Optical biosensors for real-time measurement of analytes in blood plasma

Abstract

The preparation of assemblies consisting of multiple molecular layers of bovine serum albumin (BSA), monoclonal antibodies against horseradish peroxidase (anti-HRP), and monoclonal antibodies against methotrexate (anti-MTT), as well as interaction of the assemblies with human blood plasma were observed using a grating coupler and Young interferometer (YI). The assemblies could be arranged according to decreasing amounts of nonspecific deposits bound irreversibly to them from blood plasma as follows-an adsorbed antibody monolayer saturated with adsorbed BSA, antibody multilayers linked with polycations, antibodies covalently immobilized on a BSA layer densely crosslinked with glutaraldehyde (GA), slightly crosslinked BSA double layer, slightly crosslinked antibody double layers. The occurrence of human serum albumin (HSA), human fibrinogen (Fg), IgG, and IgM in the plasma deposits was studied by binding the respective antibodies. IgG, IgM, and Fg were detected in plasma deposits on the immobilized assemblies while the composition of a plasma deposit on the unmodified sensor surface reflected roughly the plasma composition containing mainly adsorbed HSA and Fg. A crosslinked anti-HRP double layer was immobilized on a waveguiding branch of YI and a similar anti-MTT double layer was immobilized on the other branch. The sensor response to blood plasma was fairly decreased owing to a compensation of the respective optical changes in the two branches, in which a similar non-specific adsorption took place. The addition of HRP or MTT to plasma induced specific responses of the corresponding branches.

Introduction

Within the last decade, biosensors based on interaction of evanescent waves at surfaces of various optical transducers with an analyzed medium have been developed (Lukosz, 1992). The affinity binding of molecular analytes from the tested medium to specific bioreceptors immobilized on the transducer surface induces detectable changes in the transducer optics. In contrast to immunoassays currently used in practice, biosensors are capable of direct real-time sensing without additional reagents (Lundström, 1994). Thus, the sensors could be unique when a fast analysis or the permanent in situ monitoring of analyte concentrations are required. On the other hand, unlike the methods based on labeled reagents, the direct sensor detection is not able to differentiate between a sensor response caused by the binding of analytes to specific receptors and a non-specific response caused by binding other compounds from tested media to the sensor surface. The non-specific response is particularly troublesome when measurements are performed in complex biological fluids, such as blood serum, blood plasma or whole blood. Plasma proteins adsorb on solid surfaces immediately after the contact with these body fluids. The adsorption is a complex and dynamic process in which proteins can change their tertiary structure and more strongly adsorbing proteins can subsequently replace proteins adsorbed first (Scott, 1991). In addition to simple physical adsorption at the solid liquid interface, a sequence of specific processes associated with activation of the coagulation and complement systems, such as binding, activation, and release of proteins and other blood components, occurs at any surface other than that of undamaged vascular wall endothelium (Coleman et al., 1994). So far, the non-specific interaction has been a substantial barrier for sensor applications in medical diagnostics.

There have been only few scientific papers describing real-time optical sensing in serum or plasma performed in a non-competitive way, i.e. without addition of reagents to the tested fluid (Anderson et al., 1998, Ravanat et al., 1998, Otamiri and Nilsson, 1999). The detection is possible only if the response to an analyte is high enough in comparison with the non-specific adsorption. The specific response is proportional to a mass added onto the sensor surface due to the analyte binding. Thus, the concentration and molecular weight of the analyte as well as its affinity to the immobilized receptors should be high, which is not common in practice. To prevent protein adsorption on the optical transducer, its surface was coated with hydrogels, such as (carboxymethyl)dextran (Löfäs and Johnsson, 1990) or poly(ethylene glycol) (Mrksich and Whitesides, 1997), so as the access of plasma proteins to the bare surface could be prevented, and bioreceptor molecules were attached covalently to the hydrogel. In a similar way, non-specific adsorption has been decreased several times when, instead of immobilizing a receptor monolayer directly on the transducer surface, the surface was coated with a crosslinked multilayer of the antibodies. (Brynda et al., 1998). Even if direct adsorption on the transducer surface is suppressed, there is usually another non-specific sensor response due to the binding of some plasma components to the hydrogel and immobilized bioreceptors. It is believed that some non-specific response is associated with the activation of complement by immobilized antibody receptors. A smaller deposit of human serum was observed on a sensor coated with a (carboxymethyl)dextran matrix on which chicken antibodies were immobilized instead of the commonly used mouse antibodies (Vikinge et al., 1998). The observed lower adsorption of complement activating factors C1q and C3 was assumed to reduce complement activation.

The aim of this work was to gain knowledge on the interaction between blood plasma and optical sensors which use immobilized monoclonal antibodies as bioreceptors and to minimize the non-specific sensor response so that sensors capable of measuring analytes in blood plasma could be designed. Two ways were combined to reach the goal: (i) The non-specific adsorption was suppressed by immobilization of multilayer assemblies of monoclonal antibodies on sensor surfaces and (ii) The non-specific optical response of the interferometer surface coated with antibodies sensitive to an analyte was compensated by a response from a reference interferometer surface coated with an antibody insensitive to the analyte.