| Abstract |
This work focuses on the development of a multi-analyte biosensor, based on
a Surface Acoustic Wave (SAW) device. The novelty of the concept lies in the way of
achieving multiplicity: instead of the “traditional” way of a sensor element array,
multiplicity is induced by compartmentalization of a single sensor, achieved via
microfluidics (“microfluidics-on-SAW”, or “μF-on-SAW” setup).
Initially, the appropriate SAW device for the microsystem was selected among
twelve device configurations (varying in substrate, operating frequency and
waveguide thickness) upon loading with different classes of materials (mass, viscous,
viscoelastic). In particular, a dual quartz-based SAW biochip was used, operating at
155 MHz with 0.70 μm thick PMMA waveguide. Subsequently, the microfluidic
module was designed targeting flexibility and simplicity. Considering functional and
geometrical limitations imposed by the SAW biochip, the two components were
successfully assembled. The fabrication process for the microfluidic module was soft
lithography of PDMS (rapid prototyping and replica molding); 3-, 4-, and 5-channel
modules were made, all successfully tested, and the 4-channel one used in the project.
Reproducibility and sensitivity tests were carried out using aqueous glycerol
solutions, and standard protein biomolecules (neutravidin and biotinylated BSA, as
well as protein G and IgG). The standard deviation in the signal values among the
sub-areas was less than 10%, in all cases.
The proof-of-principle of multi-sample detection was achieved via four
biotinylated molecules. Each one was injected in one μF-on-SAW compartment and
interacted with pre-adsorbed neutravidin; separate detection of the analytes, kinetics
and equilibrium analysis were successfully demonstrated. Maximum multiplexity was
achieved when the two devices of the biochip were pre-functionalized with different
receptors, and four different samples were injected in each microchannel (altogether 8
probed interactions).
The final step was the application of μF-on-SAW in multi-sample detection of
clinical significance. In particular, cardiac markers were used, the detection of which
was realized via antibody-antigen interactions. The four cardiac markers (CK-MB,
CRP, D-dimer, and PAPP-A) were successfully detected individually and in various
concentrations; analytical curves were created for each biomarker and correlation to
the known physiological and pathological values was made. Eventually, by using the
μF-on-SAW it was feasible to selectively capture each marker out of a mixture or all
four, a proof that the system can potentially be used in body fluids (were many
“unwanted” species are present).
Finally, from the different groups of biomolecules detected throughout the
project, interesting results emerged concerning the interaction of acoustic waves with
biomolecules and the correlation of the acoustic signal with inherent properties of
biomolecules such as their molecular weight and viscoelastic nature.
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Development of a multi-analyte acoustic biosensing platform for clinical diagnostics
