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6 Examples of Applications of Solid-Phase Immobilization Chemistry

6 Examples of Applications of Solid-Phase Immobilization Chemistry

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OH

Glycoprotein



OH

O



HO



OH



O

HN



IO4–



O

O



O



Glycoprotein



O



H

N



OH

O

N

H



OH



O



OH

NH2



H2N(CH2)2N

NaBH4



OH

Glycoprotein



O



Glycoprotein



N



Gly-Tyr

NaBH4

Glycoprotein

HOOC



N2+



OH

O



H

N

O



N

H



OH



Glycoprotein



HOOC



OH

O



H

N



N

H



OH



Application of Chemical Conjugation to Solid-State Chemistry



O



O



O



OH



N



N



OH



FIGURE 14.18  Immobilization of glycoproteins through oxidation and reductive amination of the carbohydrate moiety.



423



424



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



to the Fc region of antibodies have been utilized in affinity chromatography. Generally, a ligand is

immobilized on a solid support, which is packed into a column. The selective affinity of an analyte

for the ligand enables it to be isolated in pure form. For instance, protein A is used for the purification of IgG, IgM, IgA, and IgD. In some cases, the ligands themselves need to be purified, which

may be difficult and costly. Their purity is an important factor in the success of selective isolation

of analytes. In addition, their stability determines the life span of the absorbent material. Because

of all these factors, synthetic ligands that combined the selectivity of the natural ligand with the

high capacity, durability, and cost-effectiveness of the synthetic systems are being designed and

utilized. Synthetic peptides with these properties have been employed in affinity chromatography.116

Numerous functional groups on such synthetic peptides can be used for immobilization and various

coupling approaches are applicable. The peptide is generally linked by using the carboxylic group

from the C-terminal or the amino group from the N-terminal, although the side chains of any of the

amino acids can also be used, taking the precaution that immobilization does not affect its binding

capability or selectivity.

The selective interactions between antibodies and antigens are of great interest in affinity chromatography. When antibodies or antibody-related reagents are used as ligands for the purification of

antigens, the process is referred to as immunoaffinity chromatography.117 Immunoaffinity columns

can serve as selective online precleanup steps for the isolation of a group of compounds, which

are captured by immobilized antibodies.118 There are numerous methods to immobilize antibodies

such as those described above, which include reductive amination, epoxy chemistry, and matrice

activation.119 Zhang et al.120 developed a novel solid support for antibody immobilization by using

acrylamide as monomer, ethylene glycoldimethacrylate as cross-linker, and bulk polymerization

as the synthetic method. The authors prepared a polymer in which the Cu(II) was embedded. The

Cu(II)-embedded polymer displayed a strong binding with antibodies and was used as a novel solid

support for antibody immobilization

When immobilized metal ions are used to isolate biomolecules, the version of affinity chromatography is referred to as immobilized-metal affinity chromatography (IMAC).121 It is based on the

known affinity of transition metal ions such as Zn2+, Cu2+, Ni2+, and Co2+ to histidine and cysteine

in aqueous solutions. The metal ions are immobilized onto a solid support to fractionate protein

solutions. Several chelating ligands are used to fix the metal to solid supports like agarose and sepharose. The common ligands are iminodiacetic acid (IDA), N,N-bis[carboxymethyl]glycine (nitrilotriacetic acid [NTA]), and tris(carboxymethyl)ethylene diamine (TED) as shown in Figure 14.19.

(A)

H2O



O



H2O

H2O



OH

N



Me



O



(B)



O





O



O–



H2O



H2O



–O



O

O



(C)

H2O



–O



O



–O



OH



H 2O



OH

O



O



O



Me



O



N

N



O



O–



H2O



Me

–O



–O



(D)



O–



H

N



N



Me



OH

N



–O



O



O

O–

O



O



FIGURE 14.19  Structures of some chelating ligands used in immobilized-metal affinity chromatography:

(A) IDA; (B) NTA; (C) TED; (D) N-carboxymethyl-2-carboxymethylglycine.



Application of Chemical Conjugation to Solid-State Chemistry



425



These chelating ligands are coupled to solid matrices though various cross-linking methodologies

described earlier. For example, sepharose is activated using epichlorohydrin and IDA is attached by

reacting its amino group with the epoxy group. Other ligand such as N,N-bis[carboxymethyl]lysine

and carboxymethyl aspartate have also been developed.121,122 However, many of the solid matrices

with chelating ligands are commercially available. There are numerous applications of IMAC for

the purification of proteins as well as immunoassays.121 The choice of the metal ion immobilized on

the IMAC ligand depends on the application. While trivalent cations such as Al3+, Ga3+, and Fe3+

and tetravalent Zr4+ are preferred for isolation of phosphoproteins and phosphopeptides, divalent

Cu2+, Ni2+, Zn2+, and Co2+ ions are used for purification of His-tagged proteins.

As discussed above, there are many matrices that have been used for affinity chromatography.123

The natural polysaccharides, such as agarose, cellulose, and cross-linked dextran, possess a high

content of hydroxyl groups available for activation and derivatization. Other organic polymers such

as polyacrylamide, polyacrylate, and polyvinyl polymers have also been used. The inorganic polymer silica is very stable and can easily be derivatized to introduce functional ligands. All these solid

matrices have their strengths and weaknesses. To improve their properties, a variety of protocols

have been developed to modify the surface of these supports by either chemical modification or

physical adsorption of polymers. Recently, monolithic materials have been developed.124 Monoliths

are continuous stationary phases that are made as a homogeneous column in a single piece. Organic

monoliths are prepared by in situ polymerization of monomers, cross-linkers, porogens, and an

initiator. Methacrylate and acrylamide-based polymers, poly(styrenedivinylbenzene), agarose, and

cryogels are examples of organic monoliths. Inorganic monoliths (silica) are prepared by the sol–gel

method or from bare silica particles. The monoliths can be molded into various shapes and forms.

Ligands are coupled onto the monolithic stationary phase by various functional groups. Monolith

with epoxide functionality can be used directly for immobilizations of ligands with amino groups.

The ligands can be attached via different spacers. For example, ethylene diamine can be used to

extend the distance from the epoxide moiety. Monoliths containing 1,2-diol groups can be oxidized

with periodate to generate in situ aldehyde groups, which are used to attach the ligand via an amino

group by reductive amination. In addition, monolith surface can be coated with various materials.

A streptavidin-coated monolith is used to immobilize biotinylated DNA.



14.6.2  Biosensors

Biosensors are devices used for the detection and measurement of biological molecules. Although

various forms of biosensors have been designed, they basically consist of two parts, a biologically

derived part and a physical part referred to as the physicochemical transducer. The biological part

is mostly based on enzymes. For example, biosensors based on acetylcholinesterase or butyrylcholinesterase principally monitor the activity of bound enzyme and change of its activity due to the

presence of an analyte and are used for detection of organophosphorus and carbamate pesticides,

nerve agents (e.g., sarin), and other natural toxins (e.g., aflatoxins).125 The biorecognition component

is coupled to electrochemical, optical, or piezoelectric transducers for measurement of the enzyme

activity. There are many methods for the immobilization of cholinesterases onto membranes such

as adsorption, entrapment, and chemical cross-linking. The chemical procedure uses bifunctional

reagents with glutaraldehyde being the most widely used.

Another common biosensor is based on GOD.126 Its use includes monitoring blood glucose for

the control of diabetes, online glucose monitoring for fermentations, and analyzing glucose concentrations in soft drinks. A variety of supports have been used for immobilization of GOD such

as cellulose, solid glass particles, porous glass particles, and nickel screens with porous glass and

cellulose being the most popular supports. Nickel oxide screens can be silanized and GOD can be

coupled by thiophosgene method. GOD can be cross-linked to solid supports with glutaraldehyde

in the presence of polyethyleneimine. Immobilization of GOD in the presence of its substrates has

been shown to protect the enzyme activity.



426



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



There are many other enzymes used in biosensors and some are genetically engineered to produce more stable structure, higher activity, broader dynamic range, and additional functional groups

for immobilization.127,128 Methylamine dehydrogenase, for example, has been engineered to improve

sensitivity of a histamine senor.129 Pyrroloquinoline quinone glucose dehydrogenase was engineered

to expand the dynamic range of a biosensor.130 A fusion enzyme between a P450 monooxygenase

and a NADPH-cytochrome P450 oxidoreductase was genetically engineered to give higher enzyme

activity than natural P450s.131 Cysteine residues have been introduced into protein sequences for the

immobilization of enzymes based on the dative binding between thiol groups and gold surfaces. For

instance, six mutants of recombinant monomeric superoxide dismutase were engineered to contain

one or two additional cysteine residues for the self-assembling on gold electrodes.132 Enzymes have

also been modified with histidine residues for their immobilization on specific metals. A hexa-Histagged acetylcholinesterase was immobilized on a Ni-NTA screen-printed electrode as discussed

for IMAC above.133 The hexa-His-tagged AChE has also been conjugated to Ni-IDA-modified magnetic beads. Polyhistidine-tagged proteins have also been specifically immobilized on to Ni-NTA

functionalized silicon nanowire for the preparation of biosensor field effect transistors.134



14.6.3  Microarrays

Microarray technology has become a fundamental tool for fast detection and analysis of biological

molecules. This rapidly growing field started with DNA microarrays for the analysis of nucleic acid

sequence information. Major applications of this technology include studying gene expression profiles and the detection of single nucleotide polymorphisms (SNPs). This informative technology has

expanded to other arrays such as protein/peptide microarrays, carbohydrate microarrays, and antibody

microarrays. In all these microarray applications, an array biochip is produced, which involves immobilization of probe molecules onto a solid support. A basic technological advance is the methodology

to immobilize all necessary probe molecules onto solid matrices. The following paragraphs will illustrate the importance of chemical cross-linking in the formulation of these microarray biochips.

14.6.3.1  DNA Microarrays

DNA microarrays and DNA chips are well developed and are commercially available for various applications.135 DNA hybridization microarrays are generally fabricated on glass, silicon, or plastic surfaces.

Other surface materials including nylon, nitrocellulose, polyacrylonitrile, polypropylene, polystyrene,

teflon, gold, polypyrrole, polyurethane, polymethylmethacrylate (PMMA), and polyethylene glycolgrafted silica surfaces and PMMA have also been used.135–138 The actual construction of microarrays

involves the immobilization or in situ synthesis of DNA probes onto the specific test sites of the solid

support or substrate material. Affymetrix has developed a photolithographic technique to carry out the

parallel synthesis of large numbers of oligonucleotides on solid surfaces.139 In this process, the glass

is first modified with a silane reagent to provide hydroxyalkyl groups, which are extended with linker

groups protected with special photolabile protecting moieties. The protecting groups are selectively

removed on photolysis, allowing the sites to be coupled by standard phosphoramidite DNA synthesis

protocols with an appropriate nucleoside phosphoramidite monomer, which is protected at their 5′ position with the photolabile 5′-(O-methyl-6-nitropiperonyloxycarbonyl) group. The 5′-terminal protecting

groups are selectively removed from growing oligonucleotide chains by controlled exposure to light for

reaction with another protected monomer. The cycles of photodeprotection and nucleotide addition are

repeated numerous times to build any given array of oligonucleotide sequences This methodology has

enabled the large-scale manufacture of arrays containing thousands of oligonucleotide probe sequences

on glass slides. Polymeric photoresist films as the photoimageable component have also been developed

for fabricating DNA arrays that utilize photolithographic methods.

Nanogen has developed an alternative DNA array technology using microelectronic chip devices

to increase the DNA hybridization rate by concentrating probe/target DNA at the test site.140 Arrays or

chips are fabricated on silicon wafers, having a base structure of silicon with an insulating layer of silicon



427



Application of Chemical Conjugation to Solid-State Chemistry



dioxide. Reversing the electric field on the test site produces an electronic stringency effect, which can

greatly improve hybridization specificity and provide the ability to discriminate point mutation and SNPs.

Chemical cross-linking has been used to attach or immobilize DNA probes onto support materials.141,142 The individual oligonucleotides are separately synthesized.138 The synthetic nucleotides

can be immobilized onto zirconylated surface based on coordination chemistry involving phosphoryl group of the oligonucleotides.143 Lane et al.144 found that poly(dG) spacer-bearing oligonucleotide probes immobilized on a zirconium phosphonate surface have a higher target capture rate

than probes with either no spacer or a different polynucleotide (polyA, poly C, and polyT) spacer

during hybridization with complementary targets. In a different approach, Wu et al.145 were able to

covalently immobilize unmodified oligonucleotides onto glass slides coated with acrylic acidco-acrylamide copolymer after activation of the copolymer coating with EDC/NHS.

In most cases, the oligonucleotides are derivatized by nucleotide chemistry processes prior to

immobilization. Various functional groups can be attached to the 3′- or 5′-end of the oligonucleotide such as mercaptoalkylation, thiophosphorylation, aminoalkylation, and silanization. The solid

support media, for example, glass slide, nylon filter, or other surfaces, are activated with bifunctional or other reagents to generate special functionalities. For example, glass slides can be silanized

with 3-glycidyloxypropyltrimethoxysilane to generate epoxy functions.146 Aminated supports can

be reacted with phenylene diisothiocyanate, disuccinimidyl carbonate or disuccinimidyl oxalate,

dimethylsuberimidate, and succinimidyl 4-(maleimidophenyl) butyrate to generate isothiocyanate-, succinimidyl-, imidoester-, and maleimido-activated surface as shown in Figure 14.20.147,148

S



H

N



N



N

H



S



S

N

O



O



(A)



N



O



N O



S



O



N

O



N

H



(C) H3C



O



N

O



NH

O



(D)

O



O



O



(B)

NH2



O



O

N

O



HN



O



CH3

NH

O



N

H



O



CH3



NH



O



N

O

O

N

H



N

O



O



FIGURE 14.20  Some examples of microchip surface activation by bifunctional reagents for the immobilization of presynthesized DNA: (A) phenylene diisothiocyanate; (B) disuccinimidyl carbonate; (C) dimethylsuberimidate; and (D) succinimidyl 4-(maleimidophenyl) butyrate.



428



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



Other bifunctional cross-linkers such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),

succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-(γ-maleimidobutryloxy)

succinimide ester (GMBS), m-maleimidopropionic acid-N-hydroxysuccinimide ester (MPS), and

N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB) have also been used.148,149 In addition, activation with NHS ester groups has been employed to activate glass surfaces.6 Many of these activated

support media are now available commercially. These reactive groups can be used to specifically

immobilize oligonucleic acid with appropriate moieties as illustrated below.

Aminoalkylated oligonucleotides can react with amino-group–directed reactive functionalities introduced onto solid supports using the cross-linkers mentioned above. For instance, aminemodified nucleic acid can be immobilized onto epoxy silane-derivatized or isothiocyanate-coated

glass slides.146,150 The epoxylated surface can also immobilize oligonucleotides with electrophilic

groups such as mercaptoalkyl-, aminooxyalkyl-, phosphoryl-, and thiophosphorylated oligonucleotides.146,151–153 Amine-modified oligonucleotides can also be immobilized onto an aldehyde-containing

surface by Schiff base formation, which can be stabilized with sodium cyanoborohydride (reductive

amination).154 A similar reaction can occur between aldehyde modified oligonucleotides and aminooxyalkylated glass surface where a stable oxime bond is formed, obviating the need for chemical reduction with sodium cyanoborohydride.155 Aminooxyalkyl groups can be generated by

UV treatment of 2-(2-nitrophenyl)propyloxycarbonyl group protected aminooxy silane. Another

approach to immobilized amine modified nucleic acids is to react with tetrafluorophenyl (TFP)activated self-assembled monolayers (SAMs) on gold-coated glass slides as shown in Figure

14.21.156 TFP-SAMs are formed by coupling thiol-containing TFP to gold surfaces. This methodology can be extended to prepare NHS-, aldehyde-, and maleimide-activated SAMs, which can

then be used to immobilized appropriately modified oligonucleotides.

Succinylated oligonucleotides can be immobilized onto aminophenyl- or aminopropyl-derivatized glass slides through amide bonds formation.157 Disulfide-modified oligonucleotides can be

immobilized onto a mercaptosilanized glass support by the thiol/disulfide exchange reaction.158

Thioalkylated oligonucleotides can be immobilized onto maleimido-activated surfaces148 or

directly on gold surfaces.159 They can also be immobilized using a heterobifunctional reagents,

N-(3-triethoxysilylpropyl)-4-(N′-maleimidylmethyl) cyclohexanamide (TPMC)160 or N-(3triethoxysilylpropyl)-6-(N-maleimido)-hexanamide (TPMH).161 The triethoxysilyl functionality of

the compounds has specificity toward a glass surface, whereas the maleimide functionality can react

with the thiol-modified oligonucleotides to form a stable thioether linkage. Immobilization of DNA

can be achieved by either reacting TPMC or TPMH with oligonucleotides to generate triethoxysilyloligonucleotide conjugates, which are then covalently attached via specific triethoxysilyl functionalities to an unmodified glass surface, or by first reacting the reagent with an unmodified glass surface

to place maleimide functionalities on the surface, which are then used for immobilization of oligonucleotides via stable thioether linkages (Figure 14.22). Oligonucleotides can also be immobilized

with N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl) cyclohexane-1-carboxamide (TPICC)162 or

F

O

G



S



F

O



5 O



F

F

F



O

L

O



D

S



F



5 O



G

H2N



3’

H2O



DNA



O

L

D



F

F



S



S



5

O



5



N

H



3’



DNA



OH



FIGURE 14.21  Coupling of amine-modified oligonucleotides to tetrafluorophenyl-activated self-assembled

monolayers on gold-coated glass slides. Hydrolysis of unreacted esters completes the reaction.



O

Si

Et

O

O

Et



N



H

N



DNA–SH

O



O



Et

OH O

Si

Si

O

OH O

Et



O

N



H

N

O



O

N



H

N



S



DNA



O

O



O

Et



DNA–SH

Et



O

Et



O

O

Si

O



N



H

N



O



S

DNA



OH

Si OH

OH



Application of Chemical Conjugation to Solid-State Chemistry



Et



O



Et

OH O

Si

Si

O

OH O

Et



OH

Si OH

OH



O



FIGURE 14.22  Coupling of thiol-containing oligonucleotides to glass slides using N-(3-triethoxysilylpropyl)-4-(N′-maleimidylmethyl) cyclohexanamide in two

different approaches.



429



430



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

DNA

N



N N



DNA



N

N



N



FIGURE 14.23  Coupling of acetylene-modified oligonucleotide to azide-terminated glass slide using click

chemistry.



N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)163 in a similar manner. Again, the triethoxysilyl functionality of TPICC and NTMTA can react with a glass

surface and the isothiocyanate functionality of TPICC, and the trifluoroethanesulfonate ester group

can react with either aminoalkyl (R-NH2) or mercaptoalkyl (R-SH) functionalities of modified oligonucleotides to form stable covalent bonds.

Many other approaches are also possible. Rozkiewicz et al.164 used click chemistry to immobilize oligonucleotides. In this approach, acetylene-modified oligonucleotides form stable covalent

bonds with a azide-terminated glass substrate as shown in Figure 14.23. Pack et al.165,166 introduced

oxa-nucleotide at the 3′- or 5′-end of the desired oligonucleotide sequences and reacted these oxanucleic acid with amine-functionalized glass slides under defined conditions as shown in Figure

14.24. The resulting microarrays were found to be highly efficient. Benters et al.167 increased reactive functionalities on glass by attaching poly(amidoamine) (PAMAM) dendrimers with 64 amino

groups in its outer sphere onto the aminated glass surface. The attached PAMAM dendrimers were

subsequently activated with 1,4-phenylenediisothiocyanate (PDITC) or disuccinimidylglutarate

(DSG) to generate a chemically reactive isothiocyanato- or succinimido-polymeric film, respectively. The resulting surface was used for the immobilization of amine-modified oligonucleotides.

In a similar approach, Hong et al.168 used a cone-shaped dendron instead of PAMAN to ensured

proper spacing between immobilized biomolecules. Alternatively, glass slides can be coated with

chitosan. The amine-rich polysaccharide is then activated with PDITC to generate reactive isothiocyanato functions, which can subsequently immobilize amine-modified oligonucleotides.169

Abramov et al.170 used nucleic acid analogs, hexitol nucleic acids (HNA), and altritol nucleic acids

(ANA), which contain an anhydrohexitol sugar moiety, as probes in DNA microarray. HNA and

ANA have high affinity toward DNA and RNA targets. The nucleic acid analog arrays are fabricated by immobilization of diene-modified oligonucleotides on maleimido-activated glass slides.

The HNA/ANA arrays could become a useful tool for nucleic acid diagnostics. Another immobilization method involves the interaction of biotin and avidin. Lim et al.171 developed a protocol

for immobilizing biotinyl-oligonucleotides onto solid support coated with PAMAM dendrimers,

which were peripherally modified with biotin and avidin. Breitenstein et al.172 used the same avidin/biotin approach to immobilize biomolecules. A glass slide was first silanized with 3-aminopropyltriethoxysilane to provide the surface with reactive amino groups that could be biotinylated

with sulfo-NHS-biotin. Neutravidin was spotted with an atomic force microscope (AFM) tip to

O

N



O

H2N



N



N



OH

O

O



NH2



O



DNA



N



N

H

H2N



OH

N

H



O



N



O

O



DNA



FIGURE 14.24  Coupling of oxa-nucleotide containing oligonucleotide to amine-terminated glass slides.



Application of Chemical Conjugation to Solid-State Chemistry



431



bind to the biotinylated surface. Biotinylated DNA was then added to bind to neutravidin. After

washing, neutravidin was deposited a second time with the same AFM tip, and then a second

biotinylated DNA was coupled by incubation. The authors claimed that the method can be used to

deposit biological molecules that can be coupled to biotin.

In addition to these modified surfaces, unactivated microscope slides may be used with activated, silanized oligonucleotides. Silanized nucleic acids can be immobilized instantly onto glass

surfaces. Kumar et al.,173 for example, prepared silanized oligonucleotides by reacting 5′-thiolated

oligonucleotides with either (3-mercaptopropyl)-trimethoxysilane followed by oxidation or 3-aminopropyl trimethoxysilane in the presence of SPDP. The trimethoxysilanized oligonucleotides can

also be prepared by reacting aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides with

glycidoxypropyltriethoxysilane.174 These silanized nucleic acids can be directly immobilized onto

unmodified glass slides. Lim et al.175 introduced distinct functional chemical groups into doublestranded DNA fragments of a random sequence using short synthetic DNA oligomers carrying

desired terminal functional groups. When a thiol group is used, these thiol-modified DNA can be

immobilized onto gold-coated or silane-modified solid substrates.

Photochemical reactions can also be used to immobilize oligonucleotides, although a number of

side reactions can occur. Kumar et al.176,177 synthesized two anthraquinone-based photosensitive heterobifunctional cross-linkers, N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide

(NTPAC)176 and N-(iodoacetyl)-N′-(anthraquinon-2-oyl)-ethylenediamine (IAED).177 The anthraquinone moiety is photosensitive and can be activated by irradiation (365 nm) to react with a variety of

carbon-containing polymers. The trifluoroethanesulfonate ester group and the iodoacetyl group of the

reagents react preferentially with aminoalkyl or mercaptoalkyl functions present in the biomolecules.

Like TPMC and TPMH discussed above, there are two ways the reagents can immobilize aminoalkyland mercaptoalkyl-oligonucleotides (Figure 14.22). They can first react with the modified nucleic acid

to form the oligonucleotide-anthraquinone conjugate and can then be photolyzed to covalently bind to

solid support. They can also be first photolyzed to couple to the solid support followed by reaction with

the modified oligonucleotides. Either method seems to give the same efficiency. Another anthraquinone photoreactive heterobifunctional reagent, 1-N-(maleimidohexanoyl)-6-N-(anthraquinon-2-oyl)

hexanediamine (MHAHD), possessing an electrophilic maleimide group, has also been shown to

efficiently immobilize thiolated oligonucleotides on a modified glass surface.178

14.6.3.2  Protein/Peptide Microarrays

Protein microarrays, which evolved after DNA microarrays, are particularly powerful for analyzing gene function, regulation, and a variety of other applications including medical diagnostics.179,180 In protein microarrays, it is important that the proteins are retained on the solid substrate

in an active state at high densities. Materials such as polystyrene, poly(vinylidene fluoride), nitrocellulose, glass microscope slides, and other matrices, to which proteins can be attached in high

densities, have been used.181 A variety of biochips has been designed, including 3D surface structures, nanowell, and plain glass biochips.182 Since polyacrylamide gel packets and agarose thin

films form highly porous and hydrophilic matrices, probe molecules can diffuse into the porous

structure and can be immobilized by chemical cross-linking to the reactive group on the matrices. These 3D matrices can be formed on glass surfaces to increase the binding capacity of the

planar surface.25,182,183

As in DNA microarrays, the challenge of constructing a protein/peptide biochip is the immobilization of proteins or peptides onto solid support surfaces. Proteins can be noncovalently adsorbed onto

solid matrices via intermolecular forces, mainly ionic bonds, hydrophobic and polar interactions.183 An

example is the use of thin poly-l-lysine films on glass slides. Another example involves coordination

chemistry where histidine residues of proteins physical bind to nickel or cobalt ions contained in a

self-assembled monolayer at the surface. Chang and Chang184 fabricated a nickel–cobalt alloy layer by

electrodeposition as a protein biochip surface. His6-tagged urate oxidase protein and penta-His biotin

were successfully immobilized on the biochip. In general, proteins/peptides are covalently linked to



432



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



solid matrices to achieve more specific binding and in such a way that the binding sites are exposed to

the sample solution. Reactive functional groups are introduced to activate the solid biochip surfaces

as discussed earlier in this chapter. Since most of the protein immobilization methodologies are presented in earlier sections, the following paragraphs will focus on demonstrating their specific use in

fabricating protein/peptide microarray biochips. MacBeath and Schreiber185 used glass slides containing aldehyde groups to print protein arrays. The aldehyde groups reacted with primary amines on the

proteins to form Schiff base linkages, which were stabilized on reduction creating stable secondary

amine linkages. To print peptides and very small proteins, the authors first attached a molecular layer

of BSA to the surface of glass slides and then activated the BSA with N,N′-disuccinimidyl carbonate.

The NHS-activated lysine, aspartate, and glutamate residues on the BSA reacted readily with surface

amines on the printed proteins to form covalent urea or amide linkages. Because typical proteins display many lysines, with ɛ-amine moieties, on their surfaces as well as the α-amine at the NH2-termini,

they can attach to the slide in a variety of orientations, permitting different sides of the protein to

interact with other proteins or small molecules in solution. In a different application, Funeriu et al.186

used hydrogel-aldehyde functionalized glass slides to prepare enzyme microarray using a commercial

microarrayer. The authors used the microarrayed enzymes to study low-molecular-weight fluorescentaffinity labels as activity probes.

The NHS chemistry, as mentioned above, is commonly used because the NHS ester can readily

react with amine or hydrazide functionalities in substances of interest. There are many methods by

which NHS-activated surfaces can be prepared. Recently, Park et al.17 developed an efficient, singlestep method for the derivatization of glass surfaces with NHS ester groups. The procedure involved

reaction of NHS-ester functionalized dimethallylsilanes with glass surface silanols in the presence

of acid catalyst trifluoromethanesulfonic acid. The NHS-activated glass surface was used to prepare

microarrays containing various substances, such as proteins, peptides, sugars, and small molecules.

Bifunctional silane cross-linkers, which have one functional group that reacts with the hydroxyl

group on glass surface and another to react with primary amine groups of proteins, can form a

SAM. For example, (3-glycidyloxypropyl)trimethoxysilane (GOPS) activates the glass surface

with an epoxide group, which reacts with primary amino groups of proteins forming a covalent

bond. Fall et al.187 immobilized allergen solutions on the GOPS-activated glass slides with a

piezoelectric arrayer for screening of allergen-specific IgE in human serum. The researchers

demonstrated that it was possible to distinguish between patients with and without elevated levels

of allergen-specific IgE.

Proteins and peptide-containing free thiol groups are specifically able to be immobilized onto

solid matrices that are activated by various thiol-directed cross-linkers as discussed in early sections

of this chapter. Both homobifunctional and heterobifunctional reagents containing N-maleimido

groups have been used. Disulfide-activated matrices, such as pyridyl disulfides, can react with proteins containing free thiols through disulfide exchange reactions leading to the formation of a new

mixed disulfide. Vinyl sulfone–modified solid matrices provide another means to immobilize proteins with free thiols groups by a conjugate addition reaction. In addition, gold-coated glass surfaces

can capture thiol-proteins through an Au-SH bond formation. Ressine et al.29 used three-dimensional gold-coated silicon chips to immobilize laccase onto an aminothiophenol SAM. Proteins can

be immobilized by Staudinger ligation between azides and appropriately substituted phosphanes.188

In this approach, alkyl azides are introduced into small molecules, peptides, and proteins, which

react with phosphane to form a chemically stable amide bond as shown in Figure 14.25. Köhn188

used this method to prepare peptide and protein microarrays where azide-functionalized N-Ras

proteins were immobilized onto phosphane-modified glass surfaces.

Photochemical reactions have also been employed to create protein/peptide, glycopeptides, and

oligonucleotide microarrys.189 Weinrich et al.190 utilized the thiolene reaction, which is a photoinduced addition of thiols to terminal alkenes yielding a thioether linkage to couple various biomolecules to silicon wafers as shown in Figure 14.26. Olefin- or thiol-functionalized biomolecules were

immobilized onto thiol-PAMAM- or olefin-PAMAM-functionalized glass slides, respectively, after



433



Application of Chemical Conjugation to Solid-State Chemistry



O



O



Biomolecule



N3



O



Biomolecule



N

H



Ph P

Ph



FIGURE 14.25  Immobilization of azide functionalized biomolecules by Staudinger ligation onto

­phosphane-modified glass surfaces.

(A)

Biomolecule



SH



h_ 365 nm



(B)

HS



Biomolecule



Biomolecule

S



S



Biomolecule



h_ 365 nm



FIGURE 14.26  Thiol-ene photoimmobilization of olefin- or thiol-functionalized biomolecules onto (A)

thiol-PAMAM- or (B) olefin-PAMAM-functionalized glass slides, respectively.



exposure to UV light at 365 nm. For example, the allyl amides of streptavidin and biotin were synthesized and immobilized on thiol-modified solid surfaces.190 Other photoreactive functional groups

have been used to immobilize proteins onto solid matrices. These include diazirine, azidophenyl,

benzophenone, dithiocarbamate, and camphorquinone as UV-reactive groups, and fluorescein,

eosin, and Rose Bengal as visible light-sensitive groups. Miller et al.191 used azidophenyl groups

for the preparation of an antibody microarray. In this process, microscope slides were coated with

poly-l-lysine, which was reacted with a second layer of N-hydroxysuccinimide-4-azidobenzoate

(HSAB). Antibodies were deposited and cross-linked to the HSAB upon UV irradiation. Kanoh

et al.192 prepared small-molecule arrays on gold using surface-bound aryl diazirine. SAMs of alkanethiolates terminated with a phenyl diazirine group are formed on gold substrates. Small organic

molecules are immobilized onto SAM via highly reactive carbene species generated from surfacebound aryl diazirine upon UV irradiation at 365 nm. Ito et al.193 prepared photoreactive watersoluble polymers containing azidoaniline or azidobenzoic acid to prepare protein microarrays.

These polymers include poly(acrylic acid), poly(vinyl alcohol), poly(phosphatidylcholine methacrylate), and PEG. To synthesize a protein microarray chip, the photoreactive polymer was mixed

with proteins and spotted on a solid surface. The solution was dried and UV irradiated to immobilize the proteins. Moschallski et al.25 also used photosensitive polymers to immobilize proteins

onto unmodified plastic surface. A terpolymer based on water-soluble dimethylacrylamide with the

photo-crosslinker methacryloyloxybenzophenone and methacrylic acid glycidylester (MAGE) was

synthesized. MAGE introduced epoxide side groups to the polymer that could bind proteins through

chemical reactions. The terpolymers were printed together with the protein onto the surface of a

plastic substrate. On irradiation with UV-light at a wavelength of 254 nm, the polymer was crosslinked and immobilized the protein to the plastic surface.

14.6.3.3  Antibody Microarrays

An antibody microarray is a special version of a protein microarray. It is an analytical array in which

antibodies or antibody mimic reagents that bind specific antigens are arrayed on solid supports.194

In a way, it is essentially an immunoassay, viz a microarray immunoassay. Like protein microarrays, in general, solid-surface chemistry is an important aspect of a robust microarray platform,



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6 Examples of Applications of Solid-Phase Immobilization Chemistry

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