Surface-immobilized Enzymes Research Articles

Investigation of heterogeneous electrochemical processes using multi-stream laminar flow in a microchannel

A multi-component microfluidic electrochemical cell is shown to be a useful analytical tool for probing complex coupled processes in electrolytic systems. We recently reported an enzymatic signal amplification phenomenon that may provide increased sensitivity when detecting bio-analytes (M. S. Hasenbank, E. Fu and P. Yager, Langmuir, 2006, 22, 7451-7453), but to fully harness this method requires an improved understanding of the underlying electrochemical and chemical processes. We use spatial control of electrolyte streams on patterned conductive substrates in a microfluidic platform to elucidate the coupling of homogeneous chemical steps to heterogeneous electrochemical charge transfer processes. Because the gold surface was observable using SPR imaging, electrochemical phenomena could be monitored optically in real time. Based on these and additional results, we propose a mechanism for the novel amplification phenomenon that involves direct electron transfer between surface-immobilized enzyme molecules and the gold surface. This improved understanding of the underlying mechanism should enable the future implementation of this phenomenon in signal amplification schemes for highly sensitive lab-on-a-chip biosensors.

Surface plasmon optical detection of β-lactamase binding to different interfacial matrices combined with fiber optic absorbance spectroscopy for enzymatic activity assays

In this study, we describe the attachment of biotin-functionalized beta-lactamase to different types of interfacial architectures. Generic biotin-NeutrAvidin binding matrices were assembled using biotin-terminated alkanethiol and poly (L-lysine)-g-poly (ethylene glycol) polymer. Quantitative comparisons were made between different matrices and binding strategies. In addition, the feasibility of regeneration was tested. Our results show that in general all matrices were well suited for the binding of the protein, although quantitative differences were observed and will be discussed. Furthermore, the results obtained by surface plasmon resonance spectrometer and optical waveguide measurements show excellent correlation. For all five matrices investigated, real time enzymatic activity assays of beta-lactamase were performed by a detection scheme that combines an affinity and a catalytic sensor. The results show that the surface-immobilized enzymes are stable and sufficiently active for highly sensitive catalytic activity measurements. The effect of surface immobilization on the catalytic activity of the enzyme is discussed.

Mean-Field Model of Immobilized Enzymes Embedded in a Grafted Polymer Layer

Two-dimensional mean-field lattice theory is used to model immobilization and stabilization of an enzyme on a hydrophobic surface using grafted polymers. Although the enzyme affords biofunctionality, the grafted polymers stabilize the enzyme and impart biocompatibility. The protein is modeled as a compact hydrophobic-polar polymer, designed to have a specific bulk conformation reproducing the catalytic cleft of natural enzymes. Three scenarios are modeled that have medical or industrial importance: 1), It is shown that short hydrophilic grafted polymers, such as polyethylene glycol, which are often used to provide biocompatibility, can also serve to protect a surface-immobilized enzyme from adsorption and denaturation on a hydrophobic surface. 2), Screening of the enzyme from the surface and nonspecific interactions with biomaterial in bulk solution requires a grafted layer composed of short hydrophilic polymers and long triblock copolymers. 3), Hydrophilic polymers grafted on a hydrophobic surface in contact with an organic solvent form a dense hydrophilic nanoenvironment near the surface that effectively shields and stabilizes the enzyme against both surface and solvent.

Stabilization of surface-immobilized enzymes using grafted polymers

We introduce a two-dimensional lattice model of immobilization and stabilization of proteinlike polymers using grafted polymers. The protein is designed to have a specific bulk conformation reproducing a catalytic cleft of natural enzymes. Our model predicts a first order denaturing adsorption transition of free proteins. On the other hand, for an immobilized protein we observe a more gradual disappearance of the hydrophobic centers accompanied by adsorption. We show that, using hydrophilic grafted polymers of proper length and grafting density, the conformation as well as the hydrophobic centers of the protein can be restored.

Effects of fabrication parameters on the enzyme loading and sensor response of enzyme-carrying conductive polymer electrodes.

Hydrogen peroxide sensors where horseradish peroxidase (HRP) is incorporated into pyrrole/3-alkylsulfonate pyrrole copolymer films deposited on an SnO2 electrode (HRP/Py-PS electrode) were investigated with regard to the effects of the fabrication parameters (electropolymerization charge, deposition current density, and electrodeposition solution pH) on the amount of surface-immobilized enzyme and the sensor response. The amount of incorporated enzyme was determined with a method recently developed by ourselves. The results suggest that the amount of entrapped enzyme increases almost linearly with the total charge passed, and strongly depends on the polymer film growth rate and the electropolymerization pH. These findings open up a way to control the amount of enzyme and the resultant response of the biosensor by modifying the preparation conditions.

Design and Characterization of Immobilized Enzymes in Microfluidic Systems

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.

Amino-silane modified superparamagnetic particles with surface-immobilized enzyme

Microfine magnetic particles of magnetite with a mean diameter of 10–15 nm, which is in the range of superparamagnetism, were prepared by coprecipitation from ferrous and ferric electrolyte solution and applied to the enzyme immobilization support. The surface of the particles was treated by a series of amino-functional silane coupling agents capable of covalently bonding with enzyme. A protease, thermolysin, was immobilized on the particles and transferred to ethyl acetate solution containing N-Cbz- l-aspartic acid and l-phenylalanine methyl ester. The dipeptide, aspartame precursor synthetic reaction was then carried out. Higher enzyme activity was found when the silane coupling agents with long spacer arms between the silane functionality and amino functionality were used such as N-(6-aminohexyl)-3-aminopropyltrimethyoxysilane, compared to conventional 3-aminopropyltrichoxysilane. Magnetic recovery of the immobilized enzyme was also demonstrated upon the application of an external magnetic field.

Total internal reflection fluorescence.

Total internal reflection fluorescence (TIRF) is an optical effect particularly well-suited to the study of molecular and cellular phenomena at liquid/solid interfaces. Such interfaces are central to a wide range of biochemical and biophysical processes: binding to and triggering of ceils by hormones, neurotransmitters, and antigens; blood coagulation at foreign surfaces; electron transport in the mitochondrial membrane; adherence and mo­bility of bacteria, algae, and cultured animal cells to surfaces; and possible enhancement of reaction rates with cell surface receptors upon nonspecific adsorption and surface diffusion of an agonist. Liquid/solid interfaces also have important medical and industrial applications: e.g. detection of serum antibodies by surface immobilized antigens; and the manufacture of biochemical products by surface-immobilized enzymes. Total internal reflection spectroscopy for optical absorption studies (called attenuated total reflection or ATR) was developed somewhat earlier than the fluorescence applications and has been widely used in surface