physiology – CNR (Pisa, I), Enea (Casaccia, Roma, I)
Herbicides are commonly used in agriculture for the control of weeds. These chemicals and their breakdown products can contaminate run-off and well waters, giving rise to
serious damage to the environment. A new sensing system for the detection of photosynthetic herbicides in water has been developed, based on the use of the Reaction Centre
(RC) isolated from Rhodobacter sphaeroides. The image in Fig.1 is a schematic representation of the photosynthetic apparatus. Photons are absorbed by the light-harvesting
complexes, and excitation is transferred to the RC, thus initiating a charge separation. The electron transfer across the membrane
produces a large proton gradient, which drives the synthesis of ATP. Fig. 2 shows a ribbon model of the Reaction Centre of the
Rhodobacter sphaeroides (RC). Some non-proteic cofactors (Bacteriochlorophyll, Bacteriopheophytin, Ubiquinone-10) are embedded in
this transmembrane protein complex.
The excitation of the RC is responsible for the electron transfer through the cofactors (see Fig. 3). The absorption of a photon
promotes the primary electron donor, i.e. the Bacteriochlorophyll dimer, to its excited state. An electron is sequentially transferred to
an accessory Bacteriochlorophyll (BA), to a molecule of bacteriopheophytin () and, lastly, to the first ubiquinone electron acceptor
(QA), which is located in a hydrophobic pocket of the protein. In the presence of the secondary ubiquinone molecule QB, the electron is
further transferred without returning to the stationary state. The return to the stationary state then takes place with a charge
Fig. 1. Schematic representation of the
recombination rate of about 1 s (yellow arrow). On the other hand, QB is loosely bound to its pocket, and can be displaced from its
photosynthetic apparatus
binding site by competitive inhibitors, such as herbicides. If the QB site is empty or occupied because of herbicides binding, the only
possible recombination path is directly from QA, (red arrow) with a life-time of about 100 ms.
pesticide solutions
The fundamental and excited states of the molecule have different absorption properties, with the fundamental state characterised by
a higher absorption at 860 nm. Therefore, replacement of the QB with herbicide can be evaluated by monitoring the absorption change
in the presence of a suitable excitation light. A light emitting diode at 860 nm and a hybrid photodetector are used as optical source
optical
fibres
and detector, respectively. Optical fibres enable the connection between a 5-cm-long optical cell that contains the RC solution and the
optoelectronic device (Fig. 4). The excitation of BChl takes place in the presence of a pulse of 2.5 s, and an equilibrium between the
excited and the steady state is then established. How this equilibrium is reached and the absorbance value at the equilibrium depend on
the concentration of the herbicides.The system was tested using atrazine solutions that contain the RC at a fixed concentration (2 M).
optical fibres
In Fig. 5, the time dependence of the absorption during the 2.5 second pulse is shown for different atrazine concentrations. The
absorbance value at equilibrium increases with an increase in the atrazine concentration, thus testifying to a progressive replacement of
QB with the herbicide. These curves can be represented by a bi-exponential model:
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ms
ms
Fig. 3. Scheme of the electron transfer processes in the Rhodobacter
Sphaeroides after light excitation
Fig. 2. Ribbon model of the Reaction Centre of
the Rhodobacter sphaeroides
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I
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t
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At
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A
t
Fig. 4. Scheme of the optoelectronic system
At = A1(e-t/1-1)+A2(e-t/2-1)
where At denotes the time-dependent absorption, 1 and 2 are the time constants from the excited state to the stationary state through the secondary (1  1 s) and primary (2
 100 ms) quinone, respectively, and A1 e A2 are related to the RC concentration with the secondary QB present or absent, respectively, in the hydrophobic pocket. The described
method has been used for the detection of five photosynthetic herbicides: diuron, atrazine, terbutryn, terbuthylazine and simazine. Fig. 6 shows the effect of the concentration
of the five different herbicides on A1 value. The obtained detection limit are 0.5 M for terbutryn, 1.0 M for atrazine and terbuthylazine and 10 M for diuron and simazine.
Future efforts will be made to immobilise the RC protein on a solid support with a proper density and to improve the detection limits.
Optical sensor for the detection of nitrogen dioxide
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10 M
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1 M
(1)
Ti(Pc)2 + NO2  (Ti(Pc)2+ NO2-)
(2)
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diuron
atrazine
terbutryn
terbutrylazine
simazine
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10
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[herbicide], M
time, ms
Fig. 5 Transient absorbance changes during the
pulse for different atrazine concentrations
Fig. 6 Effect of the concentration of different photosynthetic herbicides on A1 value
In collaboration with Prodotec (Firenze, I), Istituto di Struttura della Materia – CNR (Montelibretti, Roma, I)
The detection of nitrogen dioxide is very important in environmental applications. This compound is considered to be one of the major pollutants which potentially may exert some public
health impact on our urban and industrialised population centres worldwide. NO2 is a secondary pollutant, because is not emitted into the atmosphere in any significant quantities, but is
formed there by means of chemical reactions, especially during stagnant wintertime weather conditions. The sole precursor of the elevated NO2 levels is nitric oxide (NO), which is
emitted by motor traffic, and by stationary combustion sources such as industrial, commercial and domestic boilers fuelled by coal, gas or oil.
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A sandwich-type metal diphthalocyanine, bis(phthalocyaninato)-titanium(IV) [Ti(Pc)2], is proposed as a selective chemical transducer for the
A
neutral species
optical detection of nitrogen dioxide (NO2). Due to the exposure to NO2, two subsequent oxidations take place:
monocationic species
bicationic species
A1 and A2
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(Ti(Pc)2+ NO2-)+ NO2  [(Ti(Pc)2)++ 2NO2-]
gas-flow line
alumina
membrane
Teflon discs
Through interaction with nitrogen dioxide, this compound undergoes spectral changes in the red and green components of the visible spectrum,
optical fibre
due to changes of the absorption spectra of the neutral, monocationic and bicationic species (Fig. 1). The kinetics associated with these equilibria
is completely different, since the first one is much faster than the second one. Because of the very mild conditions requested for its reversibility,
equilibrium (2) between the monocationic and bicationic species was investigated. Good reversibility and sensitivity were observed; but the
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presence of a continuous, although slow, drift of the baseline, due to a partial reduction in the monocationic species, makes the system unsuitable
for sensing purposes over long periods. Ti(Pc)2 was immobilised on an alumina disc using a spray coating method. The treated alumina disc was
Fig. 2. The optical fibre flow-cell
positioned on the distal end of the fibre inside a suitable flow- cell (Fig. 2). The optoelectronic unit made use of a white light-emitting diode as
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source and two photodetectors, which were located in a thermostatted block capable of assuring a stabilisation of the temperature within 0.02 °C.
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A green filter and a combination of red + infrared filters in front of the two photodetectors, respectively, selected the green and red
Fig. 1. Absorption spectra of the neutral,
components of the spectrum in correspondence with which the two absorption bands of the monocationic species were located. Optical fibres
monocationic and bicationic species
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make possible the connection between the optoeletronic unit and the flow cell. The optoelectronic unit is connected to a laptop by means of a DAQ
card, and software operating in LabView is responsible for the complete drive and control of the unit. Reproducibility and reversibility of the sensor was tested by performing many
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cycles NO2N2UV light (Fig. 3). UV treatment made it possible to detach the NO2 from the Ti(Pc)2 molecule, enabling a return to its initial state. No damage to the Ti(Pc)2 molecule
from the UV treatment was observed. The response of the sensor would be too long if reaching of the steady state should be reached. For this reason, the measured quantity is the
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slope of the response curve in correspondence with every concentration. The slope was calculated by considering the linear fitting of the response curve during the first 12 minutes of
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gas flow (Fig. 4). Response of the optode to different interfering gases at different concentrations was also investigated. The optode was exposed to: 30 ppm of SO2, 100 ppm of CO,
ppm [NO2]
100 ppm of NH3 and 12 ppm of NO with an impurity of 0.6 ppm of NO2. Exposure of the membrane to 6.1 ppm of NO2 before and after the exposure of interfering agents was also
carried out (Fig. 5). The results obtained testify to the ability of the Ti(Pc)2 molecule to act as optical transducer for the detection of NO2. The selectivity of Ti(Pc)2 is extremely high
in regards to nitrogen dioxide, if compared with other phthalocyanines or with other chemical transducers for NO2 detection, which are generally characterised by many cross- Fig. 4. Logarithm of the slope (changed
of sign) vs the concentration of NO2
sensitivities. A detection limit of 0.6 ppm was obtained. Future efforts will be devoted to the improvement of the sensitivity of the sensors down to ppb levels.
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log (-slope)
Fig. 3. Response curve of the optical fibre
sensor:
exposure to nitrogen gas;
exposure to NO2; treatment by UV light
Fig. 5. Response of the optode to
different interfering gases