Surface treatment and activation protocol of gold surface:

1. Washing the gold surface with piranha solution 2. Surface’s incubation with 35 mM 11-MUA in glycerol/ethanol (1:1) for at least 48 h 3. 3 washing of surface with ethanol 95% 4. Surface’s incubation 50 mM NHS /30 mM EDAC, in recircle for 90 min in 10% ethanol  Functionalization surface:5. Surface’s incubation in 5 ppm of protein A in watery solution 6. Incubation with 1M of ethanolamine 7. Incubation with 15 ppm of polyclonal anti-Salmonella spp antibody (Pab) 8. Washing surface with PBS-T solution

Cross reactivity and hybridation of Polyclonak antibody (Pab)In this preliminary experimental work, we have tested the antibody anti-Salmonella on Salmonella entiriditis, Pseudomonas aeruginosa, Escherichia coli and Listeria monocytogenes by Elisa (Enzyme linked immune-sorbent assay).The selection of an antibody with high specificity is important when developing an immunosensor based on SPR for detection of a pathogen,in particularly Salmonella. The commercial Pab used in the current study does not exhibit any cross reactivity with other pathogen, such us E.coli and Pseudomans aeruginosa, Listeria monocytogenes.

The gold surface has been functionalized according to the protocol and handly we have immobilized the same microbial species, used with Elisa test, making a spot on the surface, and it was hybridized the Pab anti-Salmonella in 1:1000 dilution. Salmonella were spotted in triplicate at decreasing concentrations as indidated in fig. 4. afetr pab anti-Salmonella binding a further incubation with secondary antibody was performed to verify the hybridation of the pab. The secondary anti-body was conjugated with horse-radish peroxidase. The peroxidase emits a light signal when immersed in hydron peroxide and luminol, so with a photosensitive plate, the hybridation signal can be impressed.

RESULTS

SPR-BASED IMMUNOSENSOR FOR THE DETECTION OF

SALMONELLA SPP IN AQUEOUS SAMPLE

References:Zarizkij A.M. Salmonellosis.K.Zdorov’ja, 1988, 3, 160p.Cai, H.Y., Lu, L., Muckle, C.A., Prescott, J.F., Chen, S., 2005. J. Clin. Microbiol. 43 (7),3427–3430. Homola, 2008, Chem.Rev., 108(2)(2008),462-493 Barlen, B., Mazumdar, S.D., Lezrich, O., Kämpfer, P., Keusgen, M., 2007. Sensors 7,1427–1446 De Lorenzis E., Manera M.G., Montagna G., Cimaglia F., Chiesa M., Poltronieri P., Santino A., Rella R., Optics communications 2013, 294, 420-426

Salmonella is one of the most frequently occurring food borne pathogens affecting the microbial safety of food and cause great concern in the food industry. FurthermoreSalmonella infections are a serious medical and veterinary problem.Salmonella infections are zoonotic; they can be transmitted by humans to animals and vice versa. The microorganisms can be frequently found in sewage, sea, and river water and can contaminate a variety of food. Some Salmonella species are restricted to one or few animal species, whilst others have a wider host spectrum (Zarizkij A.M. Et al.,1988)Traditional methods (Zarizkij A.M. Et al.,1988, Pividori M. et al., 2003) for isolating and identifying Salmonella spp in foods relies on a multi-step process involving: preenrichment, selective enrichment in both selective and differential media, biochemical testing and serological confirmation. These cultural techniques for detection of Salmonella spp require 3 – 4 days to provide presumptive results and an additional 1 – 2 days for further biochemical confirmation (Zarizkij A.M. Et al.,1988). As a rule the traditional approaches which are used for the revealing of the infected organisms are time consumable, routine and demand a special laboratory conditions with the very professional staff. For overcoming of these disadvantages there is necessary to develop instrumental methods, in particularly, based on the principles of biosensorics. Depending on the basic transducer principles, there are advances in biosensing technologies that

use SPR biosensor for detection of pathogenic bacteria.  SPR is a label-free optical detection technique, which has been successfully exploited as a technique for real time monitoring of both chemical and biological species (Homola, 2008).The development of rapid, sensitive, specific and reproducible methods for detection of food-associated bacterial pathogens is very important for ensuring food safety. In this study, label-free SPR immunosensors was enveloped for their application in bacterial detection.

INTRODUCT ION

The immunosensor for detection of L.pneumophila using SPR was developed. An efficient and sensible SPR prototype has been designed and realized for the analysis of the proposed immunoassay. The robustness of the results demonstrate that the prototype can be used for the study of different immunosensors with high efficiency, , thereby demonstrating that such application is versatile and applicable to the detection of pathogens dangerous to human health. 

CONCLUSIONS

MATERIALS & METHODS

Fig. 1 schematic drawing of biosensor to recognize Salmonella in water samples. layer 1, glass substrate; layer 2, gold film; layer 3, 11-MUA; layer 4 protein A; layer 5, polyclonal antibody anti-Salmonella spp.

rotator laser He-Ne

neutral filter Beam splitter

Reflected beam Reference beam

photodiode FD1

photodiode FD2

picoammeter picoammeter

SURFACE PLASMON RESONANCE (SPR) TECHNIQUE

SF10 Glass prism

Reflected lightto detector

Incident p-polarized laser radiation

Glass slideAu filmFlow cellassembly

Biomolecular layer

q

Flow in Flow out

By an home-made experimental set-up it is possible to record the SPR

reflectance curve at different angles of incidence of light.

The occurence of a binding event between the

investigated antigen and antibody can be monitored by following the shift of

the angular minimum toward higher angles or the variation in reflectance at a fixed angle (corresponding to the maximum slope of

the curve)

633 nm

Schematic of the SPR system in the Kretschmann

configuration and of the flow cell for liquids

allowing the inlet and outlet of solutions

containing the analyte molecules

SPR measurements

5)

4)3)2)1)

5)

4)3)2)1)

Fig.3. a) schematic drawing of the elisa plate (b), in each well, there is a concentration of bacteria equal to the value indicated at the top. C) change in absorbance as a function of bacterial dilution

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Salmonella spp

E.coliPseudomonas a.

Listeria monocytogenes

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Fig 4. on left 1)1000 CFU/ml Salmonella entiriditis; 2)500 CFU/ml Salmonella enteriditis; 3)Negative control. On right 1)100 CFU/ml Salmonella entiriditis;2) 500 CFU/ml escherichia coli; 3)500 CFU/ml Pesudomonas aeruginosa

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etanolammine

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Fig. 5.Sensor diagram of surface functionalization. The procedure of the transducer preparation included several sequential steps: a) incubation with crosslinker (150 mM 11-MUA, out sensor chamber, and activation of carbossilic group of crosslinker by NHS/EDAC solution (1), (2) immobilization of protein A by covalent binding with ammine group of protein and activated carbossilic group of crosslinker, (3) blocking of activated ammine group, (4) binding of polyclonal antibody with protein A. the sensor diagram shows a shift of signal depending by molecules and their concentration.

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PBS-TPBS PBS PBS PBS

PBS

PBS-T PBS-T PBS-T1000 CFU/mlSalmonella

Negative control Pab anti-Sal

Fig. 6.Sensor diagram of Salmonella detection. In sequence, were injected into the sensor chamber, the following solutions: PBS-T, PBS buffer, 1000 CFU/ml of Salmonella entiriditis, PBS-T, PBS, antibody anti-E.coli, like negative control. In the sensor diagram it can be observed a small variation, but after a washing with a PBS-T solution, the signal came back to baseline. When was injected Pab anti-Salmonella spp, the sensor diagram shows a stable variation after washing with PBS-T and PBS. After the injection of regeneration solution, the signal came down quickly and then it move into the baseline when water is put in sensor chamber.

AcknowledgementsThis work was supported by grants from:ETB-2009-80, SPRAI: Surface Plasmon Resonance biosensors for pathogens detection of Agro-food Interest, Agro/Food

NANOMYC kick-off meeting – Athens, 28.1.2007

Partner 7: BIOTECGEN

NANOMYC kick-off meeting – Athens, 28.1.2007

Partner 7: BIOTECGEN

Enrico De Lorenzis1, Maria Grazia Manera2, Adriano Colombelli2,3, Fabio Cimaglia1, Maurizio Chiesa1,Palmiro Poltronieri4, Angelo Santino4, Roberto Rella2

1. Biotecgen s.r.l, Campus Ecotekne, Via Monteroni, 73100 Lecce2. IMM CNR Istituto per la Microelettronica e Microsistemi, CampusEcotekne, Via Monteroni, 73100 Lecce

3. Dipertimento di ingegneria dell’innovazione, Università del Salento, Campus Ecotekne, Via Monteroni, 73100 Lecce 4. ISPA CNR, Istituto di Scienze delle Produzioni Alimentari, Campus Ecotekne, Via Monteroni, 73100 Lecce