Métodos de transducción usados en biosensores: amperometría y fluorescencia

RESUMEN: Los biosensores tienen aplicación en una gran variedad de campos, incluyendo el análisis ambiental, biomedicina, biodefensa, alimentación y agricultura, entre otros. En este tipo de sensores, un material biológico (conocido como biomediador) reacciona con el analito y un sistema de transducción apropiado transforma dicha reacción en una señal eléctrica que puede ser procesada, almacenada o transmitida usando sistemas electrónicos. En este artículo, se describen dos métodos de transducción usados en aplicaciones bio-sensoriales: la amperometria que consiste en la medida del transferimento electrónico (corriente) del biomediador y fluorescencia que es basada en la medida de la luz re-emitida. Se enfatiza en el diseño electrónico (selección de componentes, topologia de los circuitos, problemas comunes y soluciones). Estos diseños han sido utilizados en el desarrollo de instrumentos comerciales para biosensores, caracterizados por bajos costes de producción y portabilidad.ABSTRACT: Biosensor devices have applications in a variety of fields as environmental analysis, biomedical, bio-defense, food and agriculture. On this kind of sensors, a biological material (known as biomediator) reacts with target analytes and an appropriated transduction system converts that reaction to an electrical signal that can be processed, saved and transmitted by using electronic systems. In this article, two transduction methods used for biosensing applications are described: amperometry that is based on the measurement of the electron transfer occurring inside the biomediator and fluorescence, that is based on the measurement of the re-emitted light. Emphasis has been done on the electronics design, including component selection, useful circuit topologies and common problems and solutions. Electronics has been validated for the development of biosensor-based instruments characterized by low production costs and portability

Protective role and in vitro activity of fractions extracted from Lactococcus garvieae, the lactococcosis agent in rainbow trout (O. mykiss)

Effective protocols of vaccination against lactococcosis in rainbow trout (O. mykiss) are still under investigation and the methods currently employed are based on \u201cautovaccines\u201d that are injected intraperitoneally to fish (Bercovier et al., 1997; Prearo, 2006). These vaccines allow a protection for 8 months, when integrated with adjuvants (Ravelo et al, 2006). The bacterial antigenic components involved in the protection are only partially considered by the literature. This investigation evaluated the effect of some fractions of L. garvieae (strain B05/3) in the development of a protective immune response to the infection. Extra cellular products (ECPs), bacterial whole cells (WCs) and membrane antigens (MAs) were injected intraperitoneum to 90g rainbow trouts. Fish were subsequently submitted to an intraperitoneal challenge with L. garvieae (2.6 7105 cfu/individual). The relative percentages of survival (RPS) were 95% for WCs, 35% for ECPs and 33% for MAs. These results suggest that WCs provided the best protection, but also ECPs and MAs were effective. Samples of serum collected from immunized and control fish were analysed by immunoblotting against the SDS-PAGE/Western Blotting protein profile of each bacterial fraction. The control and immunized fish sera contained immunoglobulins able to bind aspecifically the proteins having a molecular weight of 23, 48 and 102 kDa respectively. Similar findings were previously reported by Barnes et al., 2002. Moreover the respiratory burst of leukocytes isolated from rainbow trout head kidney was measured by a luminol-based microtitre plate chemiluminescence assay after stimulation with the L. garvieae extracts. The tests were performed using 20 healthy non-immunized fish, by incubating the cells and the stimulants in presence and absence of autologous serum. Preliminary results suggest an evident individual variability in the response and that the release of reactive oxygen species is strongly affected by the serum addiction. This indicates an important role of antibodies and complement in promoting the leukocyte response to different antigens (Barnes et al., 2002). The specific stimulation ability of each fraction (WC, ECPs, MA) will be discussed

Valutazione della protezione indotta da frazioni antigeniche di Lactococcus garvieae in trota iridea (Oncorhynchus mykiss)

SUMMARY \u2013 Effective procedures of vaccination against lactococcosis in rainbow trout (Oncorhynchus mykiss) are still under investigation and the methods currently employed are based on \u201cautovaccines\u201d that are injected intraperitoneally to fish. These vaccines allow a protection for 8 months, when integrated with adjuvants. The bacterial antigenic components involved in the protection are only partially considered by the literature. This investigation concerns the effect of some fractions of Lactococcus garvieae in the development of a protective immune response to the infection. Extracellular products (ECPs), bacterial whole cells (WCs) and membrane antigens (AM) were injected intraperitoneum to 90 g. rainbow trouts. Fish were subsequently submitted to an intraperitoneal challenge with L. garvieae (2.6 7105 cfu/individual). The treatments allowed the following relative percentages of survival (RPS): 95% for WCs; 35% for ECPs, 33% for AM. These results suggest that WCs give the best protection, but also ECPs and AM are effective. Samples of serum collected from immunized and control fish were analysed by immunoblotting against the SDS-PAGE/Western Blotting protein profile of each bacterial fraction. The control and immunized fish sera contained immunoglobulins able to bind aspecifically the proteins having a molecular weight of 23, 48 and 102 kDa respectively

Valutazione della risposta anticorpale e della protezione indotta da un vaccino per immersione anti Yersinia ruckeri in trota iridea (Oncorhynchus mykiss)

Vaccini costituiti da batteri della specie Yersinia ruckeri, inattivati con formalina e somministrabili tramite immersione, sono disponibili in USA e in Europa a partire dalla fine degli anni \u201970, per la profilassi della bocca rossa o \u201cEnteric Redmouth Disease\u201d (ERM). Nonostante siano state affrontate numerose prove sperimentali per indagare l\u2019esito di questo intervento di profilassi in trota iridea (Oncorhynchus mykiss) risulta ancora poco definita la correlazione fra titoli anticorpali specifici e protezione indotta dal vaccino. L\u2019obiettivo di questa indagine \ue8 stato quello di valutare in vivo la protezione indotta da un vaccino commerciale somministrato per immersione (30 secondi) a trote iridee di 5 grammi, tramite infezione sperimentale con un ceppo virulento di Yersinia ruckeri e di studiare la risposta anticorpale specifica indotta dal medesimo vaccino nelle settimane successive alla somministrazione. Tramite la tecnica di agglutinazione in micropiastra \ue8 stato possibile evidenziare la presenza di anticorpi agglutinanti nel siero dei soggetti trattati a partire da 7 settimane dopo la vaccinazione. La tecnica ELISA indiretta ha dimostrato una maggiore sensibilit\ue0, evidenziando titoli anticorpali significativamente pi\uf9 elevati rispetto ai controlli, a partire da 5 settimane dopo la vaccinazione. La percentuale relativa di sopravvivenza o RPS, collegata all\u2019efficacia protettiva conferita dal vaccino commerciale, \ue8 risultata pari al 77% in seguito a prova di infezione intraperitoneale con 3x106 UFC/sogg., effettuata a 7 settimane dalla vaccinazione. Tale riscontro consente di ipotizzare che gli anticorpi sierici prodotti in seguito alla immunizzazione tramite immersione siano protettivi nei confronti della malattia

Retrieval of spruce leaf chlorophyll content from airborne image data using continuum removal and radiative transfer

We investigate combined continuum removal and radiative transfer (RT) modeling to retrieve leaf chlorophyll a & b content (Cab) from the AISA Eagle airborne imaging spectrometer data of sub-meter (0.4 m) spatial resolution. Based on coupled PROSPECT-DART RT simulations of a Norway spruce (Picea abies (L.) Karst.) stand, we propose a new Cab sensitive index located between 650 and 720 nm and termed ANCB650–720. The performance of ANCB650–720 was validated against ground-measured Cab of ten spruce crowns and compared with Cab estimated by a conventional artificial neural network (ANN) trained with continuum removed RT simulations and also by three previously published chlorophyll optical indices: normalized difference between reflectance at 925 and 710 nm (ND925&710), simple reflectance ratio between 750 and 710 nm (SR750/710) and the ratio of TCARI/OSAVI indices. Although all retrieval methods produced visually comparable Cab spatial patterns, the ground validation revealed that the ANCB650–720 and ANN retrievals are more accurate than the other three chlorophyll indices (R2=0.72 for both methods). ANCB650–720 estimated Cab with an RMSE = 2.27 μg cm! 2 (relative RRMSE = 4.35%) and ANN with an RMSE = 2.18 μg cm! 2 (RRMSE = 4.18%), while SR750/710 with an RMSE = 4.16 μg cm! 2 (RRMSE = 7.97%), ND925&710 with an RMSE = 9.07 μg cm! 2 (RRMSE = 17.38%) and TCARI/OSAVI with an RMSE = 12.30 μg cm! 2 (RRMSE = 23.56%). Also the systematic RMSES was lower than the unsystematic one only for the ANCB650–720 and ANN retrievals. Our results indicate that the newly proposed index can provide the same accuracy as ANN except for Cab values below 30 μg cm!2, which are slightly overestimated (RMSE=2.42 μg cm!2). The computationally efficient ANCB650–720 retrieval provides accurate high spatial resolution airborne Cab maps, considerable as a suitable reference data for validating satellite-based Cab products