1. 9 4 t h C ANADI AN C HEM I S TRY C ONF ER ENC E AND EXHI B I TI ON
PA L A I S D E S C O N G R È S D E M O N T R É A L , Q U É B E C
Analytical Chemistry (AN3-510d)
THURSDAY, JUNE 9, 2011
Marco Polo G. PALACIOS 1, 2, 3
Dominic ROCHEFORT 1, 3
François BERTRAND 2, 3
dominic.rochefort@umontreal.ca
1. 2. 3.

2. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Paper based biosensors
General goal Platforms
• Creation of paper based
biosensors with commercial
applications in food, health
and security domains
Receptor: Signal: Detector:
antibodies chemical electrode
aptamers thermal thermostat
cells optical camera
enzymes acoustic resistor
nanoparticles magnetic microphone
polymers electric
tissues
virus

3. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Paper based biosensors
General goal Platforms
• Creation of paper based
biosensors with commercial
applications in food, health
and security domains
Large scale
Specific goal conditions
• Immobilize biomolecules
by using techniques that
are compatible with paper
Paper sheet
industrial procedures.

4. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Paper based biosensors
General goal Challenges
• Creation of paper based • Immobilization of enzymes on
biosensors with commercial paper
applications in food, health • Conservation of the activity
and security domains through large-scale conditions
Large scale
Specific goal conditions
• Immobilize biomolecules
by using techniques that
are compatible with paper
Paper sheet
industrial procedures.

5. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Immobilization of enzymes on paper
by microencapsulation
Our group has demonstrated that microencapsulation provides
an efficient platform for enzyme immobilization on paper1.
Diameter2
Laccase 5 nm
Microcapsules 60 µm
Mw Cut off 2000 Da
Pore of paper 1-10 µm
Activity of laccase deposited on paper1
Before After
washing Washing
Free Laccase 100 % 6%
Encapsulated laccase 100 % 94 %
1. Kouisni, L.; Rochefort D., Journal of applied Polymer Science 2009, 111 (1), 1-10.
2. Zhang, Y.; Rochefort, D., Journal of Microencapsulation 2010, 27 (8), 703-13.

6. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Microcapsulation by polycondensation
of polyethyleneimine (PEI)
Positively
charged
Sebacoyl chloride
PEI Mn:1200, 50 % wt in water
Amines: 25% primary, 50 %
secondary, 25 % tertiary
Microcapsules modified with
Fluorescein isothiocyanate
observed by fluorescence
microscopy.
(λex= 495, λem= 525 nm). Schotten-Bauman reaction.
(a) PEI; (b) sebacoyl chloride

7. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Large-Scale Immobilization
Suspension characteristics Blade Coating
• 60 % modified microcapsules Paper
• 40 % starch Roll
• Hydroxylated starch
neutrally charged
• Viscosity 11880 cP
Blade
Coating parameters
Suspension
• Gap blade/paper: 635 µm
• Paper size : 0.75 x 3 m Blade coating is the most widely
• Roll speed : 500 m/min used industrial technique for
• Drying Power : 36 kW applying suspensions to paper base1.
• Drying time : 0-30 s 1. Iliopoulos, I.; Scriven, L. E.,
Physics of fluids 2005, 17 (12) 12701.

8. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Analysis of the coated paper
Enzymatic activity was evaluated with a
colorimetric assay based on the oxidation
of p-phenylenediamine (PPD).
PPD
Images obtained from a scanner allow us to
analyze the change in color intensity of several
samples at the same time.
Intensity (I)
Enzymatic paper
Control
0s 466 s
Time (s)

9. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Analysis of the coated paper
Enzymatic activity was evaluated with a
colorimetric assay based on the oxidation
of p-phenylenediamine (PPD).
PPD
Images obtained from a scanner allow us to
analyze the change in color intensity of several
samples at the same time.
Intensity (I)
Enzymatic paper
Control The activity of the enzyme
0s 466 s immobilized on paper is calculated
Time (s)
from the slope of the portion of
curve.

10. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of drying exposure
Activity of paper modified with encapsulated enzyme.
Not difference is founded in the activity
for the papers dried at rates from 0 – 30 s.
Confidence interval of 95 %.
Laccase from Trametes versicolor
(EC 1.10.3.2) is reported to lost half
of the initial activity after 20 min of
incubation at 70 °C.
Enzymatic activity (I/s) normalized for the surface coverage (U/m2) :
6.10 x 10-4 I.m2/s.U. Infrared lamp power: 36 kW.

11. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of microencapsulation
Activity of paper modified with free and encapsulated enzyme.
This difference can be explained
by the restricted diffusion of PPD
to the laccase in the capsules.
Enzymatic activity (I/s) normalized for the surface coverage (U/m2) :
6.92 x 10-4 I.m2/s.U for free laccase and 6.10 x 10-4 I.m2/s.U for encapsulated laccase.

12. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of thickness suspension
Activity of paper coated with microencapsulated enzyme.
Two paper sheets were coated with similar enzyme coverage, 264 U/m2 and
224 U/m2, although they are from suspensions with different starch
concentrations, 11.07 g/m2 and 7.24 g/m2 (dry weight), respectively.

13. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of thickness suspension
Activity of paper coated with microencapsulated enzyme.
The suspension doesn't affect the
activity. Confidence interval of 95 %.
Two paper sheets were coated with similar enzyme coverage, 264 U/m2 and
224 U/m2, although they are from suspensions with different starch
concentrations, 11.07 g/m2 and 7.24 g/m2 (dry weight), respectively.

14. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of capsule concentration
Minimal concentration capable to
reach the maximum of color intensity.
Change in color intensity of encapsulated enzymes coated on paper with different
amounts of microcapsules filled with laccase (1859 U/g).

15. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of capsule concentration
Activity of encapsulated enzymes
coated on paper at different amounts
of microcapsules.
Change in color intensity of encapsulated enzymes coated on paper with different
amounts of microcapsules filled with laccase (1859 U/g).

16. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of inhibitors addition
(a) Cl- 100% represents the activity of paper in
absence of any inhibitor. Note the different
concentration scales for chloride inhibitors.
(c) N3-
(b) F-
Normalized activities of (▲) free and (o) encapsulated enzymes in presence
of different concentrations of inhibitors: (a) NaCl, (b) NaF and (c) NaN3.

17. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Effect of storage time
4 days
The residual activity after 6
28 days months was 15% and 66% for
free (non-encapsulated) and
encapsulated
laccase, respectively.
t½ of encapsulated enzymes
is 85% higher than non
encapsulated enzymes.
Activity of non encapsulated (free) and encapsulated
enzymes when stored several days at room temperature.

18. Conclusions
• Microencapsulation is an efficient method of immobilization for
coating enzymes in conditions similar to industrial ones.
• Free and microencapsulated enzymes can be applied on paper by
using blade-coating technique.
• Enzymes coated on paper by large-scale conditions remains actives
even after drying procedure.
• The microcapsules prevented the inhibition of laccase by azide yet
did not offer any beneficial effect for chloride and fluoride
inhibition.
• Microencapsulation increases the storage time of enzymes.

19. Challenge
Pilot coater plant of the Centre Integré des Pâtes et Papiers (CIPP)
at the Université de Québec à Trois Rivières

20. Your
biomolecules
here

21. Acknowledgments
• Industry Partners:
• Government Partners:
• Other Industries:
Daniel Matte Marie Ortman
Nancy Jacobs Katherine Rogers-Vallée
Martin Dubé Patrice Mangin
Contact: Dominic Rochefort Presenter: Marco Polo G. Palacios
dominic.rochefort@umontreal.ca g.marcopolo@gmail.com

22. References
• Kouisni, L.; Rochefort, D., Confocal microscopy study of polymer microcapsules for
enzyme immobilisation in paper substrates. Journal of Applied Polymer Science
2009, 111 (1), 1-10.
• Zhang, Y.; Rochefort, D., Comparison of emulsion and vibration nozzle methods for
microencapsulation of laccase and glucose oxidase by interfacial reticulation of poly-
(ethyleneimine). Journal of Microencapsulation 2010, 27 (8), 703-13.
• Iliopoulos, I.; Scriven, L. E., A blade-coating study using a finite-element simulation.
Physics of fluids 2005, 17 (12), 127101.
• Hildén, K.; Hakala, T.; Lundell, T., Thermotolerant and thermostable laccases.
Biotechnology Letters 2009-08-01, 31 (8), 1117-1128.

23. Laccase structure and inhibition

24. Laccase as model
Laccase is regarded as one of the most studied and available enzymes1.
Thermostable enzyme:
• Optimal temperature: 45-80 °C Uses:
• Lost half of the initial temperature • Delignification
after 20 min of incubation at 70 °C • Wine clarification
• Ethanol production
Substrates:
• Decolouration of dyes
• Aromatic compounds such as:
• Oxidation of toxic compounds
benzenediols, polyamides,
(e.g. aflatoxins and benzo[α]pyrene)
aminophenols and lignines
1. Hildén, K.; Hakala, T.; Lundell, T., Biotechnology Letters 2009-08-01, 31 (8), 1117-1128.

25. Microcapsule Advantages
• Distribution of the biomolecules on the surface

26. Microencapsulation
Crosslinking emulsion
Enzymes
(Wall Formation)
0,5 ml sebacoyl/chloride /
10 ml enzyme buffer
10 1 ml PEI
+ ml cyclohexane
Chang et al. Procedures for
microencapsulation of
Enzymes
enzymes, cells and genetically
engineered microorganisms. 50 ml cyclohexane
Molecular Biotechnology + 0,5 ml span 85
(2001)

27. Immobilization of enzymes on paper
by microencapsulation
• Our group has demonstrated that microencapsulation provides an efficient
platform for enzyme immobilization on paper1.
diameter: 5 nm
Gardco Drawdown Machine.
Pore diameter
10 µm
1) Kouisni, L.; Rochefort, D., Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper
substrates. Journal of Applied Polymer Science 2009, 111 (1), 1 - 10.

28. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Immobilization of enzymes on paper
by microencapsulation
Our group has demonstrated that microencapsulation provides an
efficient platform for enzyme immobilization on paper1.
Means sizes of the implied molecules2
Diameter
Laccase 5 nm
Microcapsules 60 µm
Pore of microcapsules 2.6 – 5 nm
Poly(ethyleneimine) Microcapsules Pore of paper 1-10 µm
2. Zhang, Y.; Rochefort, D. 2010.

29. Paper Biosensor – Microencapsulation – Blade Coating – Paper Activity
Laboratory assessment of modified paper
Paper modified with microencapsulated laccase cost 65 % more than paper
modified with free enzyme. Thereof the half life of modified paper for
encapsulated laccase is 85 % higher than the half life of free laccase