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Label-free biodetection by optical diffraction

Label-free biodetection techniques are the matter of intense research field in bioassays area. We have developed a label-free detection method based on optical diffraction. Probes molecules are immobilised on functionalized glass slides by soft lithography to generate molecular gratings that efficiently diffract light from a laser beam. Diffraction efficiency increases with the thickness of the molecular gratings. According to this principle, the measured diffraction intensity is found to increase when probe-target interactions take place at the surface of the molecular gratings. To collect and compare diffracted intensity before and after interaction, we have developed a dedicated scanner to measure the 1st order diffracted beam.

 diff.png

Advantages:

  • Label-free biodetection
  • Low-cost fabrication

Publications:

  • Amandine M.C. Egea, Laurent Mazenq, Emmanuelle Trévisiole, Vincent Paveau and Christophe Vieu, Optical label free biodetection based on the diffraction of light by nanoscale protein gratings, Microelectronic Engineering, 2013, V.111, p.425-427

Patents:

  • Patent : Jean-Christophe Cau, Helene Lalo, Jean-Pierre Peyrade, Childerick Severac and Christophe Vieu Method of seeking at least one analyte in a medium likely to contain it, 2010, WO 2010/029139

Thesis:

  • H. Lalo, Thèse INSA, Toulouse, 2009
  • J.C. Cau, Thèse INSA, Toulouse, 2009
  • J. Foncy, Thèse INSA, Toulouse, 2013

Smart Paper

Keywords : paper, soft lithography, bio-functionnalisation, paper-based lab on chip

Paper-based microfluidic devices have enjoyed rapid development since Georges Whiteside1  introduced the mPADs concept. A series of hydrophilic/hydrophobic microstructures on paper substrates were fabricated to construct mPADs using a variety of processing techniques. Compared to conventional microfluidic chips made of glass and polymer substrates, mPADs possess many unique advantages including, low-cost, ease of fabrication, strong capillary action and good biological compatibility for applications in clinical diagnosis and environmental monitoring. 
 
We develop some easy to use techniques to pattern smart paper with biomolecules and produce paper-based lab on chip.

 Fig: Soft lithogrphy of Streptavidin patterns Alexa 790nm (yellow) et Alexa 680 nm (magenta) on paper.Capture_décran_2016-07-19_à_15.29.41.png

1: Andres W. Martinez, Scott T. Phillips, and George M. Whitesides, Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices, Anal. Chem. 2010, 82, 3–10 

 

Nanostructured mold replication - Low cost

Soft lithography at nanoscale requires a nanostructured silicon or resist master mold generated by advanced and expensive lithography and complex transfer techniques like ion etching. Such a fabrication is prohibitive for the industrial use of polydimethylsiloxane (PDMS) stamps in soft lithography. Our work focuses on a straightforward – low cost – technology to duplicate silicon master molds with nanoscale structures. Hence, master silicon molds patterned with nanometer scale lines can be replicated into epoxy resist and polyurethane by the following process.

replication.png

 Principle of the duplication method

The PDMS stamp made from a silicon master mold is used as template to copy this original mold into epoxy resist or polyurethane by UV nanoimprint lithography and demonstrated a faithful replication of the silicon mold nanostructures.Moreover, the biomolecule gratings patterns obtained by µCP using the PDMS stamps molded on epoxy resist or polyurethane replicated molds are identical to those obtained with the original silicon master mold.

Epoxy resist or polyurethane are very attractive and cost-effective substrates to reproduce at large scale PDMS stamps initially made on a nanostructured silicon master mold.

Publications:

  • Amandine M.C. Egea and Christophe Vieu, Microcontact printing of biomolecular gratings from SU-8 masters duplicated by Thermal Soft UV NIL, Microelectronic Engineering, 2011, V.88, p.1935-1938.
  • Julie Foncy, Jean-Christophe Cau, Carlos Bartual-Murguia Jean Marie François, Emmanuelle Trévisiol and Childérick Sévérac, Comparison of polyurethane and epoxy resist master mold for nanoscale soft lithography, Microelectronic Engineering, 2013, V.100, p.183-187

Microfluidic to manage reliable molecular interactions

Low density microarrays for diagnosis require automatized and reproductible protocol interactions that ensure homogeneous repartition of the solutions over the spots of the microarray and that can be removed easily for allowing optical reading. We have developed a generic and removable microfluidic interface using magnetic field to ensure a uniform and sealed contact between the microfluidic cartridge and the microarray. This microfluidic platform was validated for automated immunodiagnosis on microarray. In addition, this approach allows a reduction of volumes provided by miniaturization. 

microflu

 View of the microfluidic interface reversibly sealed to a microarray. 

This approach is versatile, easy to produce and provides an effective and automated platform for multiplexed immunodiagnosis. 

Advantages:

  • Label-free biodetection
  • Low-cost fabrication

Publications:

  • A. Ali-Cherif, S. Begolo, S. Descroix, J.-L. Viovy, L. Malaquin, Programmable magnetic tweezers and droplet microfluidic device for high-throughput nanoliter multi-step assays, Angewandte Chemie International Edition, 2012, 51, 10765
  • Saliba, A.E. ; Saias, L.; Psicharia, E.; Minc, N.; Simon, D.; Mathiot, C.; Bidard, F.C.; Pierga, J.Y.; Fraisier, V.; Salamero, J.; Saada, V.; Farace, F.; Vielh, P.; Malaquin, L.; Viovy, J.L. Microfluidic Sorting and High Content Multimodal Typing of Cancer Cells in Self-Assembled Magnetic Arrays, Proceedings of the National Academy of Sciences, 2010, 107(33), 14524-9
  • M.L.Y. Diakité, J. Champ, S. Descroix, L. Malaquin, F. Amblard, J.-L. Viovy, A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR, Lab on Chip, 2012, 12, 4738
  • S. Miserere, G.Mottet, V.Taniga, S.Descroix, J.-L.Viovy, L.Malaquin, Fabrication of thermoplastics chips through lamination based techniques, Lab on Chip  2012, 12, 1849
  • Begolo, S.; Colas, G.; Viovy J.L.; Malaquin, L., New Family of Fluorinated Polymer Chips for Droplet and Organic Solvent Microfluidics, Lab on Chip 2011, 11, 508
  • J. Autebert, B.Coudert, F.-C. Bidard, J.-Y.Pierga, S. Descroix, L.Malaquin, J.-L.Viovy, Microfluidic: an innovative tool for efficient cell sorting,  Methods 2012, 57,297
  • Saias, L; Autebert, J; Malaquin, L; Viovy J.-L., Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidic systems, Lab on Chip 2011, 11, 82

Posters

Magnetic clamped microfluidic device for automated immunodiagnosis on microarrays, E. Crestel, J. Foncy, JP. Borges, A. Estève, JC Cau, C. Vieu, L. Malaquin, E. Trévisiol. Micro and Nano Engineering 2015.

Microfluidic interface for microarray diagnostic

Keywords : microfluidics, reversible magnetic clamp, multiplexed immunoassay, allergen microarray 

Reference: Reversible magnetic clamp of a microfluidic interface for the seric detection of food allergies on allergen microarrays, J. Foncy, E. Crestel, J-P Borges, A. Estève, J-C Cau, C. Vieu, L. Malaquin and E. Trévisiol, Microelectronic Engineering (2016).

To provide a robust platform for fluid handling, most microfluidic devices usually involve irreversible bonding methods to achieve a leak free interface between the microchannels and the holding substrate. Such an approach induces a major drawback when biological interactions are performed on a microarray format as it is difficult to recover the biochip for further fluorescence scanner analysis. This work describes an automated microfluidic platform using a reversible magnetic clamp for multiplexed immunodiagnostis. The microfluidic device is composed of a magnetic PDMS layer (containing iron powder) coated by PDMS, which is reversibly clamped to an epoxysilane glass slide containing an array of various antigens. The microfluidic device was validated for in vitro diagnosis of food allergies on an allergen microarray after serum interaction. The statistical analysis of spot intensities(Signal to noise ratios) on the microarray displayed excellent reproducibility. In addition to the reduction of volumes provided by miniaturization, this approach is versatile, easy-to- produce and provide an effective platform for multiplexed immunodiagnosis based on conventional fluorescent detection schemes. 

diagnostic 2

Fig 1: Schematic view of the microfluidic device. Magnetic PDMS cartridge and the microarray are hold together by magnetic force using array of magnets under the device. a) Microfluidic cartridge composed by PDMS and a magnetic PDMS layer,  b) microarray composed by an epoxysilane glass slide containing an array of various antigens (85 spots), c) the microfluidic cartridge and the microarray reversibly sealed by a magnetic field to ensure a conform and hermetic contact, d) section view of the device, e) View of the microfluidic interface. 

  • Reversible magnetic clamp of a microfluidic interface and a glass slide spotted with a microarray of allergens for serum detection of food allergies

  • The PDMS microfluidic cartridge is transparent above the channels allowing optical imaging in the microfluidic device while the reversibility of the magnetic clamp allows the reading of the microarray after microfluidic removal using a conventional fluorescent scanner.

  • No leakage was observed below 150 mBar with the magnetic clamp.

  • The functionality of the device was validated for in vitro diagnosis of food allergies in the serum of a patient. 

diagnostic 1

Fig 2:  a) Microarray fluorescence image of the interacting spots after removal of the microfluidic reversible chip, b) signal to noise ratio quantification after food allergy immunodiagnostic using the microfluidic device. The patient was allergic to cow milk, coat milk and yolk eggs. 

Low cost 2.0 diagnostics

For more than a decade labs-on-a-chip are described as ultimate tools for personalized healthcare and point-of-care diagnostics. Biosoft scientists agree with this affirmation and work on such a motivating topic. However it is clear that there is no such technology wide sprayed today due to technical and regulatory difficulties. Lab-on-a-chip will certainly be a reality tomorrow but today medical laboratories still use 1.0 methods or expensive 2.0 protocols.

While working on long-terms innovative diagnostics tools, Biosoft also develops a full chain of technological tools to manufacture a new generation of biochips, to manage a reliable interaction with serums and to analyze with simple instrumentation and efficient software solutions the biological response of the biochip.

Our aim is to adapt our available technologies to bring in no-time to the market a low-cost platform compliant with CE-IVD standard and targeting high potential applications for medical challenges. One of the best example is certainly the allergy detection. Prick tests are still widely used (clearly 1.0 methods) with the acknowledged risk of anaphylactic shock and the wide interdiction for food-allergic patients to eat a global family of aliments. More accurate tests are available but they are expensive and non-reimbursed by health systems. Low density biochips manufactured with our novel low-cost methods on a very small surface associated with a reliable management of simple fluidic systems and instrumentation based on conventional low-cost components is able to change habits in allergy detection. Such a low-cost biochip will bring personalize medicine to the allergy specialist and will deal with Prick tests, increasing security and accuracy.

Then low-cost 2.0 diagnostics are seen by Biosoft scientists as a bridge between conventional medicine and future wide-sprayed point-of-care tools. It should address the challenges of cost competitiveness with standard methods, enhancing reliability and accuracy, a fast transfer to the market, a regulatory compliance and an understanding of the needs of the medical staffs.