1

Nanoparticles with Immobilized Biosensors for Bioactive Papers

Shunxing Su, Razvan Nutiu, Carlos D. M. Filipe, Yingfu Li, Robert Pelton

McMaster Centre for Pulp and Paper ResearchDepartment of Chemical Engineering

McMaster University1280 Main St. W., Hamilton, Ontario, Canada L8S 4L7

E-mail: peltonrh@mcmaster.ca

Imagine a Imagine a BioactiveBioactive Paper TowelPaper Towel

That would change color to tell us when our kitchen counter top was contaminated with dangerous bacteria.That would change color to warn us of the presence of anthrax spores.

Imagine a Bioactive MaskImagine a Bioactive Mask

Which will:owarn the user of

viral contamination.ocapture and

deactivate the virus.

Imagine a Bioactive Water FilterImagine a Bioactive Water FilterSpecifically bind small, soluble (i.e. difficult to remove) toxinsCapture and kill all bacteria and virus.Warn user of contamination and when the cartridge must be changed.

SENTINEL Bioactive Paper NetworkBioactive Paper Network

a Network of Canadian AcademicsNetwork of Canadian Academics~ 28 professors, 11 Universities, ~ 70 total students,

PDFs and profs.~ $2.5 million / year

o 75% from Canadian Government’s Natural Science and Engineering Research Council (NSERC Network)

o 25% cash from industrial consortium and Ontario Government.

The The SENTINEL VisionVision

Bioactive Paper - inexpensive paper products which detect and repel or deactivate waterborne and airborne pathogens.

Key Elements of Key Elements of SENTINEL VisionVision

υ Paper giving instant visible indication of pathogens.

υ High speed manufacture – coating or printing.

Bioactive Paper Bioactive Paper –– a a ““TopTop”” ideaidea

New York Times, Sunday Magazine Dec 9, 2007Described the Sentinel Bioactive paper concept as one of the top 70 in “The 7th

Annual Year in Ideas”

““detectdetect”” pathogenspathogens

Instant, visible indication of pathogens without instrumentationTechnologies do not exist – this is the “killer application” for bioactive paperA large part of the SENTINEL research

Sentinel Biodetection PlatformsSentinel Biodetection PlatformsAgents which specifically recognize(capture) a targetSentinel researchers are developing four technologies in parallelo Antibodieso Bacteriophageo DNA aptamerso Enzymes

BiodetectionAgent

Target

Applying Biodetection Agent to Paper

υ To fibers before papermaking processυ Coating υ Printing on paper

Approach 1 Approach 1 –– Simply Print a SolutionSimply Print a SolutionCase 1 – no adhesion to paper surface

Example: DNA aptamers on celluloseExposure to target or developing solutions causes biodetection polymer to come off paper Su, et al. Adsorption and Covalent Coupling of ATP-Binding DNA Aptamers onto Cellulose. Langmuir 2007, 23, (3), 1300-1302

Case 2 – very strong adhesion with surface

Example: any anionic protein on wet strength resin coated paperEnzymes and antibodies may denature on surface

Paper

Paper

Approach 2 Approach 2 –– Cellulose Binding Cellulose Binding DomainsDomains

Cellulase – family of enzymes which degrade celluloseTwo parts – binding domain, catalytic domain (CBD)Sentinel researchers have genetically engineered CBD onto antibodies and phageGives spontaneous binding to pure cellulose with controlled orientationWhat about surfaces coated with size, wet strength resin, retention aids, and/or fillers?

catalyticdomain

bindingdomain

Paper

Approach 3 Approach 3 –– Carrier ParticlesCarrier ParticlesAttach biodetection molecules to carrier particles which can be formulated into inkThe particles isolate the detectors from the paper surface chemistryThree Sentinel initiatives:– Porous silica particle

Brook/Brennan– Microcapsules Rochefort/Paice– Microgels Pelton/Filipe/Li/Hall

Paper

CarrierParticle

Microgel Carrier Particles

17

Microgel Preparation

• Essentially a surfactant-free emulsion polymerization

• Particles form because polymer is insoluble at high temperature

• Electrostatically stabilized – sulfate from KPS.• Surfactant will give smaller particles

PNIPAM microgel

NH

CH 2

O

NH

O

ONH

CH3C H3

BA NIPAM

Water, 70 C

18

All Microgel Properties Depend Upon T

Below 33oC• Microgels• about 90% water• transparent

Above 33oC• latex particle• about 20% water• white

19

T.E.M.

υ Pelton and Chibante, Colloids and Surfaces 20, 247 (1986)

~500nm

Microgels for Bioconjugation

υ Require functional groupsυ We chose carboxyl groupsυ The pioneers:

– Kawaguchi (1992) - first microgel protein interactions– Pichot & Elaissari - microgel with grafted oligo-DNA

EDCStreptavidin

Biotin-Aptamer

Biotin-

IgG

SP-MG COOH

COOH

COOH

COOH

COOH

COOHAPT-MG COOHCOOH

COOH

IgG-MG

COOHCOOH

COOH

COOH

MG

• IgG or Aptamer still functions in solution after coupled onto microgel surface.

Microgel Derivatization

Microgel Swelling

200

300

400

500

600

700

2 4 6 8 10pH

Hyd

rody

nam

ic D

iam

eter

(nm

)

MG

SP-MGAPT-MG

IgG-MG

RB-MG

υ ~ 0.2 mg/m2 streptavidin on microgel surface

Electrophoretic Mobility

MG

SP-MG

APT-MG

IgG-MGRB-MG

DNA Aptamer Detecting ATP

υ Synthetic short DNA chains which fold to capture a specific target

ATP added

QDNA added

GTP added

ATP-MG in Binding Buffer

+ATP

ATP

Nutiu R., Li Y., J. Am. Chem. Soc., 2003, 125, 4771-4778

Schemes of the Set-up for Detection

Microgel

Sample solution

Microgel

Sample to be detected

Elution

Scheme 1 Scheme 2

Filter paper

Blank Filter paper

Filter paper with MG

Before Elution After Elution

“Spotted” Microgels on Filter Paper

• Microgel penetrates into filter paper about 1/3 of the thickness.

• Microgels do not move with elution

Inkjet Microgel Printing

υ Dimatix Fujifilm Printer

Printing IgG-MG or APT-MG onto Filter Paper Surface

Ag-Per

IgG-MG

Ag-PerIgG-MGPer

IgG-MG-control

ATP GTP

Quenched APT-MG

• IgG or Aptamer coupled on microgel can survive the printing process.

• Promising for making microgel-based bioinks. Biomacromolecules 2008, 9, (3), 935-941.

Cationic Paper

υ DNA aptamer is denatured when directly applied to cationic paper.

υ Microgel supported aptamer functions on cationic paper.

Aptamer Directly Applied to PAE Treated Paper

APT-MG on PAE Treated Paper

Summary

υ Microgels are immobilized on filter paperυ Microgel supported antibodies and DNA aptamers

retain activity on filter paperυ Microgel protects aptamer from cationic polymer

impregnated wet strength paper

That’s All

Bioactivepaper.comPapersci.mcmaster.ca

32

Microgel Superstar

υ B.Eng, Queensυ Ph.D. McMaster 2006υ MIT PDF 2006-2008υ New Faculty at McMaster July 2008

Todd Hoare

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Microgel Microstructure Models

VAA-NIPAM(Surface Model)

-

---

-

-

--

--

--

-

-

--

-

-

-

--

--

--

-

- -

-- -

- ---

----- -

--

-

----

--

MAA-NIPAM(Inverse Core-Shell

Model)

-

-- -

-

- - - -

-

--- --

---

-- -

-- --

---

--- -

-

-

-

- -

--

-

-

-

-

- - - -

rNIPAM = 16.7 rVAA = 0.002 ≈ 0

r/ro0 1

VAA-NIPAM

r/ro0 1

AA -NIPAM

r/ro0 1

MAA-NIPAM

-CO

OH

Con

cent

ratio

n

-- -

-- - - - - - - --

---

---

-- -

--

-

-

-

-

-- - - ----

----

-

- -

--

-

-

---

-

-CO

OH

Con

cent

ratio

n

-CO

OH

Con

cent

ratio

n

AA-NIPAM(Core-Shell Model)

rNIPAM = 0.20±0.08rMAA = 2.8±0.4

rNIPAM = 0.57±0.07

rAA = 0.32±0.04

Direct correlation between monomer reaction kinetics and resulting COOH distributions

Hoare, T.; McLean, D., J. Phys. Chem. B 2006, 110, (41), 20327-20336.

34

Chain Transfer with Vinylacetic Acid

• Slow propagation kinetics and chain transfer ability of VAA expect chain end, surface-localization of functional groups

A “one-step” method of making functionalized “hairy” microgels?

R + CH2 CH

CH2

COOH

propagation

CH2

CHCH2

COOH

R

chain transfer

CH2 CH

CHCOOH

CH

CHCOOH

CH2

+ R(terminated)

effective chain transfer(reinitiation)

degradative chaintransfer (H-transfer)

end-capping (termination)

M+CH

CHCOOH

CH2

Mpropagation

+ R

CH

CHCOOH

CH2

RCH2 C

H

CH2

COOH

resonance

propagation

Dominant in allylic radical systems

.

.

..

.

.

Propagation Chain TransferNIPAM

FunctionalMonomer

NIPAMFunctionalMonomer

Hoare, T.; Pelton, R., Macromolecules 2004, 37, (7), 2544-2550.

Detection of Specific Target by APT-MG (Scheme 2)

0.5 mM GTP

0.5 mM ATP

2 mM GTP

2mM ATP

APT-MG

• APT-MG was mixed with the quencher before applying.

• Paper strips were eluted in ATP or GTP solution.

• Elution in ATP, specific target, enhanced the fluorescence intensity.

PAE Structure and Reactionsυ Cationic, reactive

water soluble polymerυ Reacts with carboxyl

and amine groupsυ Requires heat to drive

reactionsυ Produces positively

charged paper

Obokata, T.; Isogai, A. Colloids and Surfaces 2007, 302, (1-3), 525-531.

PAEPAE

37

Microgel Swelling

υ gel swelling decreases at LCST.

υ ~ 10 timesdecrease in volume

υ Swelling sensitive to ionic strength

0.1M

0.01M

0.001M

CaCl2

ColloidallyUnstable

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DNA Aptamer with Built-in Reporting υ Structure-switching signaling

FŠTCACTGACCTGGGGGAGTATTGCGGAGGAAGGT5’

Q-GTGACTGGACCC 5’

FDNA

QDNA

Target

Non-targetNutiu R., Li Y., J. Am. Chem. Soc., 2003, 125, 4771-4778

FITC-BSA

Fluorescein-DNA

MGMG

Protein or DNA oligos will Pass Through the Microgel on Paper Surface

MG

FTIC-BSA

• Proteins have molecular level contact with microgel

Detection of Antigen by IgG-MG (Scheme 2)

Developed in Ag-Per

IgG-MG

IgG-MGDeveloped in Per Developed in

Ag-Per

IgG-MG-control

A: (1.6 µg/ml Ag-Per or Per)

B: (0.16 µg/ml Ag-Per or Per)

Detection of Antigen by IgG-MG (Scheme 1)

Ag-Per

IgG-MG

Ag-Per

IgG-MGPer

IgG-MG-control

• Antigen was conjugated with peroxidase.

• Further elution in OPD, substrate for peroxidase, gave out color signals.

• Only the strip with antigen showed brown color