1. Integration of miniaturized complex diagnostic tests : from the macro sample to the test reading Nanoforum 2005 Nanotechnology in BioDiagnostics and Analytics (NBDA) 29-30 June, Grenoble Frédéric Ginot

2. Nanoforum 2005. Frédéric Ginot - 2 Why miniaturization of Diagnosic Tests ? For in vitro diagnostic, the expected advantages of miniaturization are usually the following : less sample consumption, less reagent consumption, hence lower cost and smaller volume of hazadeous waste, higher speed, higher sensitivity, better integration and automation, field or near patient testing.

3. Nanoforum 2005. Frédéric Ginot - 3 Main Challenges Macro  Micro For infectious diseases, the sample must be large enough to be relevant, often 1-10 ml. How to make the whole sample enter the micro world ? Sensitivity When dimensions go smaller and smaller, everything else being equal, the signal to noise ratio get smaller too, and the sensitivity can be degraded. Biochemical complexity For DNA chip based tests, complexity of biochemistry is important. Miniaturization in itself does not help to integrate and automate the tests from the biochemical point of view.

4. Nanoforum 2005. Frédéric Ginot - 4 From macro to micro Sample volume for infectious diseases : –Sepsis : 2 x 10 ml of blood –Respiratory diseases : ~ 1 ml –Food : 0.1 – 1 g of transformed food –Sterile air : 1 –10 m 3 After a typical sample preparation procedure : 10 – 100 µl of purified nucleic acids. A micro system : 1 –100 nL, or even less.  Use of a solid support to make the whole sample enter a microsystem. Otherwise, only a small part of the sample is analyzed.

5. Nanoforum 2005. Frédéric Ginot - 5 From macro to micro How to use a solid support to enter a micro system ? Either the solid support is inside the microsystem; it captures the sample as it flows through the microsystem. Column-like system. Or the solid support consists of micro or nano particles introduced in the sample before entering the microsystem, e.g. output of the sample preparation stage. Magnetic concentration-like system.

6. Nanoforum 2005. Frédéric Ginot - 6 Principle of micro concentration Phase 2 : Magnetic sedimentation in a pre-filled micro concentrator Phase 1 : Standard capture of the sample onto magnetic nanoparticles, in a tube Phase 3 : Magnetic transportation in a micro chamber, 0.1 µl

7. Nanoforum 2005. Frédéric Ginot - 7 With a good combination between the particles, the buffer, and the microsystem surface, we can get : a smooth sliding of the pellet at the surface, a possible passage through bottlenecks, a negligible loss by physisorptions of the particles, A brownian dispersion of the particles when removing the magnet Magnetic pellet behavior Pellet transport and dispersionPellet plasticity

8. Nanoforum 2005. Frédéric Ginot - 8 An other benefit of nanoparticles : the magnetic division Verre PDMS ou PC The magnetic micro concentration can also be used to split a sample into several analytical channels. Principle : Apply the sample Apply the magnetMove the magnet The sample is split Division is done !

9. Nanoforum 2005. Frédéric Ginot - 9 Magnetic division Advantages : –No fluidics, –Same system for every number of channels, –No precise alignment needed. Very accurate division, CV < 5%. Use of a fluorescent pellet for quantification Results : No great impact of the shape of the entry port, self organizing pellet.

10. Nanoforum 2005. Frédéric Ginot - 10 An other benefit of micro concentration : target concentration Indeed, magnetic micro concentration can be a generic tool for several things : –As already shown, to enter a macro sample in a Lab On A Chip, with no loss of biological material. –To speed up reactions by increasing the concentration ~100 - 1000 times. –To increase detection sensitivity, for instance by enzymatic revelation. This requires to isolate the micro chamber (no diffusion, no convection)

11. Nanoforum 2005. Frédéric Ginot - 11 Bubble Valves for micro chamber Isolation 1. Air is trapped in bubble chambers during filling 2. Air expands upon heating, isolating the micro chamber 3. Air retracts back upon cooling

12. Nanoforum 2005. Frédéric Ginot - 12 Example of a micro concentrator Sample input port 0.1 µl chamber Bubble valves

13. Nanoforum 2005. Frédéric Ginot - 13 Six channels Micro concentrator

14. Nanoforum 2005. Frédéric Ginot - 14 ELOSA in a micro concentrator Principle : if the enzymatic revelation in an ELISA or ELOSA takes place in 100 nL instead of 100 µl as usual, enzymatic product concentration will increase 1000 times faster for the same number of target molecules. Elo(i)sa on particle  micro concentration  enzymatic revelation M E target S P M E 1 E  20 pM 100 E  2 nM Readable by fluorescence

15. Nanoforum 2005. Frédéric Ginot - 15 ELOSA in a micro concentrator 5.10 5 copies on 7.5.10 6 particules Real-time reading of the fluorescence The fluorescence slope depends on the copies number in the sample Enzymatic revelation in a micro chamber

16. Nanoforum 2005. Frédéric Ginot - 16 ELOSA in a micro concentrator Enzymatic kinetic versus molecule number Detection limit : a few thousands of molecules

17. Nanoforum 2005. Frédéric Ginot - 17 Integrated reading of a DNA chip We have shown how magnetic nanoparticles can introduce a macroscopic sample in a micro system, and how they bring other benefits like an easy division or an increased target concentration At the other extremity of the analytical chain, there is the reading of DNA chips. Some label less techniques do exist for such reading, but they suffer from a lack of sensitivity compared to fluorescence labeling, which is the gold standard of the field. Fluorescence reading, in the visible light, is not easy to integrate. Hence, we have chosen luminescence (no light source, no filter, only the detector).

18. Nanoforum 2005. Frédéric Ginot - 18 Optronic reading of a DNA chip Basic Principle : Manufacture the DNA chip directly at the surface of a photodetector array, e.g. an Active Pixel Sensor Use an enzymatic label, Use an enzymatic substrate which is converted into a luminescent product

19. Nanoforum 2005. Frédéric Ginot - 19 A standard VGA monochrome image sensor was used Image format640 x480 Pixel size5.6 x 5.6 µm APS chips APS chips were kindly provided by ST Microelectronics, Imaging Division.

20. Nanoforum 2005. Frédéric Ginot - 20 Enzymatic substrate for chemiluminescence Experimental set up Chip put on the electronic board for the reading Glob top (protection of connection wires) Ceramic holder Silicon surface Functionnalization / spotting

21. Nanoforum 2005. Frédéric Ginot - 21 Specific Non specific 100 pM1 pM Specific Non specific 0.1 pM Hybridization « Optronic » Reading Target : biot-Oligo 30 min hybridization Reading : 2 min At least as sensitive and reproductible as fluorescence reading Biosens Bioelectron. 2005 Mar 15;20(9):1813-20

22. Nanoforum 2005. Frédéric Ginot - 22 Complex protocol and micro fluidics Between sample entry in the micro system and integrated DNA chip reading, several complex biochemical steps must take place. Micro Fluidics has a major role to play for that : –Add, mix reagents, incubate, –Move the sample for new steps, –Purification of the sample, separations, –…

23. Nanoforum 2005. Frédéric Ginot - 23 Complexity illustration Typical process overview for a DNA chip based test Complexity challenge Using state-of-the-art reagents kits, a typical test requires more than 20 liquid reagents !

24. Nanoforum 2005. Frédéric Ginot - 24 Micro fluidics challenges Integration of different functions Each function has been demonstrated individually, but still few examples of integration of several steps, specially including reagent addition. Work with detergents In « real test », biologists often use detergents Fluidics packaging We can loose most of the advantages of miniaturization if the problem of macro  micro connection for the reagents is not treated intelligently. Robustness and low cost devices after this integration …simplification of the biochemical process remains a key of the success.

25. Nanoforum 2005. Frédéric Ginot - 25 Micro fluidics solutions Today we can distinguish two major families of micro fluidics solutions : –Fluidics in channel – see M. Palmieri presentation –“Digital Fluidics” : film du Leti ?

26. Nanoforum 2005. Frédéric Ginot - 26 Conclusions As shown in this presentation, but not exclusively, there are proven and efficient solutions for the entry and for an integrated reading of a DNA chip. And, more generally, for every unitary function needed. The technical bottleneck is now on microfluidics and in chaining several biochemical reactions requiring reagents addition, in an integrated way in an affordable device (packaging). Note that this conclusion relates to the particular field of diagnostic tests for infectious diseases based on DNA chips.

27. Nanoforum 2005. Frédéric Ginot - 27 Acknowledgments The work presented here was obtained by bioMerieux (France), the Leti (CEA, France), and the CNRS (France). Part of the work was supported by the French government (grants 98 T 258 and 03 2 90 6079 ). The APS chips came from ST Microelectronics, Imaging division (France).

28. Nanoforum 2005. Frédéric Ginot - 28 Presentation Content Some generalities about diagnostic miniaturization From the macro to the micro world using nanoparticles Other benefits of nanoparticles for integration Integrated optronic detection for DNA chips Complex biochemistry and microfluidics

29. Nanoforum 2005. Frédéric Ginot - 29 réserve

30. Nanoforum 2005. Frédéric Ginot - 30 Transportation and plasticity of a magnetic pellet

31. Nanoforum 2005. Frédéric Ginot - 31 Micro DNA Chip Small chamber of the microconcentrator. 1 mm 2, 0.1 µl. The small chamber of the micro concentrator could also be a DNA chip. If the sample is concentrated 100 to 1000 times, the hybridization is 100 to 1000 times faster for very dilute analytes.

32. Nanoforum 2005. Frédéric Ginot - 32 Packaging : the eLab card Macro to micro pour les réactifs liquides Pb du packaging/challenge