Hermanson G. Bioconjugate Techniques, Second Edition
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570 13. Silane Coupling Agents
used to couple amine-containing proteins and other molecules via amide bond formation.
In a second activation strategy, the aminopropyl groups on the surface were activated with
1,4-phenylenediisothiocyanate (PDITC) to create terminal isothiocyanate groups for coupling
amines. Both methods resulted in the successful coupling of amine-dendrimers to silica surfaces
for use in arrays (see Chapter 7, Section 4).
Amine-surfaces prepared using an aminosilane compound can be modifi ed to contain carbox-
ylate groups using the following protocol involving the reaction with anhydride ( Figure 13.5 ).
The carboxylates then can be used to couple amine-containing molecules using a carbodiimide
reaction with EDC plus sulfo-NHS (Chapter 3, Section 1.2).
Protocol
1. Dissolve glutaric anhydride (Chapter 1, Section 4.2) in DMF at near saturation (use a
fume hood). Add triethylamine to a concentration of at least 1 mg/ml to function as a
proton acceptor (base).
Figure 13.3 The deposition of APTS on inorganic substrates results in the formation of a covalent coating con-
taining primary amine groups.
Figure 13.4 APTS-modifi ed surfaces may be further derivatized with amine-reactive crosslinkers to create
additional surface characteristics and reactivity. Modifi cation with NHS–PEG
4
-azide forms a hydrophilic PEG
spacer terminating in an azido group that can be used in a click chemistry or Staudinger ligation reaction to
couple other molecules.
2. Functional Silane Compounds 571
572 13. Silane Coupling Agents
2. Apply the anhydride solution to the aminosilane-modifi ed surface (typically done by
emersion) and mix by stirring.
3. React 2–4 hours at room temperature.
4. Wash the carboxylated surface with DMF and then with water. Dried surfaces are stable
indefi nitely.
The carboxyl groups on the surface may be activated to an NHS ester for coupling with
amine-containing biomolecules according to the following protocol, based on the method of
Benters et al ., 2002.
Protocol
1. In a fume hood, dissolve N-hydroxysuccinimide (NHS) in DMF (highly pure and dried
over molecular sieves) at a concentration of 115 mg/ml (1 M) along with 206 mg/ml
(1 M) N , N 9-dicyclohexylcarbodiimide (DCC; Chapter 3, Section 1.4).
2. Add the NHS/DCC solution to the carboxylated substrate to immerse fully the surface.
Mix by stirring.
3. React for 1–2 hours at room temperature.
4. Wash the surfaces with DMF. The NHS-activated surface may be used to couple amine-
containing molecules in a buffer at physiological pH (7.2–7.4) using 50 mM sodium
phosphate.
Aminosilane surfaces also may be activated by use of a bifunctional crosslinker to contain
reactive groups for subsequent coupling to biomolecules. In one such reaction, N , N-disuc-
cinimidyl carbonate (DSC) was used to react with the amines on a slide surface and create
terminal NHS-carbonate groups, which then could be coupled to amine-containing molecules
(Niemeyer, 2004) ( Figure 13.6 ). The following procedure is based on this method.
Figure 13.5 Modifi cation of an APTS surface with glutaric anhydride creates terminal carboxylates for cou-
pling of amine-containing ligands.
Protocol
1. Dissolve 1.5 m of DSC (Chapter 4, Section 1.7) and 5 ml of diisopropylethylamine
(DIEA) in 145 ml of dry acetone (in a fume hood).
2. Add the DSC solution to the aminosilane-slides (or other aminosilane-modifi ed surface)
to immerse fully the substrate in the solution and mix by stirring.
3. React for 2 hours at room temperature.
4. Wash the modifi ed surfaces with dry acetone to remove excess reactants and reaction
by-products. Dry under nitrogen. The NHS-carbonate groups on the surface are stable to
storage under desiccated conditions (package under nitrogen with a desiccant).
5. Amine-containing molecules, such as proteins or amine-modifi ed oligonucleotides, are
coupled to the NHS-carbonate activated surface by dissolving them in 50 mM sodium
phosphate, pH 7.4, at a concentration of at least 1–10 mg/ml (for proteins). Spotting of bio-
molecules onto the activated surface may be done to create an array. Since this process usu-
ally is done using only microliter quantities or less of solution per spot, the activated surface
should be kept in a humidity chamber to prevent evaporation during the coupling reaction.
6. React for 2–4 hours at room temperature.
7. Wash the slides with coupling buffer to remove excess reactants.
2.2. Carboxyethylsilanetriol
Silane coupling agents containing carboxylate groups may be used to functionalize a sur-
face with carboxylic acids for subsequent conjugation with amine-containing molecules.
Carboxyethylsilanetriol contains an acetate organo group on a silanetriol inorganic reactive end
(Gelest). The silanetriol component is reactive immediately with inorganic OH substrates with-
out prior hydrolysis of alkoxy groups, as in the case with most other silanation reagents ( Figure
13.7 ). This compound is supplied in a 25 percent aqueous solution as the monosodium salt, and
it can be used just by diluting into water or buffer.
Figure 13.6 APTS-modifi ed surfaces can be activated with DSC to form amine-reactive succinimidyl carbonates
for coupling proteins or other amine-containing molecules.
2. Functional Silane Compounds 573
574 13. Silane Coupling Agents
Carboxyethylsilanetriol has been used to add carboxylate groups to fl uorescent silica
nanoparticles to couple antibodies for multiplexed bacteria monitoring (Wang et al., 2007)
(see Chapter 14, Section 5). This reagent can be used in similar fashion to add carboxylate
functionality to many inorganic or metallic nano-materials, which also will create negative
charge repulsion to maintain particle dispersion in aqueous solutions. Covalent coupling to the
carboxylated surface then can be done by activation of the carboxylic acid groups with a car-
bodiimide to facilitate direct reaction with amine-containing molecules or to form intermediate
NHS esters (Chapter 3, Section 1).
The following protocol for using carboxyethylsilanetriol is based on the method of Wang
et al., 2007. Other metallic particles or surfaces may be modifi ed with this silane compound in
like manner.
Protocol
1. Suspend 10 mg of silica particles in 1 ml of 10 mM sodium phosphate, pH 7.4, with
gentle mixing.
2. Add 20 l of the carboxyethylsilanetriol (as the 25 percent aqueous solution) to the
particle suspension with mixing.
Figure 13.7 Carboxylethylsilanetriol can be used to modify an inorganic substrate to containing carboxylate
groups for coupling amine-containing ligands.
3. React at room temperature for 3–4 hours with mixing.
4. Wash the modifi ed particles several times with buffer using centrifugation for separa-
tion. A fi nal wash with 10 mM MES, pH 5.5, is done to prepare the sample for coupling
amine-containing molecules using a carbodiimide reaction. For suggested protocols, see
Chapter 14, Section 4.3, Coupling to Carboxylate Particles.
2.3. N -(Trimethoxysilylpropyl)ethylenediamine triacetic acid
Another useful silanation reagent containing carboxylates actually is an effective chelator of
metal ions. N-(Trimethoxysilylpropyl)ethylenediamine triacetic acid (TMS-EDTA) contains a
trimethoxy group for coupling to OH groups on inorganic substrates and an EDTA group
for coordinating metals (minus one carboxylate, which instead functions as the hydrocarbon
arm attached to the silicon atom). This compound can be used to coat substrates and provide a
metal-chelating functional group for use in affi nity separations.
Immobilized metal affi nity chromatography (IMAC) has been used for decades as an affi nity
method for targeting certain functional groups in proteins or other biomolecules (for reviews see
Porath, 1992; Hermanson et al., 1992; Winzerling et al., 1992; Lopatin and Varlamov, 1995;
Hage, 1999). Various metal ions can be chelated by EDTA affi nity groups to provide directed
targeting of biological groups such as phosphate modifi cations or histidine-rich areas in pro-
teins. Thus, phosphorylation sites in proteins can be bound using a silane-EDTA modifi ed sur-
face containing, for instance, gallium (Posewitz and Tempst, 1999). Alternatively, if the chelating
groups are charged with nickel or cobalt, the binding of His-tagged proteins can be done.
The preparation of particles or surfaces that are able to capture specifi cally a fraction of
he proteome using metal affi nity separations makes possible analysis of distinct protein
2. Functional Silane Compounds 575
576 13. Silane Coupling Agents
Figure 13.8 TMS-EDTA can be used to modify an inorganic substrate to containing EDTA chelating groups for
complexation with metal ions.
populations by mass spec (Zhou et al., 2000). The reactions and use of TMS-EDTA in modi-
fying inorganic surfaces and coordinating metals for affi nity chromatography is shown in
Figure 13.8 .
The coating of surfaces with TMS-EDTA can be done using the general protocol described
previously in this chapter entitled Aqueous/Organic Solvent Deposition. The acidifi ed water/
organic solvent environment hydrolyzes the methoxy groups to silanols, which then polymerize
and covalently link to inorganic surface OH groups. After a surface has been modifi ed with
a chelating ligand, it is washed thoroughly with metal-free water or buffer. Finally, an aqueous
solution of the appropriate metal salt is contacted with the surface and the EDTA groups will
bind the metal for use in affi nity separations.
It should be noted that the chelator group in TMS-EDTA is a pentadentate ligand, which
will coordinate fi ve bonds with an associated metal ion. Common IMAC ligands use tridentate
or tetradentate ligands that have a greater number of available coordination sites for interac-
tion with target molecules. Iminodiacetic acid-based chelators interact with metal ions through
three bonds, typically leaving three coordination sites left for affi nity binding. Nitrilotriacetic
acid-lysine (NTA) chelating groups interact with metal ions through four coordination
bonds, leaving two sites available for affi nity interactions. However, the TMS-EDTA ligand
holds metal ions through fi ve coordination bonds, thus typically having only one remaining
for interacting with other molecules. This property may result in lower affi nities with biologi-
cal molecules and change the capture behavior for specifi c metal interaction sites on proteins.
Careful testing should be done for each application envisioned for this metal-chelating silane
coupling agent.
2.4. 3-Glycidoxypropyltrimethoxysilane and 3-Glycidoxypropyltriethoxysilane
Two very useful silane modifi cation agents are glycidoxy compounds containing reactive epoxy
groups. Surfaces covalently coated with these silane coupling agents can be used to conjugate
thiol-, amine-, or hydroxyl-containing ligands, depending on the pH of the reaction (Chapter 2,
Section 4.1). Thus, 3-glycidoxypropyltrimethoxysilane (GOPTS) or 3-glycidoxypropyltriethox-
ysilane can be used to link inorganic silica or other metallic surfaces containing OH groups
with biological molecules containing any three of these major functional groups. GOPTS has
been used most often in bioconjugation applications, but the triethoxysilane compound also
may be used in similar protocols.( Figure 13.9 )
The reaction of the epoxide with a thiol group yields a thioether linkage, whereas reaction
with a hydroxyl gives an ether and reaction with an amine results in a secondary amine bond.
The relative reactivity of an epoxy group is thiol amine hydroxyl, and this is refl ected by
2. Functional Silane Compounds 577
578 13. Silane Coupling Agents
Figure 13.9 Epoxy-containing silane coupling agents form reactive surfaces that can be used to couple amine-,
thiol-, or hydroxyl-containing ligands.
the optimal pH range for each reaction. In this case, the lower the reactivity of the functional
group the higher the pH required to drive the reaction effi ciently.
GOPTS has been used to create a high-density PEG surface on glass slides for use in arrays
(Piehler et al., 2000). It also has been used in the development of a high-throughput analyzer
using biochip technology on aluminum oxide sheets (FitzGerald et al., 2005) and in the activa-
tion of glass surfaces for detection of antibodies specifi c for hepatitis B and C viruses (Duan
et al ., 2005).
The following protocol is based on these methods. All operations should be done in a fume
hood, including wearing proper protective clothing.
Protocol
1. Prepare glass slides by washing with acid (5 percent HCl) for several hours to remove
non-binding metal ions, especially sodium, potassium, and calcium. Treatment with a
mixture of 25 percent sulfuric acid and 15 percent hydrogen peroxide (piranha solution)
for about 30 minutes is done to create 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.
2. Prepare a GOPTS solution in o-xylene or 95 percent ethanol at a concentration of 2 per-
cent (v/v). If the organic solvent is used, add 2 mg/ml N-ethyldiisopropylamine (DIPEA)
as base.
3. Immerse the glass slides in the GOPTS solution and mix by stirring.
4. React at 37°C (for the ethanol solution) or 55°C (for the o-xylene solution) for at least
5–6 hours with mixing.
5. Wash slides thoroughly with solvent and then dry in an oven at 135°C for 1 hour (explo-
sion-proof oven). The slides are now ready to couple ligands through their epoxy groups.
For protocol suggestions on conjugation to epoxy groups, see Chapter 2, Sections 1.7 and
4.1. Also, see Chapter 14, Section 4.11, Coupling to Epoxy Particles, for a method to attach
affi nity ligands to surfaces that are activated with epoxide groups.
2.5. Isocyanatopropyltriethoxysilane
Isocyanate groups are extremely reactive toward nucleophiles and will hydrolyze rapidly in
aqueous solution (Chapter 2, Sections 1.2 and 4.7). They especially are useful for covalent cou-
pling to hydroxyl groups under non-aqueous conditions, which is appropriate for conjugation
to many carbohydrate ligands. Isocyanatopropyltriethoxysilane (ICPTES) contains an isocyanate
group at the end of a short propyl spacer, which is connected to the triethoxysilane group useful
for attachment to inorganic substrates. Silanation can be accomplished in dry organic solvent to
form reactive surfaces while preserving the activity of the isocyanates.
An isocyanate reacts with amines to form isourea linkages and with hydroxyls to form car-
bamate (urethane) bonds. Both reactions can take place in organic solvent to conjugate mol-
ecules to inorganic substrates ( Figure 13.10 ). The solvent used for this reaction must be of high
purity and should be dried using molecular sieves prior to adding the silane compound.
2. Functional Silane Compounds 579