PCR Microfluidics

MICRODroplets

Belayneh Getahun BekeleMaster Program in Nanoscience, Materials and Processes: Chemical Technology at the Frontier

1. Introduction

2. Motivations for PCR Microdevice: Miniaturization

3. Design of the Methods

4. PCR in Microdroplets

5. Detection Methods5. Detection Methods

6. Current Developments: Examples

7. Summary

8. References

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1) INTRODUCTION

Microfluidics refers to the behavior and control of liquids constrained to volumes near the μL range.

Microfluidic Devices

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Giouroudi, I., and Keplinger, F. Int. J. Mol. Sci. 2013, 14, 18535-18556

Microfluidic Devices

Comparisons

Conventional PCR Microfluidic PCR

the thermal mass is large,

which leads to lengthy PCR reactions

Offline sample preparation

Low thermal mass

Rapid heat transfer

Integration of

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Offline sample preparation

Consumption of expensive reagent

Short, chimeric products

Integration of

sample preparation,

separation

amplification

detection,

On a single chip

Less reagentLiu, C.,et al. Anal. Chem. 2006, 78, 5474-5479

2. Motivation for PCR Microdevice: Miniaturization

Low High

Cost of fabrication and use,

analysis time

portability,

integration,

parallel amplification,

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analysis time parallel amplification,

acceptable disposal

Zhang, C., et al. Biotechnology Advances 2006, 24, 243–284

3) DESIGN OF THE METHODS

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Link, D. , et al. Lab Chip 2011,11, 2156−2166

PCR fluid in a microsystem of a chip

MicroPCR devices

well-based PCR chips continuous-flow PCR chips

the PCR mixture in well

the whole chip is heated and cooled.

moves the sample through fixed temperature zones

smaller thermal inertia

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and cooled.

Higher thermal inertia and time

Auroux, P., et al. Lab Chip 2004, 4,534–546 Cao, Q., et al. PLoS ONE 2012 , 7, e33176

Continuous-flow PCR

(A) Serpentine channel (B) Spiral channel-based (C) the circular channel-based

Zhang, C., and Xing, D. Nucl. Acids Res. 2007,35, 4223-4237

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Microfluidic PCR: Limitations

1) interactions between the surface

PCR Inhibition and Carry-over

Limitations similar to Conventional PCR

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2)Preference for short segmentschimeric products

Zhang, Y., and Ozdemi, P. Analytica Chmica Acta 2009, 638, 115-125

Limitations similar to Conventional PCR

4) PCR IN MICRODROPLETS

In vitro compartmentalisation of reactions in water-in-oil droplets

(as in emulsion PCR for sequencing)

reduces recombination between homologous fragments:

Avoids synthesis of short, chimeric products.

Assay carry-over and inhibition is minimized Assay carry-over and inhibition is minimized

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Zhang, Y., and Ozdemi, P. Analytica Chmica Acta 2009, 638, 115-125

6.DETECTION METHODS

TheAnalytical performance,

Real-time vs end point detection

Zhu,Y. , and Fang, Q. Analytica Chimica Acta 2013, 787, 24– 35

Analytical performance,Available probesof each analytical technique

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DNA extractionIn-situ Analyzer

Schematic view of the microfluidicstructure for continuous DNAextraction.

7. CURRENT DEVELOPMENTS: EXAMPLES

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Karle, M., et al. Lab Chip 2010, 10, 3284–3290

Cell lysis,DNA purification, PCR,

and optical detection

In-situ Analyzer

Microfluidic Device for IISA-Gene.

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Schematic illustration

PCR, and optical detection

Fukuba,T.,et al, RSC Adv. 2011,1, 1567-1573

8. SUMMARY

Microfluidic PCR is a better alternative to conventional PCR

Droplet-based Microfluidics is shown to allievate some problems of Microfluidic PCR.

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9) REFERENCES

1. Auroux, P., et al. Lab Chip 2004, 4, 534–546

2. Cao, Q., et al. PLoS ONE 2012 , 7, e33176

3. Fukuba,T,. et al, RSC Adv. 2011,1, 1567-1573

4. Giouroudi, I., and Keplinger, F. Int. J. Mol. Sci. 2013, 14, 18535-18556

5. Hataoka, Y., et al. Anal Sci 2005, 21, 53–56

6. Karle, M., et al. Lab Chip 2010, 10, 3284–3290

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6. Karle, M., et al. Lab Chip 2010, 10, 3284–3290

7. Link, D., et al. Lab Chip 2011,11, 2156−2166

8. Liu, C., et al. Anal. Chem. 2006, 78, 5474-5479

9. Zhang, C., and Xing, D. Nucl. Acids Res. 2007,35, 4223-4237

10. Zhu,Y. , and Fang, Q. Analytica Chimica Acta 2013, 787, 2435

THANK YOU VERY MUCH FOR THE OPPORTUNITY AND

YOUR ATTENTIONS

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FOR THE OPPORTUNITY AND YOUR ATTENTIONS