Recent improvements in genomics and proteomics connected to the great improvement of micro and nano-technologies open very interesting perspectives in the rich and strategic field of therapy and diagnostic for biomedical, pharmacological and agro-industrial applications. Therefore, the development of highly integrated devices able to rapidly and automatically perform thousands of complex biochemical analysis is strongly needed.
The global objective of this research activity is the development of advanced microfluidic devices (Lab-On-a-Chip, LOC) for genomics, post-genomics, proteomics, cells and molecular analysis. The solutions we design introduce great improvements compared to the existing ones in terms of sensitivity, cost reduction, speed, predisposition to automation, reliability and repeatability.
In particular, we are developing different modules for extraction/purification of DNA, amplification through PCR and hybridization/detection through traditional fluorescence approach or through innovative approaches (integration with micro-cantilevers or Surface Electromagnetic Waves, as an example). These modules have been developed, at an early stage, with traditional microelectronic and micromechanical materials such as silicon and glass, eventually with integrated polymeric interconnections . Nevertheless the cost of producing systems in silicon or glass is driving the seeking of other materials. Commercial manufacturers of microfluidic devices see many benefits in employing polymers that include reduced cost and simplified manufacturing procedures, particularly when compared to glass and silicon. An additional benefit that is extremely attractive is the wide range of available polymer materials which allows the manufacturer to choose materials properties optimised for their specific application.
As a consequence current improvements are involving several aspects of our first solutions:
- application of polymeric materials (PDMS, COC, PC, PMMA, ...) in substitution of the standard silicon/glass pair, to be patterned and defined in their properties through hot embossing  or casting in situ on master devices produced thanks to the facilities of the Group;
- introduction of more advanced (in terms of sensitivity and resolution) detection techniques based on three solutions actually under study: microcantilevers to be used as microbalances, Organic ElectroChemical Transistors (OECTs) and enhanced photonic devices, for instance through the integration of nanostructured waveguides for fluorescence DNA analysis (Surface Electromagnetic Waves);
- improved control and modification of the surface properties through wet or dry processing.
In the following a short description of the main LOC R&D activities is reported.
LOCs for genomic assay preparation
In the last decade polymer microtechnology has become robust and reliable enough to enable the design and fabrication of all-polymeric LOCs. In this framework, PDMS is one of the most preferred materials for polymeric LOCs, having gained attention for microfluidic applications since the end of the nineties. The use of PDMS elastomer for miniaturized bioassays provides unmatched advantages over silicon and glass: it is a low-cost, flexible, and optically transparent material. Additionally, a major advantage of PDMS is the simple casting processing by which it can be fabricated into complex shapes without the need for time consuming and expensive cleanroom facilities. Moreover, it can be easily bonded to other substrates of the same material, or those made of silicon and silicon-like surfaces. This last property is something unique in microtechnology, actually representing a key point for the fabrication of low-cost disposable Lab-On-a-Chips (LOCs).
Sample preparation for a genomic assay (namely extraction, purification and amplification) is still the main issue and bottleneck for a full success of a POC device based on LOC technology. DNA purification is hardly performed on chip and when reported it usually requires chaotropic salts or magnetic beads. With regards to such an issue we demonstrated an innovative way to take advantage of the non-specific interaction of PDMS with biological samples. We showed that the nonspecific absorption of DNA on PDMS surface occurs in such a way that the DNA chain is directly available for further PCR reaction, without requiring detachment. Purification systems based on PDMS usually rely on a solid-state purification, namely cellular lysis, adsorption and elution. According to the aforementioned properties we developed an innovative purification LOC requiring only cellular lysis and DNA adsorption on a PDMS surface, without elution . The purified DNA is suitable and abundant enough for a PCR reaction to be performed directly on it.
The PDMS chip was successfully designed for DNA purification from complex biological samples like whole blood and direct amplification in a two-steps protocol in the same microfluidic chamber. DNA purification was performed directly on microdevice surface without external agents (like chaotropic salts or magnetic beads), and the amplification reaction was achieved directly on the immobilized DNA
An extensive characterization of PDMS chips was performed, both chemically and morphologically, in order to better understand the results. Moreover, using a different polymer base to curing agent ratio in fabricating the PDMS devices, we obtained different PCR yields. This work shows a way to design PDMS surfaces able to perform solid phase extraction without the need of devices having complex, expensive and technologically challenging layouts.
The as developed LOC was successfully tested on different assays and biological models: amplification of a region part of the hemochromatosis gene (HFE), amplification of a gene involved in cystic fibrosys, multiplex PCR of 3 genes (hlg, hla and hld) of Staphylococcus aureus, RNA retrotranscription to cDNA and nested-PCR of the 5’UTR region of the Hepatitis C virus (HCV).
The efficiency and selectivity of PCR is strictly related to the execution of fast thermal transitions among the three temperatures required by the reaction. Reducing the amount of material that must be thermocycled during PCR protocol is the key concept that drove the development of LOCs for DNA amplification. Miniaturized devices with reduced mass exhibit a lower thermal inertia than traditional bench-top instruments, increasing the amplification efficiency. Despite polymeric LOCs promising characteristics, their low thermal conductivity is the main problem to face to design disposable PCR LOCs, being rapid temperature transitions and uniform steady state temperature difficult to achieve. Polymeric composites, in the case of filling with thermally conducting nanomaterials, can show a dramatic improvement in thermal response with respect to traditional polymers. Among the possible choices, PDMS matrix filled with carbon nanotubes (CNTs) has recently shown great potentialities in different application fields
With regards to this we fabricated and optimised a stationary PCR device made of PDMS/CNT nanocomposite with different multi-wall CNTS (MWCNTs) content . LOCs for PCR protocol were fabricated, both with pure PDMS and with different filler loadings. We characterized the thermal response of the materials, both by means of laser flash measurements and directly on-chip during PCR thermocycling. Finally, the devices were tested performing two PCR protocols for Human Actin Gene and Human β-Actin amplification. The better thermal behavior of the nanocomposite materials is directly converted in an increase of the PCR efficiency, a reduction of cycles number to achieve amplification and a reduction of the time required by each of the denaturation, annealing and extension steps.
Among the other topics, we are currently working on the implementation of PCR and genomic assay preparation on advanced polymeric photocurable materials [5, 6, 7, 8, 9].
LOCs for mutations detection
In recent years, many efforts have been spent to step forward personalized medicine. Accurate diagnosis for a specific patient is often possible only by finding out the DNA signature of the potential disease. In order to gain such information at the patient bed, doctors need a powerful and efficient technique together with a ready to use diagnostic kit running in a simple portable system. The most important hereditary diseases are caused by single nucleotide mutations in a specific DNA segment of the human gene, the so called Single Nucleotide Polymorphisms (SNPs). Only few assays are suitable to operate SNPs detection on a microarray glass slide. Among these, the Arrayed Primer EXtension (APEX) of DNA strands on a microarray chip is a promising technique able to ensure high efficiency and very quick response.
A LOC, fabricated through MEMS technology employing a LIGA-like process, was developed to execute a genetic protocol for the SNPs detection [10, 11, 12]. The LOC was made in PoliMetilMetAcrilate (PMMA) and has two levels: the lower one for the insertion and mixing of the reagents, the upper one for the interfacing with the DNA microarray chip. The hereditary hearing loss was chosen as case of study, since the demand for testing such a particular disorder is high and genetics behind the condition is quite clear. The vast majority mutations causing such a disorder are located in the Connexin 26 gene and the microarray chip was designed to contain the most relevant mutations from the same gene. The Arrayed Primer EXtension (APEX) genetic protocol was implemented on the LOC to analyze the SNPs. A low density (for detection of up to 10 mutations) and a high density microarray chips (for detection of 245 mutations in 12 genes), containing the primers for the extension, were employed to carry out the APEX reaction on the LOC. Both the microarray chips provide a signal to noise ratio and efficiency comparable with a detection obtained in a conventional protocol in standard conditions.
Our results demonstrate that a LOC approach is effective for a relatively complex genetic analysis and open the chance for a massive screening of genetic diseases, like the hearing loss studied in this work. Accordingly, it is possible to imagine the use of the low density chip, along with the LOC cartridge, on a Point-Of-Care (POC) system at the patient location, while employing the high density microarray and the same LOC cartridge inside an automated system at hospitals or laboratories for a full analysis.
Fluorescence in situ hybridization (FISH) represents a major step in the analysis of chromosomal irregularities in cancer. It allows for the precise detection of specific rearrangements, both for diagnostic and eventually prognostic purposes. In the framework of a scientific collaboration with Tethis S.p.A. (http://www.tethis-lab.com/), we succeeded in contributing to the development of a miniaturized FISH method performed on fresh and fixed hematological samples. The procedure has been developed on a platform based onto a PDMS microfluidic device that integrates cluster-assembled nanostructured TiO2 (ns-TiO2) as a nanomaterial promoting hematopoietic cell immobilization in conditions of shear stress.
As a result of the miniaturization, FISH can be performed with at least a 10-fold reduction in probe usage and minimal cell requirements, creating the possibility of using FISH in genetic screening applications. The miniaturized FISH implementation demonstrated comparable performance to standard FISH, indicating that it is suitable for genetic screenings, in research, and in clinical settings for the diagnosis of samples from onco-hematological malignancies.
FISH technology has remained substantially unmodified since its introduction approximately 20 years ago and its widespread utilization was mainly hampered by its cost. To reduce costs and improve assay performance, microfluidics can provide a means for miniaturization through the engineering of polymeric microchannels in devices wherein reagents can be loaded in small volumes, and cellular samples can therefore be concentrated. However, relevant technical challenges have to be overcome: due to the micrometric section of channels in such tools, flowing fluids cause intense shear stress on cells. This can cause them to be easily disrupted or detached, which can therefore compromise the assay.
One of the main competences of Tethis is the research and the characterization of biomaterials and coatings with properties promoting cell adhesion: a cluster-assembled nanostructured TiO2 coating (ns-TiO2) was demonstrated being able to trigger a rapid and efficient immobilization of both living and fixed hematopoietic cells, even in the presence of prolonged shear stress. To fully exploit this feature, we contributed to the engineering of a simple device, based on microfluidics, to set up a miniaturized FISH approach. The device consists of a polymeric microfluidic pad made of polydimetylsiloxane (PDMS, Sylgard 184; Dow Corning, Midland, MI, USA) at a 10:1 ratio, with a straight microchannel. The PDMS microfluidic pad has been manufactured according to standard replica molding procedures. The mold feature defining the microchannel was carefully machined to avoid the formation of surface defects and keep roughness well below the micrometric scale on the top of the PDMS microchannel. This limited light scattering, allowed microscopical inspection of the cellular sample through the microchannel and prevented the formation of bubbles during the steps of the FISH protocol. The microfluidic pad was manually assembled on top of a glass slide previously coated with 50 nm ns-TiO2.
The efficiency of the approach was tested by Tethis, by performing FISH on a panel of cultured hematopoietic tumor cells as well as on bone marrow from normal donor, prepared from fresh samples.
The described approach yields several improvements when compared to a standard FISH protocol, while preserving the same level of quality:
(i) the assay cost has been substantially reduced by decreasing the amount of probe required;
(ii) rare cells can be processed and evaluated;
(iii) the protocol is suitable for automation and increased throughput.
LOCs for cells analysis
The cell migration mechanisms play a fundamental role in physiological adult cells processes as well in cancer metastasis as in embryogenesis. The cells move in a 3D environment and are subjected to different kinds of stimuli. However, our ability to understand their formation, function, and pathology has often depended on two-dimensional (2D) cell culture studies or on animal model systems. The employing of microfluidic platforms for cell culture and analysis leads to a better mimic of a 3D environment since it allows for a more accurate cell monitoring, fluids handling and instauration of chemical concentration gradients. In vitro 3D tissue models provide a third approach that bridges the gap between traditional cell culture and animal models. New results could be obtained transferring 2D assays for angiogenesis and embryonic stem (ES) cells. Moreover, microfluidic platforms ensure a high throughput testing due to miniaturized wells integration in a small area of the substrate.
We are currently working on the development of a microfluidic multiwell platform to analyze chemotaxis mechanisms on ES cells. These cells, which could be induced to differentiate in about 220 adult cells types, represent a well-known model to be studied for a widespread number of applications.
A PDMS microfluidic platform was designed and fabricated using standard MEMS (Micro Electro Mechanical System) technology. A parallel microchannels configuration was chosen to bring stimulating or inhibiting liquids to the micro-chambers. A micro array of pillars allows for the interface with the micro-chamber containing the spheroid. These microstructures act as a barrier that maintains the liquid in contact with the micro-chamber and let the reagents diffuse from the mainstream avoiding any mixing with the reagents in the parallel micro channel. The spheroids (size: 500 µm in diameter), containing the ES cells, were maintained in a hydrogel and pipetted in the micro-chamber.
The PDMS device was obtained from a SU-8 master and bonded on a cover slip glass slide. The inlet and outlet of the chip were obtained by using a PMMA counter mold, which is mechanically aligned on the SU-8 pattern.
In order to analyze the response, the ES cells were engineered to express a fluorescent protein when a specific subtype was obtained from a given induced stimulus.
LOCs for controlled drug delivery
Since many years liposomes, i.e. lipid vesicles formed by phospholipids bilayer membranes, are widely studied as they represent good candidates for a reliable and efficient drug delivery. Since the '90s, several liposome-based drugs have been approved for clinical applications and are already commercially available. Many other liposomal formulations are constantly under evaluation, and currently the interest in the field is still growing. In particular, clinical research has demonstrated the improved efficiency of drugs administered by liposome carriers in a number of cancer therapies, such as pegylated liposomal doxorubicin (Doxil®) for the treatment of advanced ovarian cancer, metastatic breast cancer and AIDS-related Kaposi's sarcoma. The main properties making liposomes advantageous as nanocarriers are, among others, their biocompatibility, the ability to entrap both hydrophilic drugs in the inner area and hydrophobic drugs into the membrane, their selectivity with respect to the target (i.e. the tumor where the drug should be released) and the wide range of tunability of size and available surface charge.
Despite undeniable advantages, there is a large demand for new and reliable technologies providing in vitro real-time monitoring of several different liposome properties such as surface chemistry and charge, drug loading and release or stability under different conditions (temperature, salt concentrations, pH, etc.). In particular, monitoring of liposome formation and dynamics could be very useful both for pharmaceutical manufacturing and for the quality assurance assays applied to liposomal formulations.
With regards to this, we developed a microfluidic system based on Organic ElectroChemical Transistors (OECTs) fabricated by IMEM-CNR researchers for liposome sensing , well suited to detect the presence of liposomes and liposome-based nanoparticles in solution. OECTs show a dynamic range starting from 10−5 mg/ml, suited for in-vivo application of liposomes. The sensor operating at low voltages (Vg=0.6 V), shows a lowest detection limit in realtime monitoring of liposome evolution as low as 10−7 mg/ml.
We are currently working on an evolution of the microfluidic/OECTs system to integrate the capability to detect drug release as a consequence of liposome lysis.
Tel. +39 011 090 7392
- "Evaluation of different PDMS interconnection solutions for silicon, pyrex and COC microfluidic chips", G. Canavese, E. Giuri, S.L. Marasso, D. Perrone, M. Quaglio, M. Cocuzza, C.F. Pirri, J. Micromech. Microeng., 18 (2008) 055012
- “Cost efficient master fabrication process on copper substrates”, S. Marasso, G. Canavese, M. Cocuzza, Microelectronic Engineering, Vol. 88(8), August 2011, Pages 2322-2324, doi:10.1016/j.mee.2011.02.023
- "Solid phase DNA extraction on PDMS and direct amplification", L. Pasquardini, C. Potrich, M. Quaglio, A. Lamberti, S. Guastella, L. Lunelli, M. Cocuzza, L. Vanzetti, C. F. Pirri, C. Pederzolli, Lab Chip, 2011, 11 (23), 4029 - 4035, DOI: 10.1039/c1lc20371a
- "Elastomeric nanocomposite based on Carbon Nanotubes for Polymerase Chain Reaction device", M. Quaglio, S. Bianco, R. Castagna, M. Cocuzza, C.F. Pirri, Microelectronic Engineering, Vol. 88(8), August 2011, Pages 1860-1863, doi:10.1016/j.mee.2011.01.032
- "Photopolymerization of a perfluoropolyether oligomer and photolithographic processes for the fabrication of microfluidic devices", A. Vitale, M. Quaglio, M. Cocuzza, C.F. Pirri, R. Bongiovanni, Eur Polym J, 2012, 48 (6), pp. 1118 - 1126, doi:10.1016/j.eurpolymj.2012.03.016
- "Siloxane photopolymer to replace polydimethylsiloxane in microfluidic devices for Polymerase Chain Reaction", A.Vitale, M. Quaglio, S. Turri, M. Cocuzza, R. Bongiovanni, Polymers for Advanced Technologies, 24, 2013, pp. 1068–1074, DOI: 10.1002/pat.3189
- "Direct Photolithography of Perfluoropolyethers for Solvent-Resistant Microfluidics", A. Vitale, M. Quaglio, S. L. Marasso, A. Chiodoni, M.Cocuzza and R. M. Bongiovanni, Langmuir, 2013, 29 (50), pp 15711–15718, DOI: 10.1021/la402755q
- "Blue and UV combined photolithographic polymerization for the patterning of thick structures", E. Fantino, A. Vitale, M. Quaglio, M. Cocuzza, C.F. Pirri, R. Bongiovanni, Chemical Engineering Journal, 2015, Vol. 267, pp. 65-72, DOI: 10.1016/j.cej.2014.12.088
- "Oxygen Inhibition Lithography for the Fabrication of Multi-Polymeric Structures", A. Vitale, M. Quaglio, A. Chiodoni, K. Bejtka, M. Cocuzza, C.F. Pirri and R. Bongiovanni, Advanced Materials, 2015, Vol.27(31), pp. 4560-4565, doi:10.1002/adma.201501737
- "APEX protocol implementation on a Lab-on-a-chip for SNPs detection", S. Marasso, G. Canavese, S. Lobartolo, M. Cocuzza, A. Ferrarini, E. Giuri, D. Perrone, M. Quaglio, A. Ricci, I. Vallini, Microelectronics Engineering, 85 (2008), 1326-1329 (doi:10.1016/j.mee.2007.12.024)
- "A Multilevel Lab On Chip platform for DNA analysis", S. L. Marasso, E. Giuri, G. Canavese, R. Castagna, M. Quaglio, I. Ferrante, D. Perrone, M. Cocuzza, Biomedical Microdevices, Vol. 13, Issue 1 (2011), pag. 19 (doi: 10.1007/s10544-010-9467-5)
- "A polymer Lab-on-a-Chip for genetic analysis using the arrayed primer extension on microarray chips", S.L. Marasso, D. Mombello, M. Cocuzza, D. Casalena, I. Ferrante, A. Nesca, P. Poiklik, K. Rekker, A. Aaspollu, S. Ferrero, C.F. Pirri, Biomed. Microdev., accepted for publication (2014), DOI: 10.1007/s10544-014-9869-x
- "Liposomes sensing and monitoring by Organic Electrochemical Transistors integrated in microfluidics", G. Tarabella, A.G. Balducci, N. Coppedè, S. Marasso, S. Barbieri, M. Cocuzza, P. Colombo, R. Mosca, F. Sonvico, S. Iannotta, Biochimica et Biophysica Acta (BBA) - General Subjects, 1830, 2013, pp. 4374-4380, 10.1016/j.bbagen.2012.12.018
Partners & Collaborations
Fondazione Bruno Kessler – Trento
IMEM-CNR - Parma
Università degli Studi di Catania - Dipartimento di Scienze Microbiologiche e Ginecologiche
Università degli Studi di Genova - Dipartimento di Fisica
Università degli Studi di Milano - Dipartimento Scienze Cliniche “L. Sacco” - Istituto di Malattie Infettive e Tropicali
Center for Advanced Biomaterials for Healthcare at CRIB (CABHC@CRIB), Italian Institute of Technology (IIT) - Naples
Università degli Studi di Torino – Dipartimento di Scienze Oncologiche
Istituto per la Ricerca contro il Cancro (IRCC) – Candiolo (TO)