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Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

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PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

173

either the initiation of peptide synthesis or the immobilization of peptides. It was often demonstrated that the surface-bound peptides’ accessibility to the proteins or enzymes used in screening is a critical factor. Insertion of a spacer between the peptide and the surface is an effective way to circumvent this drawback (Figure 7.11B). Generally, all linker molecules introduced to transform a given surface function into a functional group suitable for amino acid or peptide attachment can be considered as spacers. Such spacers can improve the efficiency of peptide/ligand interaction on surfaces as demonstrated with FLAG epitope peptides recognized by the monoclonal anti-FLAG M2 antibody [581]. The signal increased with the length of spacer introduced between the epitope and the surface. Additionally, for protein tyrosine kinase p60c-src it was demonstrated that only incorporation of the long and hydrophilic 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid building block spacer allowed effective phosphorylation of the glass surface-bound peptides [143]. Moreover, insertion of hydrophilic dextran structures between the surface and the presented peptides (Figures 7.12 and 7.13) was described as necessary for efficient enzyme substrate interaction [337].

The loading of glass surfaces is too low for several biochemical reactions (Tables 7.1, 7.2). Several different approaches for generating dendrimeric spacers/linkers are used to increase the density of immobilized peptides (Figure 7.11C, 7.12–7.16). Alternatively, the effective surface per area can be increased by the use of porous silicon [306, 347, 462]. Examples for one-step generation of dendrimeric structures (Figure 7.11C) are represented by poly-lysine coated glass slides (Table 7.2) or aziridine polymerization onto aminopropylsilylated glass surfaces (Figure 7.16) [85, 257, 388]. An interesting approach for increasing the density of reactive functions is surface modification by adsorption of structured α-helical peptides [332] or proteins (bovine serum albumin, BSA; [338]) yielding amino modified polymers. The BSA amino functions were transformed into active esters by treatment with N,N-disuccinimidyl carbonate (Figure 7.15).

More sophisticated procedures for preparing multiple linker structures by multistep functionalization of glass surfaces have been reported (Figures 7.14, 7.17) [31, 33, 197, 242, 258, 302, 410]). Alternatively, surface loading could be improved by coatings employing three-dimensional layers such as hydrogels (Rubina et al., 2003), semi-wet gels [258], agarose films [5], acrylamide gel pads [367, 430, 565] or gelatine pads [136].

7.2.2. Generation of Micro-Structured Surfaces

Micro-structured or micro-patterned surfaces represent an alternative way to achieve spatially addressed deposition of molecules. This could be useful for generating microreactors [604] or improving array regularity. Two major principles are used for micropatterning: contact printing techniques (see Section 7.2.4.1.) and photolithografic technologies. The use of micro-contact printing for micro-patterning [36, 107, 225, 226, 240, 283, 284, 349, 507] has been reviewed extensively [60, 365, 366, 512, 601, 602] and is therefore not discussed in detail. Dip-Pen nanolithography/scanning probe lithography [51, 67, 249, 256, 302, 385, 519, 577, 580, 598] and microfluidic channel networks [83, 103, 105, 398] were used for nanoand micro-patterned immobilization of biomolecules such

174

ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

i

O

O

OH

O

O

HN

O

O

ii

 

H2N

O

O

N

HO

H

iii

O

OH

O

OH

O

OH

OO

O

OH

O

 

 

 

OH

 

OH

 

O

O

O

 

 

HOH

N O

O N

H

O

O

iv

OH

O

OH

O

OH

OO

O

OH

O

 

 

 

NH2 HN

 

OH

 

O

O

O

HOH

N O

O N

H

NH2 O

FIGURE 7.12. Transformation of porous polypropylene into hydrophilic matrix suitable for enzyme profiling [337]. i = oxidation using Cr2O3/H2SO4/H2O, 54:54:80 (wt,wt,wt), ii = 5% oxalylchloride in CH2Cl2, 2 h, room temperature followed by treatment with 10% 2,2’-(ethylenedioxy)diethylamine in CH2Cl2, iii = carboxymethylated dextran, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in water, 12 h, room temperature, iv = carbonyl diimidazole, acetonitrile, 30 min, 0.3 M diaminopropane in acetonitrile, 1 h.

TABLE 7.2. Selected suppliers of modified surfaces

surface function

world wide web

dimension [mm]

material

amino

www.aims-scientific-products.de

80

× 120

cellulose

amino

 

100

× 150

cellulose

amino

 

235

× 250

cellulose

streptavidin

 

100

× 150

cellulose

amino

 

240

× 240

polypropylene

amino

 

80

× 120

polypropylene

amino

 

240

× 240

polypropylene

amino

 

80

× 120

polypropylene

bromide

 

100

× 150

cellulose

carboxyl

 

100

× 150

cellulose

Fmoc-ß-Alanine

 

100

× 150

cellulose

Fmoc-Proline

 

100

× 150

cellulose

mercapto

 

100

× 150

cellulose

hydroxyl

 

240

× 240

polypropylene

hydroxyl

 

80

× 120

polypropylene

hydroxyl

 

240

× 240

polypropylene

hydroxyl

 

80

× 120

polypropylene

epoxy

www.quantifoil.com

75

× 25

glass

aldehyde

 

75

× 25

glass

epoxy

www.noabdiagnostics.com

75

× 25

glass

aldehyde

 

75

× 25

glass

NHS ester

 

75

× 25

glass

aldehyde

www.aat-array.com

75

× 25

glass

amino

www.sigmaaldrich.com

75.5

× 25

glass

poly-L-lysine

 

75.5

× 25.5

glass

carboxyl

www.nuncbrand.com

76

× 25

heat stable polymer

amino

 

76

× 25

glass

epoxy

 

76

× 25

glass

aldehyde

 

76

× 25

glass

amino

www.eriesci.com

n.a.

 

glass

poly-lysine

 

n.a.

 

glass

epoxy

 

n.a.

 

glass

loading

400 nmol/ cm2

400 nmol/ cm2

400 nmol/ cm2

2 nmol/ cm2

80 nmol/ cm2

80 nmol/ cm2

600 nmol/ cm2

600 nmol/ cm2

800 nmol/ cm2

500 nmol/ cm2

800 nmol/ cm2

600 nmol/ cm2

600 nmol/ cm2

150 nmol/ cm2

150 nmol/ cm2

1500 nmol/ cm2

1500 nmol/ cm2 n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

(cont.)

DISCOVERY DRUG AND PROTEOMICS IN ARRAYS PEPTIDE

175

TABLE 7.2. Continued

surface function

world wide web

 

dimension [mm]

material

loading

 

 

 

 

 

 

amino

www.arrayit.com

76

× 25

glass

8 pmol/cm2

amino

 

76

× 25

glass

8 pmol/cm2

aldehyde

 

76

× 25

glass

8 pmol/cm2

poly-L-Lysine

 

76

× 25

glass

n.a.

acrylic

 

76

× 25

glass

8 pmol/cm2

carboxyl

 

76

× 25

glass

8 pmol/cm2

cyanato

 

76

× 25

glass

8 pmol/cm2

epoxy

 

76

× 25

glass

8 pmol/cm2

mercapto

 

76

× 25

glass

8 pmol/cm2

amino

www.xenopore.com

75

× 25 or 22 × 40

glass

16 pmol/cm2

aldehyde

 

75

× 25 or 22 × 40

glass

16 pmol/cm2

epoxy

 

75

× 25 or 22 × 40

glass

16 pmol/cm2

maleimide

 

75

× 25 or 22 × 40

glass

16 pmol/cm2

nitrilotriacetic acid

 

75

× 25 or 22 × 40

glass

n.a.

streptavidin

 

75

× 25 or 22 × 40

glass

n.a.

biotin

 

75

× 25 or 22 × 40

glass

n.a.

mercapto

 

75

× 25 or 22 × 40

glass

16 pmol/cm2

amino

www.corning.com/lifesciences

75.5 × 25.3

glass

n.a.

substituted antraquinones

www.exiqon.com

75

× 25

injection molded polymer

n.a.

amino

www.greinerbioone.com

75

× 25

glass

n.a.

aldehyde

 

 

 

glass

n.a.

streptavidin

 

 

× 25 or 24 × 60

glass

n.a.

epoxy

www.asperbio.com

75

glass

n.a.

amino

 

75

× 25 or 24 × 60

glass

n.a.

isothiocyanate

 

75

× 25 or 24 × 60

glass

n.a.

epoxy

www.genescan.com

75.7 × 25.4

glass

16 pmol/mm2

amino

www.perkinelmer.com/

75

× 25

glass coated with modified acrylamide

n.a.

 

areas/proteomics/chem2.asp

active surface: 12 × 40 or

polymer

 

 

 

12

× 12 (two pad)

 

 

176

SCHUTKOWSKI MIKE AND MERGENER-SCHNEIDER JENS REINEKE, ULRICH

epoxy

www.mwgbiotech.com

75

× 25

glass

n.a.

mercapto

www.ApogentDiscoveries.com

75

× 25

glass

n.a.

animo

www.csem.ch

75

× 25

glass

n.a.

carboxyl

 

22

× 20 available for print

metal oxides

4.2 pmol/mm2

maleimide

 

 

 

metal sulfides

3.4 pmol/mm2

mercapto

 

 

 

silicon

 

 

 

 

 

silicon nitride

 

 

 

75.5 × 25

plastics

 

amino

www.us.schott.com

glass

n.a.

isothiocyanate

www.picorapid.de

76

× 26

glass

n.a.

amino-reactive

www.ucb-group.com

75

× 25

glass

n.a.

 

 

variable size

cellophane

n.a.

 

 

variable size

polypropylene

n.a.

amino

biolink@bellatlantic.net

120 × 80

highly crosslinked, polyaminated,

15 nmol/mm2

 

 

 

× 25 (slide)

polyurea coated polypropylene fleece

 

amino

www.pall.com

75

positive charged nylon membrane bound

n.a.

 

 

60

× 22 (membrane)

to glass

 

aldehyde

www.pall.com

Ultrabind US450 membrane roll

modified polyethersulfone

n.a.

 

 

3000 × 320

 

 

amine reactive

www.accelr8.com

75

× 25

glass or silicon

n.a.

thiol-reactive

 

 

 

 

n.a.

biotin

 

 

× 25

 

n.a.

NHS ester

www1.amershambiosciences.com

75

glass

50 fmol/mm2

 

 

 

 

 

 

n.a. not available

DISCOVERY DRUG AND PROTEOMICS IN ARRAYS PEPTIDE

177

178

ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

i X Y

R HN

CF3

ii

 

 

 

 

X Y

 

 

S

HN

 

 

O

HN

 

CF3

 

OH

 

 

 

 

 

 

OH

O

O

 

 

 

OH

O

 

 

 

 

 

 

 

 

OH

O

 

 

 

 

 

 

 

NH

OH

 

 

S

OH

O

 

 

 

OH

 

 

 

NH

O

 

 

 

 

 

 

 

OH

 

 

 

OH

O

 

 

 

OH

 

 

 

 

CF3

N

N

FIGURE 7.13. Photobonding to and functionalization of surfaces such as glass, polystyrene, silicon nitride or polyurethane membranes. i = photochemical coupling of N-substituted m-3-(trifluoromethyl)diaziridin-3- yl-anilines, R = 4-maleimidobutyryl residue [91, 442]; ii = Optodex treatment [71].

as proteins, but no applications for the creation of peptide arrays have been described so far.

Surfaces modified by amino groups protected by photosensitive protection groups can be used to generate microstructures simply by irradiation through a lithographic mask [480, 605]. A very similar approach [613] starts from the derivatization of glass surfaces with a photolabile self-assembled monolayer (Figure 7.18). Irradiation led to release of hydrophobic moieties yielding hydrophilic, carboxy modified areas (B) within a hydrophobic (A) environment.

Alternatively, photoresist coatings can be used in combination with photolithographic masks to create micro-patterns. Following irradiation, areas protected by photoresist coatings will not undergo modification upon treatment with tridecafluoro- 1,1,2,2-tetrahydro)trichlorosilane in anhydrous toluene (Figure 7.19). After removal of the

PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

179

H2N

H2N

i

O

OO

N

N

O

O

H

 

O

H

O

N

N

 

OO

O

ii

OO

 

NH2

 

H2N

HN

N

 

PAMAM

H

 

 

 

HN

H

H2N

N

 

NH2

 

OO

N

S

 

 

 

 

C

 

O

O

 

S

 

NH

 

 

 

 

 

 

 

 

 

 

N

NH

HN

 

 

N

H

 

 

 

 

H

 

PAMAM

 

 

 

H

 

 

 

 

H

NH

HN

 

 

N

N

 

 

S

 

NH

O

O

 

S

 

 

C

S

 

N

 

O

O

 

 

 

 

 

 

 

NH

 

 

 

 

 

 

HO

3

N

iii

 

 

 

 

H

 

 

 

 

 

 

 

 

HO

 

H

 

 

 

3

N

 

 

NH

 

 

 

 

O

O

 

N

 

S

 

S

O

O

 

C

NH

 

S

 

 

 

 

N

NH

HN

 

 

N

H

PAMAM

 

 

H

 

 

 

 

H

 

 

 

 

H

NH

HN

 

 

N

N

 

 

 

 

 

 

S

 

NH

O

O

 

 

 

 

C

S

 

 

 

 

 

 

N

 

 

 

 

 

FIGURE 7.14. Glass surface modification/activation via starbust dendrimer coating [33]. i = treatment of aminosilylated glass with homobifunctional disuccinimidylglutarate in CH2Cl2 containing 1% diisopropylethylamine, 2 h; ii = 10% PANAM starbust monomer, 12 h; iii = activation and intermolecular crosslinking using homobifunctional 1,4-phenylenediisothiocyanate in CH2Cl2 containing 1% pyridine, 2 h.

protecting layer the newly exposed glass surfaces can be transformed into hydrophilic areas by aminopropylsilylation and acylation. Butler and coworkers produced so-called surface tension arrays [70] for the on-chip synthesis of oligonucleotides using a similar principle (Figure 7.20). The resulting hydrophilic areas (B), surrounded by hydrophobic areas (A) with a very low surface tension, represent micro-reactors holding the reaction solvent in area B due to differences in the wetting characteristics. The perfluorosilane modified areas A have wetting properties similar to Teflon. Alternatively, Brennan used a laser for

180

ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

H2N

i

OO

NN H

O

 

 

 

ii

 

NH2

 

 

H2N

 

O

 

 

BSA

N

N

 

H

H

H2N

NH2

i

O

O

O

 

N

 

 

 

 

 

N

O

NH

O

 

 

 

O

 

 

 

O

HN

 

 

 

 

 

 

 

O

 

BSA

N

N

 

 

 

H

H

NHN

O O O NH

N

O O

FIGURE 7.15. Multistep functionalization of glass surfaces [338]. i = N,N-disuccinimidyl carbonate, diisopropylamine, dimethylformamide, 3 h, room temperature; ii = PBS pH 7.5, 1% bovine serum albumin (BSA), 12 h, room temperature.

PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

ii

HO

i

 

NH2

 

 

 

H2N

NH2 HO

 

 

 

 

H2N

HO

 

 

H2N

 

H2N

NH2

 

 

 

H2N

 

HO

 

 

NH2

 

H N

 

H2N

 

 

2

 

NH HO

 

H2N

H2N

 

2

 

 

 

 

H2N

 

NH2 HO

 

H2N

 

 

H N

H2N

H N

NH

2

2

H2N

2

 

H2N

HO

 

H2N

 

 

H2N

 

 

 

HO

 

 

H2N

 

H2N

NH2

 

H2N

 

H2N

H2N

HO

 

 

 

 

H N

HN

NH2

 

2

 

 

 

NH

NH HO

 

 

2

2

 

 

H2N

 

 

 

H2N

 

 

181

 

CH3

H2N

Si

 

O

 

CH3

iii

NH2

N

CH3

N Si

O

CH3

N

NH2

FIGURE 7.16. Aziridine-polymerization [85, 257, 388] and poly-lysine coating. i = the glass surface is coated by covering it with poly-L-lysine solution and drying the film; the polymer film is bound to the surface by electrostatic interactions between silyl-OH functions and positive charged amino groups in the polymer; ii = ethoxydimethylaminopropylsilane/toluene, iii = aziridine / methylene chloride / acetic acid.

ablation of fluorosiloxanes (Figure 7.21) to create micro-patterned surfaces starting from a uniformly hydrophobic modified glass slide [61]. Furthermore, photolithographic techniques have been reported for the micro-patterning of films ([188, 268, 381, 606]; reviewed in [186, 305]). Micro-mirror mediated patterning of biomolecules has been reported [303, 304, 510].

182

 

 

ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

 

 

 

O2N

O

 

O

 

 

 

 

 

 

 

 

 

 

 

i

O

N

ii

nN

 

 

 

 

H2N

N

 

 

 

 

 

 

 

 

 

H

 

H

H

 

H2N

 

 

 

 

 

 

 

 

 

iii

 

O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N

iv

H

H

 

O

 

 

 

H

 

 

 

 

 

 

 

N

N

 

 

 

 

 

 

H2N

N

 

N

N

 

 

 

 

 

H

 

H

H

 

 

 

 

 

v

 

 

 

 

 

 

O

O

O

O

 

 

 

 

 

 

 

 

 

 

 

 

N

N

N

N

 

 

 

 

 

N

N

 

 

 

 

 

H

 

 

H

 

 

 

 

 

 

O

O

 

 

 

 

 

 

vi

 

 

 

 

H

H

 

 

H2N

O

N

N

O

NH2

 

 

2

 

 

2

OO

 

 

 

O

 

 

O

 

H N

O

N

N

N

N

N

 

N

N

 

2

 

2 H

H

 

 

H

 

 

 

 

 

O

O

 

 

 

 

H2N

O

2 N

N

O 2

NH2

 

 

 

 

H

H

 

 

FIGURE 7.17. Multistep functionalization of aminoalkylsilylated glass surfaces or plasma-aminated polypropylene surfaces [31]. i = 4-nitrophenylchloroformiate, diisopropylamine, CH2Cl2, 2 h; ii = diamine, dimethylformamide, 12 h; iii = acryloylchloride, diisopropylamine, dimethylformamide, 24 h, iv = tetraethylenepentamine, dimethylformamide, 12 h; v = acryloylchloride, diisopropylamine, dimethylformamide, 24 h; vi = 1,4-bis(3-aminopropoxy)-butane, dimethylformamide, 12 h.

7.2.3. Peptide Array Preparation

Peptides are usually synthesized step by step using appropriate side chain (permanent protecting group) and N-terminal protected (temporary protecting group) amino acid derivatives (Figure 7.22). Generally, fluorenyl-oxy-carbonyl- (Fmoc), tert-butyl-oxy-carbonyl- (Boc) and nitroveratryl-oxy-carbonyl (Nvoc) moieties are used as temporary protecting groups (PG) while preparing peptide arrays. Subsequent to amino acylation of surfacebound amino functions, the PG of the surface-bound amino acid derivative is removed by base (Fmoc), acid (Boc) or light (Nvoc) treatment. Repeated amino acylation and deprotection reactions yield a surface-bound target peptide in the side chain protected state. Final