imprinted polymer

Molecular Imprinting PolymersMolecular Imprinting Polymers

MIPsMIPs

IntroductionIntroduction

In chemistry, In chemistry, molecular imprintingmolecular imprinting is a is a technique to create template-shaped technique to create template-shaped cavities in polymer matrices with memory cavities in polymer matrices with memory of the template molecules. of the template molecules.

Schematic of molecular imprintingSchematic of molecular imprinting

Templateassembly

(binding)Template

a

c

b1- add cross-linker2- polymerise(in porogenicsolvent)

Template

re-binding

templateremoval

recognition site

History of Molecular ImprintingHistory of Molecular Imprinting Molecular imprinting was used as early as the 1930's by Molecular imprinting was used as early as the 1930's by

MV PolyakovMV Polyakov to selectively capture various additives in to selectively capture various additives in a silica matrix.a silica matrix.

In the 1940's In the 1940's Linus PaulingLinus Pauling hypothesized that a process hypothesized that a process similar to molecular imprinting could be responsible for similar to molecular imprinting could be responsible for the selectivity of antibodies to their respective antigens.the selectivity of antibodies to their respective antigens.

Biological systems Biological systems

Molecular recognition plays an important role in Molecular recognition plays an important role in biological systems and is observed in between receptor-biological systems and is observed in between receptor-ligand, antigen-antibody, DNA-protein, sugar-lectin, ligand, antigen-antibody, DNA-protein, sugar-lectin,

RNA-ribosome, etcRNA-ribosome, etc AntigensAntigens

AntigenAntigen

Antigen-binding site

History of Molecular ImprintingHistory of Molecular Imprinting

The concept of molecular imprinting was revived in the The concept of molecular imprinting was revived in the 1970's when Günter Wulff discovered that highly 1970's when Günter Wulff discovered that highly crosslinked organic polymers could also be used to crosslinked organic polymers could also be used to make molecular imprints with high specificity.make molecular imprints with high specificity.

In more recent years, imprinted polymers have been In more recent years, imprinted polymers have been

used to capture everything from steroids to TNT.used to capture everything from steroids to TNT.

Imprinting methodologiesImprinting methodologies

CovalentCovalent Reversible covalent linkageReversible covalent linkage

OH

OH

R1

R2

+ B R3HO

HO O

B

OR1

R2

R3

- 2 H2O

+ 2 H2O

Molecular Imprinting: CovalentMolecular Imprinting: Covalent

O

OO

O

O OH

B

B

O

OO

O

O OH

B

B

OHHO

OH

OH

B

B

O

OO

O

polymerize

- galactose

+ galactose

Wulff & Schauhoff J. Org. Chem., 1991, 56, 395-400.

Imprinting methodologies Imprinting methodologies advantagesadvantages and and disadvantagesdisadvantages

Covalent ImprintingCovalent Imprinting Ability to fix template in place during polymerisation - Ability to fix template in place during polymerisation -

lower dispersity in binding siteslower dispersity in binding sites Can be carried out in any solvent flexibilityCan be carried out in any solvent flexibility Can be difficult to remove template from polymer - Can be difficult to remove template from polymer -

low recovery of valuable templates and low number low recovery of valuable templates and low number of binding sitesof binding sites

Limited number of chemistries for fixing template to Limited number of chemistries for fixing template to polymer reversibly - reduction in number of templates polymer reversibly - reduction in number of templates that can be imprintedthat can be imprinted

Poor kinetics of re-bindingPoor kinetics of re-binding  

Imprinting methodologiesImprinting methodologies

Non-covalent

Monomer-template complexes

+R1

O

OHN

R3

R2

R1

O

OH

R2

R3

N

Molecular Imprinting: Non-covalentMolecular Imprinting: Non-covalent

OO

H

O

O

H

CH3

O

OO

O

HN

NH

H3C

HN

H

O

O

O

OH

HO

O

CH3

O

OO

O

HN

NH

H3C

HN

O

OH H

OO

OH

O

H

O

O

O

O

H

EGDMA,polymerize

Non-covalent template-monomer complex Imprint is spatially and functionally complemetary to template

Ramström et.al. Tetrahedron: Asymmetry, 1994, 5, 649-656.

Imprinting methodologies Imprinting methodologies - - advantagesadvantages and and disadvantagesdisadvantages

Non-covalent imprintingNon-covalent imprinting Easy to remove template from polymer- good Easy to remove template from polymer- good

recovery of valuable templates and accessible recovery of valuable templates and accessible binding sitesbinding sites

Very large number of templates amenable to non-Very large number of templates amenable to non-covalent imprintingcovalent imprinting

Rapid kinetics of re-bindingRapid kinetics of re-binding Inability to fix template in place during polymerisation Inability to fix template in place during polymerisation

- polydispersity in binding sites, poor definition- polydispersity in binding sites, poor definition Generally requires low-polarity aprotic solvents - Generally requires low-polarity aprotic solvents -

incompatible with aqueous polymerisationsincompatible with aqueous polymerisations  

Imprinting methodologiesImprinting methodologies

Sacrificial spacer (semi-covalent) Covalent link during synthesis

Non-covalent rebinding

R1 R2O

O

OR1

O

O ClR2

OH

+

R1

OH

HO

R2

Molecular Imprinting: Spacer ApproachMolecular Imprinting: Spacer Approach

O

OO O O

O

O

H

OH

O

H

O

OO

O

AIBN, 65oC, 24h

1M NaOH/MeOH reflux

Cholesterolin n-hexane

Whitcombe et al. J. Am. Chem. Soc., 1995, 117, 7105-7111.

CVPC

Imprinting methodologies Imprinting methodologies - advantages and disadvantages- advantages and disadvantages

Sacrificial spacer methodSacrificial spacer method Ability to fix template in place during polymerisation - Ability to fix template in place during polymerisation -

lower dispersity in binding siteslower dispersity in binding sites Can be carried out in any solvent flexibilityCan be carried out in any solvent flexibility Rapid kinetics of re-bindingRapid kinetics of re-binding Can be difficult to remove template from polymer - Can be difficult to remove template from polymer -

low recovery of valuable templates and low number low recovery of valuable templates and low number of binding sitesof binding sites

Limited number of chemistries for fixing template to Limited number of chemistries for fixing template to polymer reversibly - reduction in number of templates polymer reversibly - reduction in number of templates that can be imprintedthat can be imprinted

Target molecules & Imprinting Target molecules & Imprinting matricesmatrices

Target moleculesTarget molecules

Small organic molecules:, pesticides, amino acids, Small organic molecules:, pesticides, amino acids, nucleotide bases, steroids ,sugars ,metal ion peptides, nucleotide bases, steroids ,sugars ,metal ion peptides, proteins and drug,…proteins and drug,…

Imprinting matricesImprinting matrices

Acrylic and vinyl polymersAcrylic and vinyl polymers

Organic polymersOrganic polymers

Other imprinting matricesOther imprinting matrices

Preparation MIPsPreparation MIPs

Generally MIPs have been prepared as monoliths using ‘bulk’ polymerization of vinylic monomer mixtures by free radical initiation;

Consequently, the material requires grinding before use (sieving is often also employed to fractionate by particle size)

Preparation MIPsPreparation MIPs

In situ polymerization. In order to avoid the grinding and packing of HPLC

columns the polymer is formed inside a column as a porous monolith.

Coated silica particles. A polymerizable group was first attached to the

silica surface and polymerization was then carried out using template, cross-linker and functional monomer.

Molecular imprinting of theophylline immobilized onto a solidsupport: immobilized template with monomers (1), composite material after polymerization (2), imprinted polymer after dissolution of the support.

1 2 31

Preparation MIPsPreparation MIPs

Precipitation polymerization.

Precipitation polymerization can be performed with similar prepolymerization mixtures as for bulk polymers, except that the relative amount of solvent present in the mixture is much higher. When polymerization progresses, imprinted nano- or microspheres precipitate instead of polymerizing together to form a polymer monolith.

Preparation MIPsPreparation MIPs

W/O emulsion polymerization. Binding sites confined at the interior surface of voids

within an organic polymer can be created by polymerization of the continuous (oil) phase of a water-in-oil (W/O) emulsion stabilized with an amphiphilic functional surfactant complexed with the template molecule at the water–oil interface.

and…

Applications of imprinted polymers Applications of imprinted polymers

SeparationSeparationChromatographyChromatographyCapillary electro chromatographyCapillary electro chromatographySolid phase extractionSolid phase extraction

Pseudo immunoassays

Synthesis & CatalysisSynthesis & Catalysis

SensorsSensors

Separation: Separation: chromatographychromatography

MIPs as stationary phases:MIPs as stationary phases:

Imprinted enantiomer retained on columnImprinted enantiomer retained on column

Separation:Separation: Solid phase extraction

Pseudo immunoassays

An An immunoassayimmunoassay is a biochemical test that measures is a biochemical test that measures the concentration of a substance in a biological liquid, the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its advantage of the specific binding of an antibody to its

antigen.antigen.

A promising application area for MIP development is as replacements for biological receptors such as antibodies in analogues of immunoassays.

Imprinted polymers-antibody binding site mimicsImprinted polymers-antibody binding site mimics

N

N N

N

O

H3C

O

CH3

H O

OH+

O

OH

H

CH3

O

H3C

O

N

NN

N

HO

O

HO

O

EGDMApolymerize

O

OH

H

CH3

O

H3C

O

N

NN

N

HO

O

HO

O

O

OH

extract rebind

HO

O

HO

O

HO

O

HO

O

O

OH

N

N N

N

O

H3C

O

CH3

CH3

N

N N

N

O

H3C

O

CH3

CH3

X

Theophylline

Caffeine

Comparison of MIPs and Comparison of MIPs and antibodiesantibodies

In vivo In vivo preparationpreparation

Limited stabilityLimited stability

Limited applicabilityLimited applicability

Higher costsHigher costs

In vitro In vitro preparationpreparation

Unlimited stabilityUnlimited stability

General applicabilityGeneral applicability

Lower costsLower costs

Antibodies MIPs

SensorsSensors

a chemical sensor selectively recognizes a a chemical sensor selectively recognizes a target molecule in a complex matrix and target molecule in a complex matrix and generates an output signal using a transducer generates an output signal using a transducer that correlates to the concentration of the that correlates to the concentration of the analyte .analyte .

Sensor performanceSensor performance selectivity,selectivity, sensitivity, sensitivity, stability,stability,

MIPs have unique properties that MIPs have unique properties that make them especially suitable for make them especially suitable for sensor technology. They exhibit sensor technology. They exhibit good specificity for various good specificity for various compounds of medical, compounds of medical, environmental, and industrial environmental, and industrial interest; and they have excellent interest; and they have excellent operational stability. Their operational stability. Their recognition properties are recognition properties are unaffected by acid, base, heat, or unaffected by acid, base, heat, or organic phase treatment making organic phase treatment making them highly suitable as them highly suitable as recognition elements in chemical recognition elements in chemical sensors.sensors.

MIP Sensors

Sensor typeSensor type

Quartz-crystal microbalance-based sensing devices

Optical sensors

Electrochemical Sensor

and…

Optical sensors : Fluorescent Sensor

F = fluorescent tag

Weakly fluorescent strongly fluorescent

Typical set of fluorescence emission spectra of D-fructoseimprinted polymer at different concentrations of D-fructose (λex=370nm)

REFERENCESREFERENCES

J. Mol. Recognit. 2006; 19: 106–180

Analyst, 2001, 126, 747–756

Analytica Chemica Acta 534(2005) 31-39

Anal Bioanal Chem(2006) 386 1235-1244