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Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

Lim CY, Owens NA, Wampler RD, Ying Y, Granger JH, Porter MD, Takahashi M, Shimazu K - Langmuir (2014)

Bottom Line: The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer.This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh.This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemical Engineering, ‡Chemistry, §Bioengineering, and ∥Pathology and the ⊥Nano Institute of Utah, University of Utah , Salt Lake City, Utah 84112, United States.

ABSTRACT
N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

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Related in: MedlinePlus

Kinetic plot for aminolysis of the DSP-derivedadlayer in 500 mMethylamine in 50 mM borate buffer (pH 8.50).
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fig9: Kinetic plot for aminolysis of the DSP-derivedadlayer in 500 mMethylamine in 50 mM borate buffer (pH 8.50).

Mentions: Thekinetic plot for the data in Figure 8 is shownin Figure 9. Due to the weakness of amide bands,this analysis tracked the decrease in the NHS carbonyl mode at 1748cm–1. To obtain the heterogeneous aminolysis reactionrate constant, the combined second-order rate laws for the two reactionsoccurring in parallel can be written as7where ka is thesecond-order heterogeneous rate constant for aminolysis (M–1 s–1) and [NH2] is the deprotonatedamine concentration in bulk solution (M). The corresponding integratedrate law is given by eq 8:8


Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

Lim CY, Owens NA, Wampler RD, Ying Y, Granger JH, Porter MD, Takahashi M, Shimazu K - Langmuir (2014)

Kinetic plot for aminolysis of the DSP-derivedadlayer in 500 mMethylamine in 50 mM borate buffer (pH 8.50).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222659&req=5

fig9: Kinetic plot for aminolysis of the DSP-derivedadlayer in 500 mMethylamine in 50 mM borate buffer (pH 8.50).
Mentions: Thekinetic plot for the data in Figure 8 is shownin Figure 9. Due to the weakness of amide bands,this analysis tracked the decrease in the NHS carbonyl mode at 1748cm–1. To obtain the heterogeneous aminolysis reactionrate constant, the combined second-order rate laws for the two reactionsoccurring in parallel can be written as7where ka is thesecond-order heterogeneous rate constant for aminolysis (M–1 s–1) and [NH2] is the deprotonatedamine concentration in bulk solution (M). The corresponding integratedrate law is given by eq 8:8

Bottom Line: The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer.This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh.This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemical Engineering, ‡Chemistry, §Bioengineering, and ∥Pathology and the ⊥Nano Institute of Utah, University of Utah , Salt Lake City, Utah 84112, United States.

ABSTRACT
N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

Show MeSH
Related in: MedlinePlus