Molecularly Imprinted Polymers (MIPs) as Customizable ... - CoSMoS

Molecularly Imprinted Polymers (MIPs) as Customizable Stationary Phases Ken Shimizu Professor Department of Chemistry and Biochemistry

CoSMoS 2012

Outline

I. Introduction to Molecularly Imprinted Polymers II. Examples III. Characteristics IV. Limitations V. Recommendations VI. Future

The molecular imprinting process

482

B. Tse Sum Bui, K. H

ig. 1 The molecular imprinting principle [4]. a functional monomers, b cross-linker, c template molecule, 1 assembly of the prepolymeris omplex, 2 polymerisation, 3 extraction, 4 rebinding

a. Functional monomers (hydrogen bonding, ionic groups)

MIPs have attracted great interest (>70%) during recent years b. Crosslinker ecause they have certain advantages over immunosorbents, Template resistance to elevated uch as mechanicalc.robustness, emperatures and pressures, an improved inertness to strong cids, bases and organic solvents, as well as low cost, ase of preparation, long shelf-life and reproducibility. Consequently, MIPs have been widely used in applications

MIP preparation and evaluation Selection of the synthesis protocol

In its simplest form, a typical MIP synthesis prot contains theal.template, one or more(2010) functional Haupt et Anal Bioanal Chem 398, monome 2481 cross-linker, a polymerisation initiator and a solvent.

I. Molecularly Imprinted Polymers (MIPs) MIPs: Highly crosslinked synthetic organic polymers that can be tailored with specificity for a template molecule. Tunable molecular recognition platforms Antibodies

synthetic molecular receptors

MIPs

preparation time

3-6 months

1-6 months

1-7 days

average affinities (M-1)

106 - 109

102 - 106

102 - 103

inexpensive to prepare

no

no

yes

easy to tailor specificity

yes

no

yes

thermal/chemical stability

no

yes

yes

Versatile molecular recognition platform

Chromatographic Stationary Phases chiral stationary phases (CSPs) HPLC, capillary electrophoresis (CE), GC Solid-Phase Extraction (SPE) SupelMIP™ Molecularly Imprinted Polymer SPE Cartridges Biotage® AFFINILUTE™ MIP Sensors optical, electrochemical, luminescent, QCM sensor arrays - “electronic noses/tongues” Catalysis (“plastic antibodies”) Polymer Drug (colesevelam•HCL) - Macromolecules (2001), 34, 1548

Applications

Chromatographic Stationary Phases chiral stationary phases (CSPs) HPLC, capillary electrophoresis (CE), GC Solid-Phase Extraction (SPE) SupelMIP™ Molecularly Imprinted Polymer SPE Cartridges Biotage® AFFINILUTE™ MIP Sensors optical, electrochemical, luminescent, QCM sensor arrays - “electronic noses/tongues” Catalysis (“plastic antibodies”) Polymer Drug (colesevelam•HCL) - Macromolecules (2001), 34, 1548

The molecular imprinting process

482

B. Tse Sum Bui, K. H

ig. 1 The molecular imprinting principle [4]. a functional monomers, b cross-linker, c template molecule, 1 assembly of the prepolymeris omplex, 2 polymerisation, 3 extraction, 4 rebinding

a. Functional monomers (hydrogen bonding, ionic groups)

MIPs have attracted great interest (>70%) during recent years b. Crosslinker ecause they have certain advantages over immunosorbents, Template resistance to elevated uch as mechanicalc.robustness, emperatures and pressures, an improved inertness to strong cids, bases and organic solvents, as well as low cost, ase of preparation, long shelf-life and reproducibility. Consequently, MIPs have been widely used in applications

MIP preparation and evaluation Selection of the synthesis protocol

In its simplest form, a typical MIP synthesis prot contains theal.template, one or more(2010) functional Haupt et Anal Bioanal Chem 398, monome 2481 cross-linker, a polymerisation initiator and a solvent.

II. L-Phenylalanine anilide imprinted polymer

FM

FM•template complex

template

crosslinker O

n

+

O

O H

NH3

N

AIBN, (light or heat)

O

H

O O

O

H

O

O

O

O

O

NH2

H N

O O

O

O O H

O

H O

O O

H

wash with MeOH

NH3

N

H

O

O O H

H

O

NH2

H N O

O O

H

Mosback et al. J. Am. Chem. Soc. (1988) 110, 5853 Shimizu et al. Org. Lett. (2002) 4, 2937

Enantioselectivity

0.9

H

0.8

H

O

N

N H

T T T T T T T

0.7

(L)-PAA

0.6

Bound (mM)

H

0.5

H

N

O N H

0.4 0.3

(D)-PAA

0.2

Free

T

0.1

Bound

T T T T T

0 0

0.5

Free (mM)

1

1.5

T

2

Shimizu et al. Org. Lett. (2002) 4, 2937

Low cost and ease of preparation

1. Break Vial 2. Grind Δ or hν

Monomers Crosslinker Initiator Template Solvent

3. Extract


1. Break Vial 2. Grind 3. Extract

Δ or hν


crosslinker

functional monomer

O

O O

O

+ O

ethylene glycol dimethacrylate

O OH

or

NH2

methacrylic acid or amide


1. Break Vial 2. Grind 3. Extract

Δ or hν


Shimizu et al. J. Chem. Educ. (2005), 82, 1374 crosslinker

functional monomer

O

O O

O

+ O

ethylene glycol dimethacrylate

O OH

or

NH2

methacrylic acid or amide

HPLC stationary phases

HPLC elution profile: D-PA

O

NH2

L-PA

N H

L-phenyl alanine anilide (L-PA)

Solute: D,L-PA Stationary Phase: L-PA imprinted polymer (MAA-EGDMA) Mobile phase: MeCN-aqueous 0.05 M KP buffer (pH 4) (7:3, v/v) Flow-rate: 1 mL/min Separation factor (α): 2.1 to 13

B. Sellergren and K.J. Shea., J. Chromatogr. (1993), 635, 17

HPLC stationary phases


O

NH2

L-PA

N H

L-phenyl alanine anilide (L-PA)

Solute: D,L-PA Stationary Phase: L-PA imprinted polymer (MAA-EGDMA) Mobile phase: MeCN-aqueous 0.05 M KP buffer (pH 4) (7:3, v/v) Flow-rate: 1 mL/min Separation factor (α): 2.1 to 13 • predictable elution order B. Sellergren and K.J. Shea., J. Chromatogr. (1993), 635, 17

HPLC stationary phases 382

Anal Bioana


O

NH2

N H

L-phenylalanine anilide (L-PA)

column length: 10 cm L-PA

particles: irregular (25 - 60 µm)

Fig. 4 Scanning electron micrographs of (S)-propranolol-imprinted MIP beads obtained by dispersion polymerization in mineral oil (top) and MIP particles obtained by conventional bulk polymerization after grinding and sieving (bottom). Reprinted with permission from Ref. [38]

were performed using a well-establ employing L-phenylalanine anilide MAA and EDMA as functional m agent, respectively. In the framew optimization study [45], the effects tal conditions during the graft chromatographic performance of modified MIP-silica composites particles with a pore diameter of (0.8 nm) MIP film gave the mo graphic performance with regard to load capacity, however, increased w of the MIP layer, reaching an optim Because of the single-point attac initiator, however, solution-phase p undesired particle aggregation c suppressed. In an effort to address t resorted to dithiobenzoate-type in inherently high activity of the surf species [46]. The presence of the mixture favored reversible additi transfer (RAFT) polymerization benefit of a slower and more read located polymer growth. Under th ditions aggregation phenomena trig polymerization could be completel extended reaction times (Fig. 5). The obtained by use of this imprint homogenous MIP films with enha acteristics compared with conventio

Drawbacks: inherent thermodynam

Despite the major advances achiev chromatographically suitable polym tive MIPs do not compete with estab CSPs when challenged with rea

B. Sellergren and K.J. Shea., J. Chromatogr. (1993), 635, 17 obtained within 4 h. The applicability of this procedure,

orogen giving a macroporous polymer with high rface area so that many of the binding sites genated are close enough to a surface to be kinetically Other examples cessible. It should usually be as non-polar as ossible, especially where interactions are only T. Takeuchi, J. Haginaka / J. Chromatogr. B 728 (1999) 1 – 20

1.0 mL/min ergic stimulants. These molecularly Boc-D-Phe s could separate the corresponding he antipode. Boc-L-Phe polymer for (S)-naproxen (2derivative), a non-steroidal anti, was prepared through bulk poly-vinylpyridine and ethylene glycol 0.2 mL/min the functional monomer Boc-D-Phe and crosy, and its chiral recognition ability Boc-L-Phe sing non-aqueous mobile phases ized molecularly imprinted polypared for (S)-naproxen by a twoa redox polymerization method nctional monomer and crosslinker . HPLC chromatograms for separation of Boc-Phe on an ve with water as the suspension -co-EDMA imprinted MIP CSP. 250 ! 4.6 mm s imprintedBoc-l-Phe polymer gave similar n, Ag ofR.aseach CH 2Cl2/heptane/AcOH for 10 naproxen thatenantiomer, previously Ansell, J. Adv Drug Deliv Rev (2005), 57, 1809 "1 v/v/v)to eluent at: (a) 1 mL h:0.1 regard the comparison of min the , 30 8C; (b) 0.2 mL , 70 8C polymers (Jackson and unpublished). printed for Ansell, (S)-naproxen

7

ketoprofen ibuprofen

O

O HO

S-naproxen R-naproxen S-naproxen

Tanaka et al. Chem. Lett. (1997) 26, 555

III. Versatility R.J. Ansell / Advanced Drug Delivery Reviews 57 (2005) 1809–1835 successfully imprinted chiral templates

1815

Fig. 5. Structures of enantiomeric drugs which have been employed as templates for the production of MIP CSPs, and/or whose enantiomers have been separated on MIP CSPs.

Ansell, R. J. Adv Drug Deliv Rev (2005), 57, 1809

Materials Properties Robust stationary phase (years as effective HPLC CSP) - rigid highly crosslinked insoluble polymer - stable to aqueous and organic solvents under pressure - stable to most modifiers (pH 2 - 10) High surface areas 200-500 m2/g Porous Monolith . 12

T Takeuchi, J. Haginaka / J. Chromatogr. B 728 (1999) 1 – 20

Fig. 10. Schematic illustration of superporous three-dimensional molecularly imprinted polymers for CE and CEC separation. Reproduced from Ref. [93] with permission.

background electrolyte as a suspension of particles

sorbents because some natural ligands are not sufficiently stable for long-term storage. Molecularly imprinted polymers are capable of molecular recognition and are stable, easy to prepare and inexpensive, thus they could be considered as artificial affinity media, and applied to specific on– off separation. In practice, the effectiveness of molecularly imprinted polymers for affinity separation has been demonstrated in applications to solidphase extraction (SPE), which involves an on–off separation protocol for pre-concentration or pre-treatR. Bru ̈ggemann, M.J. Freitag, E.N. Whitcombe, J. Vulfson, J. Chromatogr. A 781 (1997) 43. ment of analytes. The first molecularly imprinted SPE (MI-SPE) was reported for pentamidine, which is used for the treatment of AIDS-related pneumonia [78]. The high selectivity of the polymer allowed the drug to be sufficiently enriched to be analyzed directly even when present in low concentration in a urine sample. In this system, 20% (v / v) phosphate buffer in

proven wrong and rapidly forgotten. On rare ever, incorrect hypotheses have value beyond rticular experimental observation or phenomenon on a life of their own. Such was the case with Fig. 1 Schematic adapted from reference 1 showing four steps (1, 2, 4, 5) of Pauling’s six step mechanism by which an antigen imprints structural proposal for how antibodies are produced in the information into an antibody molecule. response.1 The hypothesis that antigens induce or ng site within the otherwise unfolded polypeptide of s extremely compelling (Fig. 1). Indeed, the idea spiration for Dickey who reported in 1949 that dye d be imprinted into silica. This early study showed broad sense Pauling’s concept could be put into mentally.2 more or less dormant until two landmark reports of which are widely acknowledged as propelling ting forward into the modern field it is today. The f3 and Mosbach4 showed that molecular templates, 1. Easy and inexpensive to prepare noncovalently, respectively, could imprint their own ithin polymers by the process shown schematically II. Rationally customizable selectivity e noncovalent approach, wherein the functional

IV. So what is the catch?

III. Robust materials properties

merman was born in Chicago, Illinois. He obtained Prof. Ronald Breslow at Columbia University and tdoctoral work with Prof. Sir Alan R. Battersby at of Cambridge. He joined the Department of e University of Illinois in 1985 where he currently William H. and Janet G. Lycan Professor of s research interests include synthetic organic, , bioorganic, dendrimer, and polymer chemistry.

coff was born in Buenos Aires, Argentina in 1969. grated to Israel and started his chemical career at

Fig. 2 Schematic representation of the polymer imprinting process showing one binding site within the polymer matrix. Cross-linking functionality may be covalently or noncovalently linked to the template.

S.C. Zimmerman et al. Chem. Commun. (2004), 5





Only small fraction of binding sites are imprinted

“stoichiometric noncovalent imprinting” uses aof1MIPs : 1 ra Another strategy for increasing the homogeneity s well as its between the involves increasing the preequilibrium constant group shown in on Fig.the 3a. tem functional monomer to the complementary ntly strong to This can be done in a number of ways, for example by using binding Tight binding favors sites A, D, and F at the expense of B s 3 21 Only small fraction of binding sites are imprinted s in solution. contacts that are very strong (i.e., Kassoc 4 10 M ). Such This approach usually involves ionic Fig.3 and 3a.24 “stoichiometric noncovalent imprinting” uses strong a 1 : 1 ratio of hy toward functional monomer to the complementary group on the template. thout templanality (i.e., B Tight binding favors sites A, D, and F at the expense of B sites in Fig. 3a.24 This approach usually involves strong ionic hydrogen ing sites are

pam MIP (reference 15). The synthetic scheme emphasizes the need to noncovalently assemble mplate (1) in a preequilibrium step preceding cross-linking with ethylene glycol methacrylate e schematic of the resulting gel emphasizes the heterogeneity of binding sites: high affinity site ce(B)15). The synthetic emphasizes thehighest need to noncovalently as Zimmerman et trapped al. Chem.scheme Commun. (2004), 5 K. Mosbach et al.affinity Nature (1993), 361, 645 inSC macropore, (C) template, (E) embedded site, (D) site with shape nstants and populations of three classes of binding sites needed to fit binding isothermglycol (data from eequilibrium step preceding cross-linking with ethylene meth

Origins of Binding Site Heterogeneity (BSH)

polymerization Prepolymerization mixture

wash Molecularly imprinted polymer (MIP)

Effects of binding site heterogeneity

analytical window

80 70 N (umol/g)

•Extreme peak asymmetry (tailing) and poor resolution

NIP

60 50 40

•Highly concentration dependent affinity and selectivities

30 MIP

20 10 0 0

1

2

3

4

5

log K (1/M) KD Shimizu et al., Anal. Chem. (2004), 76, 1123

6

Effects of binding site heterogeneity 33

B. Sellergren, K.J. Shea / J. Chromatogr. A 690 (1995) 29-39 20

HPLC

analytical window

o

D,L-phenylalanine anilide

] .... ... k0L

80

5 nmol

15

70 N (umol/g)

60 50

2.5 nmol

NIP 10

""'0--...

40

''"0...

200 nmol

°°"0...

" ' " 0 ...... 0

30

@-'~- 0~.,0.

MIP

20 10

. . . . . . . " < > " ~ O ....... ~. ...... O "--------.----........._.

. . . . . . . .

0

! 10

. . . . . . . .

D,L-PA

0

20

18 16 14

1 •--'m .....

2 k' L

.... • ....

---~....

3

i 100

. . . . . . . . 1000

50 nmol

(nmol)

4

5

log K (1/M)

k' D

KD Shimizu12et al., Anal. Chem. (2004), 76, 1123 10

100 nmol

6 0

i

I

I

I

10

20

30

40

I~

Time (rain) Fig. 5. Elutionetprofiles of increasing of O,L-PA applied Mosbach. al. J. Org. Chem.,loads (1997) 62, 4057 on P2. Flow-rate, 1.0 ml/min; mobile phase, MeCN-0.05 M KP (pH 7) (7:3, v/v). The sample loads from right to left in

Effects of binding site heterogeneity

concentration dependent selectivity

analytical window

80

16

70

14

NIP

N (umol/g)

60

12 10

50

α

40

8 6

30

4

MIP

20

2

10

0 0

0

0.5

1

1.5

2

[L-PAA] (mM)

0

1

2

3

4

5

6

log K (1/M)

K.D. Shimizu et al. Anal. Chem., (2004), 76, 1123 KD Shimizu et al., Anal. Chem. (2004), 76, 1123

V. Solutions: a) Chromatographic Gradient Elution (Boc-D,L-Trp)

isocratic: 3% AcOH in MeCN Polymers with Enantiomeric Recognition

gradient: AcOH MeCN (0% to 7%) J. Orin g. C hem., V ol. 62, No. 12, 1997 40

acid competed with the sample molecule for the am hydrogen-bonding functional groups and reduced possibility of hydrogen-bonding interactions between sample molecule and the amide groups at the recognit sites. For any enantiomeric recognition, at least three of four groups around the chiral center must be specifica recognized. For amino acid derivatives, these th groups are the carboxyl group, the amino protect group, and the side chain. (a) We believe that, prior to polymerization, a comp forms between the free carboxyl group of the templa and the amide group of the functional monomer (Schem 1-3). Esterification of the template carboxyl gro prevents the formation of hydrogen bonds between t Figure 2. Comparison of an enantiomeric separation using and the amideAmide groupMIP of the polymer (Scheme 2 isocraticgroup or gradient elution. was made against Neither of thephase, two esters, Ac-D,Lacid -Trp-OEt or Boc-D,L-T (a) Mobile 0.3% acetic in acetonitrile; Boc-L-Trp. OMe, could be separated the made D,Lby -Trp wasMIPs injected in 20agai flow rate, 1.0Mosbach. mL/min; 40al. µg Bocet J.of Org. Chem., (1997) 62, 4057 k!L ) 2.83, R ) 3.08, RThe µL of acetonitrile; templatesk!with a free carboxyl group. amount D ) 0.92, s ) 1.97. (b) Gradient elution: solvent A, the acetonitrile; solvent B, acetic sample injected onto MIPs made against Ac-L-T

V. Solutions: a) Chromatographic Gradient Elution (Boc-D,L-Trp)

isocratic: 3% AcOH in MeCN Polymers with Enantiomeric Recognition

α = 3.08, Rs = 1.97

gradient: AcOH MeCN (0% to 7%) J. Orin g. C hem., V ol. 62, No. 12, 1997 40

acid competed with the sample molecule for the am hydrogen-bonding functional groups and reduced α = 2.69, Rs = 2.51 possibility of hydrogen-bonding interactions between sample molecule and the amide groups at the recognit sites. For any enantiomeric recognition, at least three of four groups around the chiral center must be specifica recognized. For amino acid derivatives, these th groups are the carboxyl group, the amino protect group, and the side chain. (a) We believe that, prior to polymerization, a comp forms between the free carboxyl group of the templa and the amide group of the functional monomer (Schem 1-3). Esterification of the template carboxyl gro prevents the formation of hydrogen bonds between t Figure 2. Comparison of an enantiomeric separation using and the amideAmide groupMIP of the polymer (Scheme 2 isocraticgroup or gradient elution. was made against Neither of thephase, two esters, Ac-D,Lacid -Trp-OEt or Boc-D,L-T (a) Mobile 0.3% acetic in acetonitrile; Boc-L-Trp. OMe, could be separated the made D,Lby -Trp wasMIPs injected in 20agai flow rate, 1.0Mosbach. mL/min; 40al. µg Bocet J.of Org. Chem., (1997) 62, 4057 k!L ) 2.83, R ) 3.08, RThe µL of acetonitrile; templatesk!with a free carboxyl group. amount D ) 0.92, s ) 1.97. (b) Gradient elution: solvent A, the acetonitrile; solvent B, acetic sample injected onto MIPs made against Ac-L-T

and hydrogen bondi analyte functional gr specific analyte reten introduced through during sample prep extraction selectivity background is obser lower detection limit sample prep techniq

V. Solutions: a) Chromatographic Non-polar subsite A Non-polar subsite B

CE

SPE (solid-phase extraction) Figure 3. Overview of a Typical SupelMIP SPE Procedure SupelMIP Solid Phase Extraction ™

1

2

3

4

Sample Load

Wash

Elution

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Did you know that of 0) for the FAPAS ( Scheme) proficiency FAPAS is the largest i testing program sinc sigma-aldrich.com

Table 1. Relative Sele

Molecularly Imprinted Polymers for the Highly Selective Extraction of Trace Analytes from Complex Matrices

Non-Selective

P

Liqu

Nilsson, Anal. Chem., (1997), 69, 1179

Hy

(-)- and (+)-galanthamine

1 Condition and equilibrate SupelMIP SPE 2 Sample Load

Biotage® AFFINILUTE ™ MIP 3 Application of a series of vigorous wash steps

C18

that will selectively retain analyte(s) of interest but elute interfering components

U. Jordis, A. Rizzi, F. Grohmann ECHET98 (1998)

Beta-blockers, Beta-agonists, 4 Analyte elution Riboflavin (vitamin B2), triazine herbicides, antibiotics, tobacco specific by-products, amphetamines, fluoroquinolones, nitroimidazoles sigma-aldrich.com/supelmip

L

NEW SupelMIP Phases and Applications

L

Achieve Lower Detection Limits

L

Reduce Time and Costs

L

Improve MS-Compatibility

L

No Method Development Required

Highly Selective

Solutions: b) Improve imprinting efficiency

polymerization Prepolymerization mixture

wash Molecularly imprinted polymer (MIP)

Solutions: b) Improve imprinting efficiency 15.2. SYNTHESIS OF MIPs

399

covalent MIP

O O O

O

O NH

O

O

HN

O

O

NH NH O NH N

HN

O HN O

N Zn N

N

HN

= carbohydrates

polymerize

Scheme 15.2 Covalent imprinting of a-phenyl-D-mannopyranoside in a divinyl benzene/4vinylphenylboronic acid matrix, via the formation of covalent boronic ester linkages between the 4-vinylphenylboronic acid and the carbohydrate. Adapted from Wulff, Vesper, et al. (1977). Copyright 1977 Wiley InterScience.

G. Wulff et al. Makromol. Chem. 1977, 178, 2817

The covalent nature of the monomer – template complex yields greater control over the imprinting process. The kinetic stability of the prepolymerization complex

Solutions: b) Improve imprinting efficiency 15.2. SYNTHESIS OF MIPs

high-affinity non-covalent MIP

399 O O O

O

O NH

O

O

HN

O

O

NH NH O NH N

HN

O HN O

N Zn N

N

HN

= carbohydrates

polymerize

Scheme 15.2 Covalent imprinting of a-phenyl-D-mannopyranoside in a divinyl benzene/4vinylphenylboronic acid matrix, via the formation of covalent boronic ester linkages between the 4-vinylphenylboronic acid and the carbohydrate. Adapted from Wulff, Vesper, et al. (1977). Copyright 1977 Wiley InterScience.

The covalent nature of the monomer – template complex yields greater control over the imprinting process. The kinetic stability of the prepolymerization complex

KD Shimizu et al. Org. Lett. 2005, 7, 963-966.

Solutions: b) Improve imprinting efficiency

‘non-covalent’ MIP

‘covalent’ MIP

Remcho et al. J. Chromatogr., A 2001, 922, 87-97.

Making imprints more homogeneous

chloride, naphthoyl chloride, an The method by which most MIPs are made all but guarantees the whose sizes correspond to th production of binding sites with different structures. Whether the wherein the post-imprinting mod differences in binding site structures produce measurable differabsence of template, showed a de ences in the binding properties will depend onfor the specific binding site size and the size of Recommendations using system, MIPs in separations but it is common to find a broad range of affinities. The classic results are similar to the molecu study of diazepam (1) and theophylline MIPs reported by Mosbach the derivatization of convention Another strategy for increa illustrates the degree of hetereogeneity possible as well as its As seen in polymer Fig. 3, the between the involves increasing the preequil origin.1.15 Must select andhydrogen templatebonding that interact diazepam template (1) andmatrix MAA should (2) is not sufficiently strong This can be done in a number of w - Polymer have innate affinity fortotemplate make assemblies such as 3should the predominant species in solution. that are very strong - Template have multiple hydrogen bonding orcontacts ionic groups “stoichiometric noncovalent im Thus, an excess of 2 is (amines, added to alcohols, push the equilibrium towardphosphate 3 and carboxylic acids, etc) functional monomer to the comp the resultant polymer contains many sites formed without templation or without the full complement of binding functionality (i.e., B Tight binding favors sites A, D, know imprinting matrix and (i.e. solvent, temperature) Fig. 3a.24 This approach usuall sites II. in Utilize Fig. 3a). Some extraordinarily tightconditions binding sites are

- Polymerization conditions have been carefully optimized for imprinting efficiency and surface area

III. Washing step is very important -can be limiting factor for highly sensitive apps IV. Be aware of the consequence of binding site heterogeneity -easily saturated -peak tailing -concentration dependent behavior

Fig. 3 (a) Synthesis and schematic representation of a diazepamS.C. MIPZimmerman (reference 15). synthetic scheme emphasiz et The al. Chem. Commun. (2004), 5 methacrylic acid (MAA) molecules around the diazepam template (1) in a preequilibrium step preceding cross-link

Future Shimizu et al., Chem. Commun. (2004) 10, 1172

I. Targeted applications (avoid BSH) -SPE -sensor arrays

II. Moving beyond commercially available monomers -covalent imprinting -high-affinity non-covalent imprinting III. Protein and Virus imprinting

K. Shea et al., J. Mater. Chem., (2011), 21, 3517

Shimizu et al., unpublished

Acknowledgments Di Song Chen Zhao Brent Dial Roger Rasberry Yagang Zhang William Carroll CJ Stephenson Nathaniel Greene Sean Wu Yizhao Chen Greg Rushton Holly Ricks Judy Lavin Yong Chong Charles Degenhardt Robert Umpleby

For more information on MIPs MIP literature database: http://www.mipdatabase.com/ Selected MIP review articles: L. X. Chen, S. F. Xu, J. H. Li. “Recent advances in molecular imprinting technology: current status, challenges and highlighted applications” Chem. Soc. Rev., 2011, 40, 2922-2942. R. J. Ansell. “Molecularly imprinted polymers for the enantioseparation of chiral drugs.” Adv Drug Deliv Rev, 2005, 57, 1809-1835. A. J. Hall, M. Emgenbroich, B. Sellergren. “Imprinted polymers” Top. Curr. Chem., 2005, 249, 317-349. S. C. Zimmerman, N. G. Lemcoff. “Synthetic hosts via molecular imprinting - are universal synthetic antibodies realistically possible?” Chem. Commun., 2004, 5-14.

Advice

The MIP ‘Rule of Six’ • • • • • •

Never use the analyte as a template unless there is absolutely no alternative Make rational choices about which regions of an analyte are likely to command the best types of interaction in a low dielectric medium (organic solvent) and then incorporate these elements in an analog of the analyte molecule Select monomers that are likely to form strong interactions in the chosen solvent (e.g., Brönsted acids or bases/H-donors or acceptors/nonpolar groups, etc.) - this will increase capacity and influence homogeneity of the binding cavities Choose templates and monomers that will be soluble in the porogenic solvent to be used in the polymerization - this may seem obvious but it sometimes requires carrying out solubility tests Ensure as far as possible that the template-monomer mixture is stable and does not undergo side reactions under the polymerization conditions Consider the nature of the matrix from which the analyte will eventually be extracted when selecting the cross-linking monomer - a range of di- or tri-unsaturated cross-linking monomers (e.g., vinylic, acrylic, methacrylic, acrylamide, etc.) with varying chemistries are available to create the porous organic network material.

MIP Technologies AB

Templates R.J. Ansell / Advanced Drug Delivery Reviews 57 (2005) 1809–1835

1815

Fig. 5. Structures of enantiomeric drugs which have been employed as templates for the production of MIP CSPs, and/or whose enantiomers have been separated on MIP CSPs.

such conditions, there may be excessive peak overlap, however, so the combination of sample load, run time and chromatographic resolution should be given particular consideration. Where these parameters have not been stated in published work, they have whenever possible been estimated by this author from the available data or published chromatograms. Although in many cases several different racemates may have been injected onto the imprinted column, besides the imprinted compound and its enantiomer,

published the separation of (R,S)-timolol (and of (R,S)-propranolol) on a (S)-timolol-imprinted polymer [41]. Itaconic acid (IA) and MAA were compared as functional monomers, and IA gave rather better chromatographic separation and resolution, although the mobile phase used with the MAA polymer may have caused some problems (straight MeCN/AcOH mobile phases give complications with basic analytes) and the excess of functional monomer/template used (6:1) may also have been sub-optimal. Nonetheless,

R. J. Ansell. “Molecularly imprinted polymers for the enantioseparation of chiral drugs.” Adv Drug Deliv Rev, 2005, 57, 1809-1835.

there are only a limited number of direct physical characterization methods for imprinted polymers. These include surface area and porosity measurements, IR spectroscopy, solid state NMR (13CPMAS spectroscopy), and swelling. The spectroscopy methods presented are best for investigating molecular level features of the MIP materials. The surface area, porosity, and swelling measurements characterize macroscopic features of MIPs; however, information provided by these on the binding site structure of MIPs is very limited. On the other hand, this data can be very useful for drug delivery applications such as controlling substrate release times, and swelling properties due to environmental factors.

mer, the type and amount of porogen, and the reaction temperature. Although binding and selectivity by MIPs in chromatographic or batch rebinding mode are not dependent on macroporosity, applications in drug delivery may rely on mass-transfer kinetics related to porosity. Surface area measurements in MIPs are primarily carried out using a nitrogen adsorption porosimeter using a BET (Brunauer, Emmett and Teller) analysis routine that is standard to all instruments. For pore size distributions in MIPs, the same nitrogen adsorption data can be analyzed using BJH (Barret, Joyner and Halenda) methods also available on porosimetry instruments. Results from this type of characterization are provided in Table 2 which compares the effect of different porogens on

Important role of solvent

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Second family of mesopores (2-50 nm) Third family of macropores (50-1000 nm)

Large aggregates of microspheres (µm)

Table 2 Surface area, pore volume, and average pore size in MIPs made with EGDMA / MAA monomers using l-phe-an as template (adapted from Ref. [12])

10-30 nm Nucleii

Microsphere (100-200 nm)

First family of micropores (