Abstract Book of EAVLD2012 - eavld congress 2012

S4 - P - 06
SEPRION-COATED MICROCANTILEVER SENSORS FOR PRP SC DETECTION
Daniela Meloni¹, Danilo Pitardi¹, Maria Mazza¹, Riccardo Castagna 2 , Ivan Ferrante 2 , Carlo Ricciardi 2 , Elena
Bozzetta¹.
¹Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Istopatologia e Test Rapidi, Turin, Italy;
2
Politecnico di Torino, Scienza dei Materiali e Ingegneria Chimica, Turin, Italy.
Cantilever, Seprion, TSE, PrP sc detection
Introduction
The current state of technology to detect animal TSEs relies upon
the post-mortem detection of prions, considering the EU trend in
the age limit for testing [1] and in order to improve surveillance
and food safety, new technologies should be developed to allow
the ante mortem tests for PrP sc proteins. The new tests should be
body fluids-based and performed on presymptomatic animals, but
to be effective they would need to detect small amounts of PrP sc
and to differentiate PrP c from PrP sc .
Mechanical biosensors appear to be one of the most promising
techniques for molecular diagnostics [2]. Dynamic-mode
mechanical biosensors are oscillating devices with a resonance
frequency that changes when molecules land on the cantilever.
Thanks to the monitoring of different resonance modes over
arrays of several cantilever-based microbalances, the selective
identification and quantification of adsorbed mass of the order of
few picograms with very high precision was recently
demonstrated [3].
Here we propose the development of a biosensing platform for
PrP sc
detection based on microcantilever arrays coated with a
selective polymer (Seprion from IDEXX Lab. Inc.).
Materials & methods
Microcantilever arrays were fabricated in a clean room making
use of standard microtechnologies such as KOH bulk etching,
optical lithography and Reactive Ion Etching; they were dipped
into coating solution for few minutes inside a laminar flow bench,
washed with PBS and MilliQ water, and finally dried on a plate
dryer.
An optical lever setup is used to monitor cantilever resonance
curve while vibrating in vacuum environment; a piezoactuator is
employed for the excitation. We calculated the arithmetic mean
and uncertainty of relative frequency deviation Δf/f over the
modes for each cantilever, and then we used the “weighted
average”method to have the best estimation of the true value Δf/f
of the array [3].
PrP sc
was extracted from scrapie positive samples, brainstems
were homogenised in lysis buffer and centrifuged at 22.000 g for
20 minutes, the supernatant was collected and incubated with
proteinase K (20 μg/ml) for 1 hour at 37° C. After centrifugation at
215.000 g for 1 hour, the pellet was dissolved in distilled water.
The coated arrays were incubated for 2h in a water-based
solution containing pathologic PrP sc or simply water as negative
control experiment (“blank”). Samples were then rinsed in DI
water and finally dried in a steam of nitrogen.
Fig. 2. Micro-cantilever array
Fig3. PrP sc capture
Results
Microcantilever arrays were successfully treated with both 0.1%
and 0.2% coating solution: the calculated mass surface densities
seemed to be rather similar over the arrays and dilution, showing
a good homogeneity of the functionalization procedure.
Preliminary data exhibited a relevant negative relative frequency
deviation Δf/f up to nearly 10 times higher respect to blank
signals, showing that the polymer remained active in immobilizing
the target molecule and thus an increment of mass occurred on
cantilever surfaces when exposed to PrP sc solutions.
Discussion & conclusions
Cantilever-based microbalances were successfully coated with
Seprion polymer to selectively immobilize pathogenic PrP sc .
Preliminary data showed that the technique looks promising, but
more focused experiments are needed to exploit its potentiality.
References
1. European Commission, Brussels, 16.7.2010, COM (2010) 384 final.
Communication from the Commission to the European Parliament and the
Council. The TSE Road map 2, a Strategy paper on Transmissible
Spongiform Encephalopathies for 2010-2015.
2. Arlett, J.L., Myers, E.B., Roukes, M.L. (2011). Comparative advantages
of mechanical biosensors. Nat. Nanotechnol. 6, 4:203-15.
3. Ricciardi, C., Fiorilli, S., Bianco, S., Canavese, G., Castagna, R.,
Ferrante, I., Digregorio, G., Marasso, S.L., Napione, L., Bussolino, F.
(2010). Development of microcantilever based biosensor array to detect
Angiopoietin-1, a marker of tumor angiogenesis. Biosens. Bioelect.,
25:1193-1198
Figure 1: Flow chart explaining the new technique