Hermanson G. Bioconjugate Techniques, Second Edition

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560 12. Iodination Reagents

8. Remove the organic solvent by evaporation using a steady, but gentle, stream of

nitrogen.

9. Dissolve the equivalent of 250 ng of a protein to be labeled in 2–2.5 l of ice-cold

50 mM sodium borate, pH 8.5.

10. Add the protein solution to the dried, iodinated Bolton–Hunter reagent.

11. React for 2 hours on ice. An overnight reaction may be done at 4°C.

12. Remove excess labeled Bolton–Hunter reagent by gel ﬁ ltration or dialysis.

The Bolton–Hunter reagent also may be used to modify a molecule prior to the iodination

reaction. In this case, an amine-containing protein or other molecule is coupled via the NHS

ester end of the reagent to form an amide bond derivative. This derivative is then iodinated

using any of the iodination reagents discussed in this section. This approach can be useful

in preparing stable Bolton–Hunter derivatives that can be stored for extended periods until

requiring iodination, eliminating the relatively short half-life of

125

I-labeled probes.

5.2. Iodinatable Bifunctional Crosslinking Agents

Bifunctional crosslinking agents containing an activated aromatic ring system may be radioio-

dinated using similar procedures as that described previously for the Bolton–Hunter reagent.

Certain conjugation compounds have been designed with the potential for radiolabeling in

mind. For instance, there are a number of photoreactive phenyl azide crosslinkers that possess

an activating hydroxyl group on their phenyl rings. The phenolic group provides sites of facile

iodination ortho and para to the hydroxyl.

The heterobifunctional crosslinker ASBA (4-( p-azidosalicylamido)butylamine) (Chapter 5,

Section 6.1) is an example of this type of iodinatable photoreactive reagent. The phenyl azide

group may be radiolabeled using any standard iodination process (described previously) before

coupling of its primary amine end to a carbonyl group on a macromolecule. After allowing the

modiﬁ ed molecule to bind to a target, the complex may be photolyzed and the covalent conju-

gate detected by its radioactivity.

The homobifunctional photoreactive BASED (Chapter 4, Section 5.1) has two photoreactive

phenyl azide groups, each of which contains an activating hydroxyl. Radioiodination of this

crosslinker can yield one or two iodine atoms on each ring, creating an intensely radioactive

compound. Crosslinks formed between two interacting molecules are reversible by disulﬁ de

reduction, thus allowing traceability of both components of the conjugate.

An extremely versatile iodinatable heterobifunctional is APDP ( N -[4-( p -Azidosalicylamido)

butyl]-3 -(2 -pyridyldithio)propionamide) (Chapter 5, Section 4.2). One end of the crosslinker

can couple with sulfhydryl-containing molecules, while the other end is a nonselective photo-

reactive phenyl azide. Again, the phenyl ring contains an activating hydroxyl group, providing

radioiodination capability. Modiﬁ cation of a sulfhydryl-containing molecule may be done after

iodination of the reagent. After the labeled molecule is allowed to interact with a target mol-

ecule, the photoreactive process can be initiated to form a covalent conjugate. Subsequently,

the crosslinks may be cleaved using a disulﬁ de reducing agent, thus transferring the radiolabel

to the second molecule.

SASD (sulfosuccinimidyl-2-( p-azidosalicylamido)ethyl-1,3’-dithiopropionate) (Chapter 5,

Section 3.2) behaves in a similar manner, except it contains an amine-reactive end that can be

coupled to proteins and other molecules. Its photoreactive end can be iodinated using any of

the radioiodination reagents discussed previously. Just as in the case of APDP, SASD crosslinks

can be cleaved by a disulﬁ de reductant to transfer the radioactive component to a second

molecule.

Finally, the small amine-reactive and photoreactive crosslinker, NHS–ASA (Chapter 5,

Section 3.1), can be iodinated to provide a non-cleavable radioactive conjugate.

Figure 12.8 shows the iodination products resulting from labeling these reagents with

125

Any of the iodination reagents described previously can be used to radiolabel these compounds

prior to their incorporation into target molecules. However, the insoluble iodination reagents

are probably the best choice, since the separation of radiolabeled compound from excess oxi-

dant simply involves removing the solution.

There are many other compounds that have been investigated for their use in indirect radio-

labeling of proteins. For an excellent overview of these chemical reactions (see Wilbur, 1992).

Figure 12.8 Some common crosslinking agents that are capable of being radioiodinated. The sites of iodination

are shown in bold.

5. Iodinatable Modiﬁ cation and Crosslinking Agents 561

562

A silane compound is a monomeric silicon-based molecule containing four constituents. Since

silicon is in the same family as carbon on the periodic table, it too is able to form covalent bonds

with four other atoms. However, it is less electronegative than carbon and undergoes reac-

tions that are unique compared to typical organic compounds. Silanes that contain at least one

bonded carbon atom are called organosilanes. Organosilanes also can have hydrogen, oxygen,

or halogen atoms directly attached to the silicon atom core. Some of these derivatives are highly

reactive and can be used to form covalent linkages with other molecules or surfaces. The more

useful organosilanes are those that contain a functional organic component in addition to one

or more silane reactive groups. The organic portion also may contain a reactive group, which

allows conjugation of the organosilane molecule to other organic compounds. Conversely, the

silane reactive groups typically are unreactive toward organic molecules, but can covalently cou-

ple to certain inorganic substrates. The advantage of this type of functional silane derivative is to

promote the bonding of an organic molecule to an inorganic particle, surface, or substrate.

The general structure of a functional silane coupling agent is shown in Figure 13.1 . The

organic arm typically has a structure that terminates in a functional group or reactive com-

ponent, which facilitates the covalent linkage to another organic molecule. The other part

Silane Coupling Agents

Figure 13.1 The general structure of a silane coupling agent includes a functional group or reactive group at

the end of an organic spacer. This alkyl chain is attached to the central silicon atom, which also has up to three

hydrolysable groups attached to it.

consists of the silane reactive groups attached directly to the silicon atom and can be of several

types. They may comprise simply a hydrogen atom (called a silicon hydride), a halogen-silicon

derivative, such as a chlorine atom (called a chlorosilane), an  OH group (called a silanol), or

groups containing methyl ether or ethyl ether organic constituents (called a methoxy- or ethox-

ysilane, respectively).

The general reactions of silane coupling agents toward inorganic substrates are illustrated

in Figure 13.2 . The reaction mechanism and formation of a reactive intermediate can be dif-

ferent from that usually encountered with organic reactive groups. Unlike most other reactive

groups discussed in this book, the most common silane coupling groups (alkoxy) often must

ﬁ rst undergo hydrolysis to form a reactive intermediate, which then couples to the substrate.

The hydrolysis of an alkoxysilane produces a silanol, which actually is the highly reactive form

necessary for coupling to inorganic surface hydroxyls.

The initial reaction that occurs in solution or near an inorganic substrate is condensation of

the silane coupling agents together to form a polymer matrix linked together by  Si O Si

bonds. Concurrently, the growing silane network interacts with the inorganic substrate through

the formation of a hydrogen bonding network with the  OH groups on its surface. Another

condensation reaction then occurs, usually requiring heat or vacuum to remove water, which

results in the formation of covalently linked organosilane polymer to the surface, forming sta-

ble siloxane linkages (or oxane bonds). The organosilane coating applied in this manner does

not result in a monolayer, but a thicker polymer layer with the reactive organic components

extending out from the surface. The thickness of the organo-siloxane layer is dependent on the

concentration of silane coupling agent used in the reaction and the amount of water present

in the solution. Gelest reports that deposition of a silane coupling agent using a 0.25 percent

aqueous solution will result in a layer appr oximately 3–5 silanes thick.

Alkoxysilanes are used for modiﬁ cation of surfaces due to the instability of silanol deriva-

tives, which will spontaneously hydrogen bond and conjugate together in solution. Forming the

alkoxy derivative acts as a stabilizer to prevent silanol polymerization. However, alkoxysilanes

typically are unreactive with substrate hydroxyl groups under ambient temperature conditions.

Ethoxysilanes are virtually unreactive to substrate  OH groups without prior hydrolysis.

Methoxysilanes are the most reactive, but only react very slowly at room temperature. However,

under the right conditions, both methoxy- and chlorosilane groups are sufﬁ ciently reac-

tive to couple directly with inorganic substrate functionalities without prior hydrolysis. For

instance, the addition of a catalyst to the reaction to increase the hydrogen bonding capability

of substrate hydroxyls has been done to increase the reaction rate for methoxysilanes (Kanan

et al., 2002). Also, chlorosilanes and methoxysilanes can be reacted in an organic solvent with-

out the presence of water (i.e., in THF, toluene, or hydrocarbon solvents), if done under reﬂ ux-

ing conditions to drive the reaction to completion. In this case, a siloxane polymer network

doesn’t form in solution, because no hydrolysis occurs to form silanols on the silane coupling

agents. Therefore, instead of a thick polymer layer forming on the substrate as in aqueous reac-

tions, a monolayer results where each organosilane is coupled directly to the substrate via a

siloxane bond. The reactive organic components stick out from this monolayer for eventual

conjugation with biomolecules or other molecules.

Therefore, functional silanes in a sense are bifunctional compounds that can be used to

attach one substance to another. They have been used for many years as adhesive agents to

promote the bonding of an organic layer to an inorganic layer. Some examples are in the cou-

pling of inorganic metals, particles, ﬁ berglass, or ﬁ llers to organic polymers, plastics, rubber,

13. Silane Coupling Agents 563

Figure 13.2 The reactions involved with the coupling of an organosilane compound to an inorganic surface

containing available  OH groups. The ﬁ rst step in the reaction involves the hydrolysis of the alkoxysilane

groups to form highly reactive silanols. The silanols undergo hydrogen bonding with other silanols in solution

and on the substrate, resulting in a network of associated organosilane derivatives. A condensation reaction

then takes place to form a polymerized coating of the organosilane on the surface of the substrate.

564 13. Silane Coupling Agents

or resins. The uses for functional silanes thus are extensive and span many industries, such as

automotive, building materials, coatings, and electronics, only to name a few. For reviews of

silane coupling agents and their uses, see Plueddemann, 1991, and VanDerVoort et al., 1996.

Some common inorganic substrates for use with silane coupling agents in approximate order

of efﬁ ciency and stability for modiﬁ cation include silica, quartz, glass, and the oxides of alumi-

num, copper, tin, titanium, iron, chromium, zirconium, nickel, and zinc. All of these substrates

can have functional inorganic  OH groups on their surface that react with the silanols on the

silane coupling agents to form siloxane bonds. Sometimes the surfaces require prior treatment

to form the  OH groups and remove contaminants, which will interfere with the silanation

process. Glass, for instance, often needs to be treated with acid (5 percent HCl) for several

hours to remove non-binding metal ions, especially sodium, potassium, and calcium, which

are ubiquitous in the environment. In addition, treatment with a mixture of 25 percent sulfuric

acid and 15 percent hydrogen peroxide (piranha solution) for about 30 minutes is done to cre-

ate a high density of hydroxyl functionalities suitable for silane modiﬁ cation. Glass slides also

can be cleaned and washed prior to modiﬁ cation with a silane with DMSO, ethanol, and water,

and then etched using 10 percent NaOH (w/w) in water for 1 hour.

The distinctive bifunctional nature of silane coupling agents has led to their application in

bioconjugate chemistry. There are many silane coupling agents commercially available that

contain functional groups or reactive groups that can be used to covalently link biomolecules

to inorganic substrates (Dow Corning, Gelest). Inorganic substrates treated with a suitable

silane coupling agent subsequently can be used to couple antibodies, proteins, oligonucleotides,

or other biomolecules containing the appropriate chemical groups for reaction. By judicious

choice of the proper organo-functional component, a silane coupling agent can be used to

design virtually any bioconjugation complex.

1. Silane Reaction Strategies

The reaction techniques that can be used with functional silane coupling agents are varied.

Reactions can be done in aqueous solution, entirely in organic solvent, organic solutions con-

taining a small amount of water, and even in the vapor phase. They also can be done at room

temperature or under elevated temperature conditions. The choice of reaction strategy often is

governed by the type of substrate initially being modiﬁ ed by the silane compound and the inor-

ganic reactive groups on the silane. For instance, if the inorganic substrate can be treated by

mixing or suspending it in a solution of functional silane, such as with particle coatings, then

this may be simplest option for silanization. Another choice may be to dip the substrate into

the silane solution, as is sometimes done with glass slides and other small surfaces that can be

handled in trays or baskets. Alternatively, complex surfaces, such as those often encountered

with devices, may be treated in a cha mber wherein the silane compound is volatilized by heat-

ing or under vacuum. This option usually results in the uniform modiﬁ cation of all surfaces

with a thin layer of functionalized silane.

The following sections describe the major organosilane reaction strategies used to modify

surfaces and provide suggested protocols, which may be used with the functional silane com-

pounds described later in this chapter. Many organosilane compounds are initially only spar-

ingly soluble in aqueous solution. Therefore, solutions containing silanes in either 100 percent

aqueous or in a water/organic mixture containing a large amount of the aqueous component

1. Silane Reaction Strategies 565

566 13. Silane Coupling Agents

often will appear as insoluble two-phase systems. As the alkoxy groups hydrolyze, the silanols

will dissolve into the aqueous solution and the solution will become clear. However, if the solu-

tion is allowed to sit long enough, the silanols will react, form siloxane linkages, and polymer-

ize in solution. At this point, the solution again will become cloudy due to these large polymers.

1.1. Aqueous/Organic Solvent Deposition

This method involves the deposition of functional silane onto an inorganic substrate using a

dilute solution of water in organic solvent. The small amount of water at acid pH facilitates

the hydrolysis of the alkoxy groups on the silane to form the reactive silanols necessary for

coupling. The method of silane solution contact with the substrate may be accomplished by

suspension stirring, dipping, spinning, or spraying the silane solution.

Protocol

All operations should be done in a well-ventilated fume hood. Use care not to inhale vapors or

get reactive silanes on your skin or in your eyes.

1. Prepare a solution containing 3–5 percent water in ethanol (v/v) and adjust the pH to

4.5–5.5 with acetic acid.

2. Dissolve a silane coupling agent in the acidic water/ethanol solution with stirring to a

ﬁ nal concentration of 2–5 percent (v/v). Allow hydrolysis to occur for 5 minutes at room

temperature to form reactive silanols.

3. Contact the inorganic substrate with the silane solution for as brief as 2 minutes to

as long as several hours, depending on the degree of organosilane polymer deposition

desired on the surface. Benters et al., (2002) performed the reaction on glass slides in a

stirring bath for 2 hours. Optimization of the reaction time should be done to determine

the best performance of the modiﬁ ed substrate in its intended application.

4. Wash the substrate several times with ethanol or the water/ethanol mixture to remove

excess silane compound.

5. Cure the organosilane-modiﬁ ed substrate by incubation at 110°C for 30 minutes or at

room temperature in a low humidity environment. Curing under vacuum also will aid in

the removal of water and the formation of siloxane bonds.

1.2. Aqueous Deposition

Functional organosilanes can be applied to substrates directly from aqueous solutions, provided

the silane compound is soluble is water.

Protocol

1. Dissolve an alkoxysilane in water at a concentration of 0.5–2.0 percent (v/v). If the com-

pound is not very soluble in water, a nonionic detergent can be added to the solution at

0.1 percent to promote solubility.

2. Adjust the solution with acetic acid to pH 5.5.

3. Contact the substrate with the silane solution from 2 minutes to as long as 1 hour,

depending on the degree of organosilane polymer deposition desired on the surface.

Optimization of the reaction time should be done to determine the best performance of

the modiﬁ ed substrate in its intended application.

4. Remove excess solution and cure at 110–120°C for 30 minutes to remove traces of water

and form the siloxane bonds.

5. Wash the substrate with water or buffer.

1.3. Organic Solvent Deposition

This method of silanation, which uses organic solvent without the addition of water, is suitable

for highly reactive silane derivatives, such as chlorosilanes, aminosilanes, and methoxysilanes.

This procedure will not work for ethoxysilanes, as these compounds are not reactive enough

without prior hydrolysis to create the silanol. This method is convenient to use for silica

particle modiﬁ cation and for the functionalization of metallic nanoparticles having the requi-

site ⎯OH groups present (see Chapter 14, Section 5).

Protocol

1. In a fume hood, dissolve the organosilane coupling agent containing chloro-, amino-, or

methoxysilane reactive groups in toluene, THF, or a hydrocarbon solvent at a concentra-

tion of 5 percent silane.

2. To assure a thin monolayer of silane modiﬁ cation on the substrate, ﬁ rst remove all traces

of water by drying it at 150°C for 4 hours. This step is especially important for nano-

particle modiﬁ cation, as the high surface area of the particle population can hold a lot of

water by hydrogen bonding. Note that if the substrate contains water, a thicker polymer

layer of silane will build up on it due to hydrolysis of the reactive groups to silanols close

to the surface of the substrate. After drying, wash the particles with solvent several times

by centrifugation before resuspending them in the silane solution. For other substrates,

submerge them in the silane solution with mixing.

3. React with gentle stirring for 12–24 hours by heating to reﬂ ux in a fume hood.

4. Cool the reaction and wash the substrate with solvent to remove excess silane reagent

and reaction by-products. The modiﬁ ed substrate may be dried or washed into aqueous

buffer for further conjugation with biomolecules.

1.4. Vapor Phase Deposition

Many silane coupling agents can be applied to substrates by volatilization in an enclosed cham-

ber under heat or vacuum. In this approach, the substrate is placed within the chamber in a

fashion to allow for vapor phase molecules to access all areas that are to be derivatized. This

method is commonly used for silanizing glass slides or substrates that are difﬁ cult to suspend in a

silane solution. Slides are often placed in racks within the chamber and all surfaces get modiﬁ ed

1. Silane Reaction Strategies 567

568 13. Silane Coupling Agents

with silane in a uniform manner. Vapor deposition also uses a very small amount of organosilane

compound compared to what may be necessary to immerse fully a substrate device in solution.

Protocol

1. In an enclosed chamber made of glass or acrylic, such as a vacuum chamber, place the

substrate to be modiﬁ ed in such a manner as to expose the surfaces to the vapor phase.

2. Place the organosilane coupling agent in a small reservoir under or next to the substrate in

the chamber. If volatilization by heating is to be done, an explosion-proof heating device

should be used to maintain the silane solution temperature at 50°C or above. Alternatively,

apply a vacuum to the chamber until the silane compound begins to volatilize. The

vacuum tubing may be clamped off to maintain vacuum within the chamber once a suf-

ﬁ cient level of evacuation has been reached. Vapor phase deposition usually requires that

the silane have at least a 5 mm vapor pressure to achieve adequate concentration in the

atmosphere within the chamber.

3. React for 4–24 hours within the chamber to result in a uniform coating of the substrate

surface with organosilane. Often surfaces are coated overnight to complete the reaction.

2. Functional Silane Compounds

Functional silane compounds containing an organo-functional or organo-reactive arm can be

used to conjugate biomolecules to inorganic substrates. The appropriate selection of the func-

tional or reactive group for a particular application can allow the attachment of proteins, oli-

gonucleotides, whole cells, organelles, or even tissue sections to substrates. The organosilanes

used for these applications include functional or reactive groups such as hydroxyl, amino, alde-

hyde, epoxy, carboxylate, thiol, and even alkyl groups to bind molecules through hydrophobic

interactions.

The following sections discuss the organosilane compounds commonly used for conjugation

to inorganic surfaces. These reagents and many other silane derivatives are available from a

number of commercial sources, which include Dow Corning, Gelest, Aldrich, and others.

2.1. 3-Aminopropyltriethoxysilane and 3-Aminopropyltrimethoxysilane

These two silane coupling agents are among the most popular choices for creating a func-

tional group on an inorganic surface or particle. Both reagents contain a short organic 3-amino

propyl group, which terminates in a primary amine. The only difference in these compounds is

the silane reactive portion that contains either a triethoxy group or a trimethoxy group. Thus,

the reagents display differences in reactivity toward substrate  OH groups related to the rela-

tive reactivity of the alkoxysilane functionalities. The trimethoxy compound is more reactive

and can be deposited on a substrate using 100 percent organic solvent without the presence of

water to promote hydrolysis of the alkoxy groups prior to coupling. In this case, the organic

solvent deposition protocol described in the previous section can be used to modify covalently

substrates with a layer of aminosilane. The advantage of this process is that a thinner, more

controlled deposition of the silane can be made to create a monolayer of aminopropyl groups

on the surface.

When using 3-Aminopropyltriethoxysilane (APTS) (the triethoxy version), the reaction

must occur in at least a partially aqueous environment. The ethoxy groups are not reactive

enough to couple spontaneously with the  OH groups on an inorganic surface without

prior hydrolysis to form silanols. This is typically done in 5 percent water in ethanol that has

been acidiﬁ ed with acetic acid to pH 4.5–5.5. The protocol described in the previous section

for aqueous solvent deposition can be used with success to modify surfaces or particles with

this reagent ( Figure 13.3 ). This process results in a layer containing about 3–8 organosilanes

in thickness, which effectively masks the underlying inorganic substrate with aminopropyl

functionalities.

Once deposited on a substrate, the alkoxy groups form a covalent polymer coating with

the primary amine groups sticking off the surface and available for subsequent conjugation.

Carboxyl- or aldehyde-containing ligands may be directly coupled to the aminopropyl groups

using a carbodiimide reaction (Chapter 3, Section 1) or reductive amination, respectively

(Chapter 3, Section 4). Alternatively, surfaces initially derivatized with an aminopropylsilane

compound can be modiﬁ ed further with spacer arms or crosslinkers to create reactive groups

for coupling afﬁ nity ligands or biomolecules. For instance, the amine groups may be deriva-

tized with an NHS–PEG

-azide compound for use in click chemistry or Staudinger ligation

reactions for linking proteins or other biomolecules (Chapter 17, Section 4 and 5). This modi-

ﬁ cation forms extremely hydrophilic PEG spacers on the surface that terminate in alkyl azide

groups for conjugation with alkyne or phosphine derivatives ( Figure 13.4 ).

Other crosslinking agents that contain an amine-reactive group on one end also may be used

to modify and activate the APTS-modiﬁ ed substrate. Surfaces may be designed to contain, for

instance, reactive hydrazine groups for conjugation with carbonyl-containing molecules, such

as aldehydes formed through periodate oxidation of carbohydrates or natively present at the

reducing end of sugars and glycans (Chapter 17, Section 2; Chapter 1, Section 4.6).

Benters et al. (2002) used two approaches to modify APTS surfaces. In one instance, the

amine groups were acylated using glutaric anhydride to create carboxylate functionalities,

which were then activated with NHS/DCC to form the NHS ester. This derivative could be

2. Functional Silane Compounds 569