Optical fibre sensors for biomedical and environmental Optical fibre sensors for biomedical and environmental applicationsapplications

resp. F. Baldini resp. F. Baldini (F.Baldini@ifac.cnr.it)(F.Baldini@ifac.cnr.it)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)

0.0 0.5 1.0 1.5 2.0 2.5 3.020

40

60

80

100

120

140 optical sensor Tonocap

pCO

2 [h

Pa]

time [h]

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

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

  required measuredMeasurement range 0-145 hPa 0–150

hPaResolution at 0 hPa 1.33 hPa 0.2 hPa

Resolution at 150

hPa 1.33 hPa 0.6 hPa

Response time (t90) 10 min < 1 min

Response time (t99) 10 min < 2 minAccuracy ±5% (±2.66

hPa) 2.5 hPa

Measurement period 24 hours 48 hours

Optical biosensor for the detection of NF-kB

In collaboration with: Institute of Clinical Physiology – CNR (Pisa, I), Oak Ridge National Laboratory (Oak Ridge, Tennessee, USA)

G e n e e x p r e s s i o n

p 5 0 p 6 5

I k B( C y t o p l a s m )

( N u c l e u s )

S t i m u l i

p 5 0 p 6 5

p 5 0 p 6 5

I k B K i n a s e( I K K )

G e n e e x p r e s s i o n

p 5 0 p 6 5p 5 0 p 6 5

I k B( C y t o p l a s m )

( N u c l e u s )

S t i m u l i

p 5 0 p 6 5p 5 0 p 6 5p 5 0 p 6 5

p 5 0 p 6 5p 5 0 p 6 5

I k B K i n a s e( I K K )

+Capillary glass

NF-kB decoy

Cellular lysate flow

Labelled p50/p65

+

Monitoring of fluorescence increasing

Competition

Regeneration

+Capillary glass

NF-kB decoy

Cellular lysate flow

Labelled p50/p65

+

Monitoring of fluorescence increasing

Competition

Regeneration

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

Scheme of the optical biosensor to be developed. NF-kB decoy is a double-stranded DNA containing a NF-kB-binding site which binds activated NF-kB. Labelled NF-kB; Inhibited NF-kB; Activated NF-kB

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.

Optical biosensor for the detection of photosynthetic herbicides

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

100 ms

100 ms

optical fibres

pesticide solutions

opticalfibres

I

A

t

t

0,00000

0,00500

0,01000

0,01500

0,02000

0,02500

0,001 0,01 0,1 1 10 100 1000 10000

[herbicide], M

A1

and

A2

diuron

atrazine

terbutryn

terbutrylazine

simazine

Fig. 1. Absorption spectra of the sensing membrane for different CO2

values

Fig. 3. Photo of the optical probe head

Fig. 4. The combined probe

monitoring of pCO2 and is characterised by long response times (30-40 minutes).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 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 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

Fig. 5. Laboratory characterisation: comparison with Tonocap.

Fig. 6. Clinical test carried out on an intensive care patient

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.

Fig. 2. Cross section of the CO2-sensitive membrane

• cytokines (IL-1, TNF, INF-, IL-6)• iNOS, COX-2• Adhesion molecules(ICAM-1, VCAM-1)• immunoreceptors(MHC class I, II)• Acute-phase proteins

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.

• cytokines (IL-1, TNF)• hypoxia/anoxia, hyperoxia• protein kinase C activators• mitogen-activated protein kinase (MAPK) activators

• phorbol 12-tetradecanoate 13-acetate(TPA);phorbol 12-myristate 13-acetate (PMA).

• bacterial products (LPS)• viral products (dsRNA or Tax protein)

• UV-radiation

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

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 NF-kB in a biological sample can be satisfied using a suitable and reusable biosensor.

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 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 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.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 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). 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:

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.

-0,030

-0,025

-0,020

-0,015

-0,010

-0,005

0,000

0 500 1000 1500 2000 2500

time, ms

At

10 M

1 M

0

Optical sensor for the detection of nitrogen dioxide

In collaboration with Prodotec (Firenze, I), Istituto di Struttura della Materia – CNR (Montelibretti, Roma, I)

A sandwich-type metal diphthalocyanine, bis(phthalocyaninato)-titanium(IV) [Ti(Pc)2], is proposed as a selective chemical transducer for the optical detection of nitrogen dioxide (NO2). Due to the exposure to NO2, two subsequent oxidations take place:

(1) Ti(Pc)2 + NO2 (Ti(Pc)2+ NO2

-) (2) (Ti(Pc)2+ NO2

-)+ NO2 [(Ti(Pc)2)++ 2NO2-]

Through interaction with nitrogen dioxide, this compound undergoes spectral changes in the red and green components of the visible spectrum, 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 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 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 source and two photodetectors, which were located in a thermostatted block capable of assuring a stabilisation of the temperature within 0.02 °C. A green filter and a combination of red + infrared filters in front of the two photodetectors, respectively, selected the green and red components of the spectrum in correspondence with which the two absorption bands of the monocationic species were located. Optical fibres 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

optical fibre

aluminamembrane

gas-flow line

Teflon discs

strength), leaching of the dye or the quaternary ammonium salt and allows for the 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 probe head is 7 mm in diameter and 9.5 mm in length. The optolectronic unit makes use of LEDs and solid-state photodetectors as optical sources and detectors, respectively; processing of the signal is performed by means of a lap-top connected to the device via an A/D card. A catheter containing a single fibre (core diameter: 0.6 mm) is connected with the front panel of the optoelectronic device, and is coupled with the probe head. The sensor was carefully characterised in laboratory and compared with Tonocap. The optical

Fig. 3. Response curve of the optical fibre sensor: exposure to nitrogen gas;exposure to NO2; treatment by UV light

Fig. 1. Absorption spectra of the neutral, monocationic and bicationic species

Fig. 2. The optical fibre flow-cell

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 cycles NO2N2UV 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 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 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, 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-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.

Fig. 5. Response of the optode to different interfering gases

ppm [NO2]0 2 4 6 8 10

log

(-sl

ope)

-2,0

-1,5

-1,0

-0,5

Fig. 4. Logarithm of the slope (changed of sign) vs the concentration of NO2

Fig. 3. Scheme of the electron tra- nsfer processes in the Rhodobacter Sphaeroides after light excitation

Fig. 1. Schematic representation of the photosynthetic apparatus

Fig. 2. Ribbon model of the Reaction Centre of the Rhodobacter sphaeroides

Fig. 4. Scheme of the optoelectronic system

Fig. 6 Effect of the concentration of diffe-rent photosynthetic herbicides on A1 value

Fig. 5 Transient absorbance changes during the pulse for different atrazine concentrations

protective coatingsensitive layercarrier

gastric juice