Lab-on-chip: PDMS microdevice for miRNAdetection from biological samples

Federica Barbaresco

Supervisors: Prof. Fabrizo Pirri and Prof. Matteo Cocuzza

Submitted and published works

• Canavese G., Ancona A., Racca L., Canta M., Dumontel B., Barbaresco F., Limongi T., Cauda

V., Nanoparticle-assisted ultrasound: a special focus on sonodynamic therapy against cancer,

Chemical Engineering Journal, 340, (2018) pp.155-172.

• Scordo G., Barbaresco F., Bertana V., Potrich C., Lunelli L., Cocuzza M., Marasso S. L.,

Pederzolli C., Pirri C. F., Lab-on-chip for precision medicine. In: 3th edition of the Nanoinnovation

Conference & Exhibition, Roma, 14-16th September, 2018., in press.

List of attended classes• 01REIRR - Terapie avanzate (nanomedicina, terapia genica e cellulare) in chirurgia

(29/06/2018,4 cfu)

• 01LXBRW - Life Cycle Assessment (LCA) (02/07/2018, 5 cfu)

• 01MQLKI - X-ray diffraction by material (exam planned, 5 cfu )

• 02LCRKG – Fisica di superfici ed interfacce (exam planned, 3 cfu)

• 01QORRV - Writing Scientific Papers in English (27/06/2018, 3 cfu)

• 01RISRV - Public Speaking (01/09/2018, 1 cfu)

• 02LWHRP - Communication (04/09/2018, 1 cfu)

Novel contributions

Addressed research questions/problems• Enhance the MiRNA purification and successive detection by increase the Surface-

to-Volume ratio in the reaction chamber.

• From ASYMMETRIC DROP to SPIRAL CHIP

• Increase of the

Surface/Volume Ratio:

ൗ𝑺𝑺𝒑𝒊𝒓𝒂𝒍

𝑺𝑫𝒓𝒐𝒑=9.6

• Lab-on-chip

Research context and motivation• Circulating microRNAs (miRNAs) are a class of endogenous small single-stranded

RNAs which emerged as key players in the post-transcriptional regulation of gene

expression. Synthesized in the cell nucleus, and active in the cytoplasm on

sequence-specific of target messenger RNAs, microRNAs can be secreted in

circulation as cell-free entities. In fact, circulating miRNAs have been detected in

every body fluid, including blood plasma, protected in the extracellular environment

by vescicles or in protein complexes.

• The regulation exerted by miRNAs affects a

variety of fundamental cellular processes

including cell development and proliferation and

cell death. Thus, MiRNAs have gained clinical

relevance as their aberrant expression has been

shown to correlate with the pathogenesis and

progession of several diseases, including cancer.

• Due to miRNAs relevance and easy accessibility, there is an increasing interest in

circulating microRNAs as potential minimally invasive biomarkers for the prognosis,

diagnosis and assessment of response to treatment in personalized medicine.

• Lab-on-chip (LOC) approach could offer several advantages to the introduction of

miRNAs analysis in the clinical practice. Microfluidic devices would allow to integrate

in a cost-effective way a whole analytical process reducing its complexity, time of

analysis, sample and reagents volumes.

Adopted methodologies• Microfluidic devices dimensions are characterized by optical and FESEM microscopy.

• Functionalization times are evaluated by observing the absorption of fluorescent synthetic MiRNA

by fluorescence microscopy.

Future work• Optimize the functionalization dynamic time on spiral device

• Measure the limit of MiRNA detection (LoD)

• Detect MiRNAs from different biological fluids (BSA, Plasma, Blood, Saliva, Urina)

• Automatize the whole process

PhD program in

Electrical, Electronics and

Communications Engineering

XXXIII Cycle

Spiral Fabrication Process

• The microdevice is formed by a polymeric reaction chamber made of PDMS sealed with a flat

silicon surface. The mold of the reaction chamber is made of SU-8 2150 deposited on a silicon

wafer by a photolitographic process and then silanizated by CH₃SiCl₃. PDMS (10:1 w/w) ispoured into the mold for casting on a hot plate at 120 °C for 15 minutes. The molded reaction

chamber is sealed with a silicon surface through a thin intermediate layer of PDMS (10:1 w/w)

spinned on silicon surface at 2000 rpm for 5s and at 4000 rpm for 60s and pre-reticulated on a

hot plate at 70°C for 6 minutes. Finally the top and the bottom parts of the device are bonded by

means of O2 plasma treatment (300W, O2 30%, 2 min) and located in oven at 90°C for 2 hours to

perform reticulation.

Functionalization Time

• Spiral device is functionalized with a silane mixtures containing APTMS (0.1% v/v) and PEG-s

silane (0.9% v/v) through a wet functionalization at 60 °C in dynamic regime (2 μL/min) at

different times to assess the best absorpion of MiRNA on microdevice.

APTMSPEG-s

PDMS PDMS