Development of dual receptor biosensors: an analysis of FRET pairs

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Abstract

The development of a dual receptor detection method for enhanced biosensor monitoring was investigated by analyzing potential fluorescent resonance energy transfer (FRET) pairs. The dual receptor scheme requires the integration of a chemical transducer system with two unique protein receptors that bind to a single biological agent. The two receptors are tagged with special molecular groups (donors and acceptors fluorophores) while the chemical transduction system relies on the well-known mechanisms of FRET. During the binding event, the two FRET labeled receptors dock at the binding sites on the surface of the biological agent. The resulting close proximity of the two fluorophores upon binding will initiate the energy transfer resulting in fluorescence. The paper focuses on the analysis and optimization of the chemical transduction system. A variety of FRET fluorophore pairs were tested in a spectrofluorimeter and promising FRET pairs were then tagged to the protein, avidin and its ligand, biotin. Due to their affinities, the FRET-tagged biomolecules combine in solution, resulting in a stable, fluorescent signal from the acceptor FRET dye with a simultaneous decrease in fluorescent signal from the donor FRET dye. The results indicate that the selected FRET pairs can be utilized in the development of dual receptor sensors.

Introduction

There is an important need in the public health and defense arenas for ultrasensitive biosensors capable of rapidly detecting dangerous biological agents (Mulchandani et al., 1998, Mulchandani et al., 1999, Anderson et al., 1998, Seo et al., 1999). For example, these sensors might be capable of quickly reporting the presence of infectious airborne, waterborne or blood-borne microorganisms or microorganism-produced toxins. A major limitation of current optical biosensors is the background ‘noise’ due to non-specific binding which may lead to false positive readings (Ferguson et al., 1991, Perez-Luna et al., 1999). Researchers have focused on improving immobilization techniques in order to help eliminate or reduce non specific adsorption (Diederick and Losche, 1997, Kroger et al., 1999). For example, surface functionalization of self-assembled monolayers continue to be studied in order to reduce the effects of non-specific adsorption (Blankenburg et al., 1989, Spinke et al., 1993). Prime investigated the protein adsorption effects in self assembled monolayers of omega-functionalized long chain alkanethiolates in the hopes of better understanding the interactions between proteins with organic surfaces, while Perez-Luna investigated binding reaction between streptavidin and biotin and their role on surface properties, in order to understand nonspecific adsorption events (Prime and Whitesides, 1991, Perez-Luna et al., 1999). To overcome the problem of non-specific binding, researchers have adsorbed blocking agents, such as bovine serum albumin, gelatin and Triton X-100 onto the substrate (Bhatia et al., 1989, Diederick and Losche, 1997). However, there was still a measurable level of nonspecific binding. To eliminate, or at least dramatically lower the adverse background, we present a technique that involves incorporating multiple receptors. When the multiple receptors bind to the target analyte, they will elicit some form of unique biological activity (i.e. conformational change) that will allow detection and help eliminate or reduce false positives.

Cell receptors are a feasible line of approach for novel biosensor development and have been developed into viable sensor systems (Canziani et al., 1999). Naturally occurring protein receptors are typically found in cell membranes and they contain structural and functional domains that penetrate the intracellular and extracellular spaces. Within the body, these receptors act as intercellular communication links by reversibly binding to specific neurotransmitters and hormones; but they may also act as binding sites for many drugs and toxins. As a result, cell receptors have been utilized in the development of a variety of biosensor systems, from nitric oxide sensors to understanding renal cellular interactions (Nakai et al., 1998, Barker et al., 1999, Newman et al., 1999). Most of these researchers exploited the affinity between the agent and a single receptor. However, in addition to single receptor systems, there are many biological agents that utilize multiple receptors that offers a unique approach for sensing when utilized with the chemical transduction system, FRET.

In order to function properly, the dual receptor method requires the integration of a chemical transducer system, FRET, with two unique protein receptors, each capable of binding to a single biological agent. Researchers have utilized FRET in structural biology and biochemistry as a method to measure protein structures (Selvin, 1995). In addition, the principles of FRET have been used in optical sensor development (Chang and Sipior, 1995, Ballerstadt and Schultz, 1997, Pearce, 1998). For example, the FRET pair of sulforhodamine 101 and Bromocresol green, was used to develop NH3 sensors (Chang and Sipior, 1995), while the FITC (fluorescein-5-isothiocyanate) and TRITC (tetramethylrhodamine isothiocyanate) FRET pair was adopted in competitive–binding assays (Ballerstadt and Schultz, 1997). Pearce utilized the FRET pair, 7-diethylaminocoumarin-3-carboxylic acid and lissamine rhodamine B sulfonyl chloride, to detect Ni(II) (Pearce, 1998). Although FRET sensors do exist, there has not been significant research in the development of FRET based dual receptor sensors.

The principles of FRET are well known and have been widely used in biochemical and biomedical analysis for many years (Wu and Brand, 1994). FRET occurs when radiation is absorbed by a fluorophore (termed the donor) on a typical 10−15 s time scale. Following the donor's energy absorption, one of two possible events may occur: (1) the energy may be re-emitted in the form of fluorescence on a 10−9 s time scale; or (2) the excited molecule (donor) can transfer its energy to another fluorophore (termed the acceptor) which subsequently fluoresces in the same order of time. This latter phenomenon is fluorescence resonance energy transfer or FRET (Chantal and Lebrun, 1998).

FRET has been explained by Förster in terms of the relative dipole orientation of the donor and the acceptor and the distance between these two fluorophores (Förster, 1948). The efficiency of energy transfer is greatly affected by these parameters. The energy transfer yield is given by Eq. (1):E=R06/(R6+R06)where R is the distance between the donor and acceptor in a biological environment. R0 is a constant for each donor–acceptor pair and is defined as the distance that energy transfer, E is 50% efficient. R0 is called the Förster critical distance. The transferred energy is depended on both the Förster critical distance, which is characterized by the FRET pairs themselves and the distance between the donor and acceptor fluorophores; hence, a distance dependent method of detection.

An additional method that is utilized to calculate transfer efficiency, E, is from the fluorescence intensities (quantum yields) using Eq. (2):E=1−(Fda/Fd)where Fda is the donor fluorescence intensity determined at a given wavelength in the presence of the acceptor and Fd is the corresponding quantity determined in the absence of the acceptor (Lakowicz, 1983). This method was used to roughly calculate the transfer efficiency of the FRET fluorophore pairs. The design of the dual receptor biosensor is depended upon achieving high transfer efficiencies; hence, a change in fluorescent intensity, induced by donor and acceptor molecular distances brought about by the agent, can be detected.

This paper focuses on investigating and optimizing the chemical transduction system, FRET, for the development of dual receptor biosensors. A number of fluorophores were tested and identified as possible pairs. The pairs that demonstrated high spectral overlap and energy transfer were then tagged to the biomolecules, avidin and biotin. Avidin and biotin were utilized as a test bed to prove the feasibility of the proposed method. The specificity of biotin binding to avidin provides the basis for developing FRET dual receptor sensor systems.

Section snippets

Materials

All reagents were reagent grade unless otherwise indicated. The dyes, fluorescein-5-isothiocyanate (FITC) and 7-dimethylaminocoumarin-4-acetic acid (DMACA) were obtained from Molecular Probes (Eugene, OR). The dye, tetramethylrhodamine isothiocyanate (TRITC) and the biomolecules, streptavidin, avidin and biotin, as well as fluorescein-tagged biotin, were obtained from Sigma Chemical (St. Louis, MO). An additional fluorophore, 7-amino-4-methyl-3-coumarinylacetic acid-N-hydroxysuccinimide,

Determination of FRET pairs for biosensor applications

A number of FRET dye pairs were examined which resulted in three FRET pairs having sufficiently high spectral overlaps. They were, DMACA/FITC, AMCA/FITC and FITC/TRITC. Fig. 1 shows the excitation and emission scans of the fluorophore pair, FITC/TRITC. FITC acted as the donor while TRITC was the acceptor fluorophore. FITC was excited at 495 nm and had an emission at 523 nm while TRITC had an excitation peak at 554 nm and an emission peak at 580 nm. The donor's emission peak overlapped the

Conclusion

This paper detailed the investigation and optimization of the FRET chemical transduction system. A number of fluorophores were tested as potential FRET pairs. The promising FRET pairs were further tested and conjugated to the biomolecules, streptavidin, avidin and biotin. These pairs were DMACA/FITC, AMCA/FITC and FITC/TRITC. This identification and fluorescent labeling was the first step in developing enhanced biosensors that utilize the FRET dual receptor method. Because of their affinities,

Acknowledgements

The project was funded through a State of Michigan Research Excellence Fund (REF) and by the Whitaker Foundation Special Opportunity Grant.

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