Integration of miniaturized complex diagnostic tests : from the macro sample to the test reading

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. 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. Main Challenges • Macro  MicroFor 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 ? • SensitivityWhen dimensions go smaller and smaller, everything else being equal, the signal to noise ratio get smaller too, and the sensitivity can be degraded. • Biochemical complexityFor 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. 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 m3 • 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. 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. Principle of micro concentration Phase 1 : Standard capture of the sample onto magnetic nanoparticles, in a tube Phase 2 : Magnetic sedimentation in a pre-filled micro concentrator Phase 3 : Magnetic transportation in a micro chamber, 0.1 µl

7. Magnetic pellet behavior • 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 Pellet transport and dispersion Pellet plasticity

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

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

10. This requires to isolate the micro chamber(no diffusion, no convection) 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.

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. Example of a micro concentrator 0.1 µl chamber Sample input port Bubble valves

13. Six channels Micro concentrator

14. 1 E  20 pM 100 E  2 nM target target P E E M M S Readable by fluorescence 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

15. Real-time reading of the fluorescence The fluorescence slope depends on the copies number in the sample ELOSA in a micro concentrator Enzymatic revelation in a micro chamber • 5.105 copies on 7.5 .106 particules

16. ELOSA in a micro concentrator Enzymatic kinetic versus molecule number Detection limit : a few thousands of molecules

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. 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. APS chips A standard VGA monochrome image sensor was used APS chips were kindly provided by ST Microelectronics, Imaging Division.

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

21. « Optronic » Reading Non specific Specific Hybridization 100pM 1pM Specific At least as sensitive and reproductible as fluorescence reading Non specific Target : biot-Oligo 30 min hybridization Reading : 2 min 0.1pM Biosens Bioelectron. 2005 Mar 15;20(9):1813-20

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. Complexity illustration 4 4 4 - - - 6 6 6 hours hours hours 1 1 1 - - - 2 2 2 hours hours hours * * * * * * * * * * * * * * * * Result Result Sample Sample * * * * * * * * Labeling Chip Scanning Chip Scanning Nucleic acid Nucleic acid Hybridization Hybridization Extraction Extraction amplification amplification Fragmentation Fragmentation of of target target to Chip to Chip Analysis Analysis of DNA/RNA of DNA/RNA Several Several in in parallel parallel Purification • 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. Micro fluidics challenges • Integration of different functionsEach function has been demonstrated individually, but still few examples of integration of several steps, specially including reagent addition. • Work with detergentsIn « real test », biologists often use detergents • Fluidics packagingWe 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. 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. 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. 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. 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. réserve

30. Transportation and plasticity of a magnetic pellet

31. Micro DNA Chip 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. Small chamber of the microconcentrator. 1 mm2, 0.1 µl.

32. Packaging : the eLab card • Macro to micro pour les réactifs liquides • Pb du packaging/challenge