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

Integration of miniaturized complex Integration of miniaturized complex

diagnostic tests : from the macro sample to diagnostic tests : from the macro sample to

the test readingthe test reading

Nanoforum 2005Nanoforum 2005

Nanotechnology in BioDiagnostics and Analytics Nanotechnology in BioDiagnostics and Analytics (NBDA)(NBDA)

29-30 June, Grenoble29-30 June, Grenoble

Frédéric Ginot

Nanoforum 2005. Frédéric Ginot - Nanoforum 2005. Frédéric Ginot - 22

Why miniaturization of Diagnosic Tests ?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.

Main ChallengesMain 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.

From macro to microFrom 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.

From macro to microFrom 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.

Principle of micro concentrationPrinciple 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

Phase 3 :

Magnetic transportation in a micro chamber, 0.1 µl

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 behaviorMagnetic pellet behavior

Pellet transport and dispersion Pellet plasticity

An other benefit of nanoparticles : An other benefit of nanoparticles : the magnetic divisionthe magnetic division

VerrePDMS ou PC

• The magnetic micro concentration can also be used to split a sample into several analytical channels.

• Principle :

Apply the sampleApply the magnetMove the magnetThe sample is splitDivision is done !

Magnetic divisionMagnetic division

• Advantages :– No fluidics,– Same system for every number of channels,– No precise alignment needed.

Very accurate division, CV < 5%.

1 2 3 4 5 6 7 8

Numéro du canal

Use of a fluorescent pellet for quantification

• Results :

No great impact of the shape of the entry port, self organizing pellet.

An other benefit of micro concentration : An other benefit of micro concentration :

target concentrationtarget 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)

Bubble Valves for micro chamber IsolationBubble 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

Example of a micro concentratorExample of a micro concentrator

Sample input port

0.1 µl chamber

Bubble valves

Six channels Micro concentratorSix channels Micro concentrator

ELOSA in a micro concentratorELOSA 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

target

target1 E 20 pM

100 E 2 nM

Readable by fluorescence

5.105 copies on 7.5 .106 particules

Real-time reading of the fluorescence

0,00E+00

5,00E+03

1,00E+04

1,50E+04

2,00E+04

2,50E+04

3,00E+04

3,50E+04

4,00E+04

0 5 10 15 20 25min

ra4,00E+03 2,00E+04 vide

1,00E+05 non spe 0 cible

The fluorescence slope depends on the copies number in the sample

Enzymatic revelation in a micro chamber

0E+00 2E+04 4E+04 6E+04 8E+04 1E+05

copies

Enzymatic kinetic versus molecule number

Detection limit : a few thousands of

molecules

Integrated reading of a DNA chipIntegrated 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).

Optronic reading of a DNA chipOptronic 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

e-Pixel APS

P1P1P1

*Pn*PnPn

e-e-Pixel APSPixel APS

A standard VGA monochrome image sensor was used

Image format 640 x480

Pixel size 5.6 x 5.6 µm

APS chipsAPS chips

APS chips were kindly provided by ST Microelectronics, Imaging Division.

Enzymatic substrate for chemiluminescence

Experimental set upExperimental set up

Chip put on the electronic board for the reading

Glob top (protection of connection wires)

Ceramic holder

Silicon surfaceFunctionnalization / spotting

Specific

Non specific

100 pM 1 pM

Specific

Non specific

0.1 pM

Hybridization

« Optronic » Reading« 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

Complex protocol and micro fluidicsComplex 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,– …

Complexity illustrationComplexity illustration

• Typical process overview for a DNA chip based test

• Complexity challengeUsing state-of-the-art reagents kits, a typical test

requires more than 20 liquid reagents !

4- 6 hours 1-2 hours

Nucleic acidamplification

Several in parallel

** ****

LabelingFragmentation

Hybridizationof target to Chip

Chip ScanningAnalysis

Extraction of DNA/RNA

4- 6 hours4- 6 hours 1-2 hours1-2 hours

Nucleic acidamplification

Several in parallel

** ****

FragmentationHybridization

of target to Chip Chip Scanning

AnalysisExtraction

of DNA/RNA

Purification

Micro fluidics challengesMicro 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 macromicro 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.

Micro fluidics solutionsMicro 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 ?

ConclusionsConclusions

• 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.

AcknowledgmentsAcknowledgments

• 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).

Presentation ContentPresentation 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

réserveréserve

Transportation and plasticity of a magnetic pelletTransportation and plasticity of a magnetic pellet

Micro DNA ChipMicro DNA Chip

Small chamber of the microconcentrator.

1 mm2, 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.

Packaging : the eLab cardPackaging : the eLab card

• Macro to micro pour les réactifs liquides

• Pb du packaging/challenge

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

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