Recognition-based biosensors capable of specifically detecting chemicals, toxins, and bio-agents in their environment are of increasing importance. An important goal in biosensor evolution is production of nanoscale assemblies capable of continuously monitoring concentrations of target species in a simple, reliable manner. This is accomplished by designing sensor components to carry out analyte recognition and binding while simultaneously producing useful output signals via an integrated signal transduction system. Optically addressed biosensors of this type often employ fluorescence resonance energy transfer (FRET) in signal transduction. FRET has been employed in carefully designed sensing systems for proteins, peptides, nucleic acids, and other small molecules.
Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation. Such sensors can either continuously monitor analyte concentrations, or easily be returned to baseline readout values by removal of analyte. Current bioassays on the market are single-use or limited-time use; they need to be replaced after each test or within a short time. This increases both test costs and the logistical demands for performing the analysis. Fielded biosensors can have complex robotics that handle the reagent storage and sensor surface replacement.
The functional simplicity afforded by biosensors, allowing autonomous and continuous monitoring of chemical species, promises to make these devices useful in chemical process monitoring, pharmaceuticals screening, patient point-of-care and environmental testing, public health, and in defense-related fields. Biosensors that utilize FRET are also attractive due to the intrinsic sensitivity of FRET to small changes in donor-acceptor distance and orientation.
The reusable biosensor described in this work easily targets analytes, like toxins or hormones, with a controllable binding affinity. The sensor can be reused for subsequent sensing events once it is washed of analyte. It can be easily adapted to target other analytes due to its modular design.
The biosensor is self-assembled and consists of two co-functional entities. The first entity is a surface tethered biorecognition element, such as a receptor protein. The second entity is a multifunctional tethered modular arm that contains a point of surface attachment, a flexible DNA linker, and a dye label. The dye label is attached to a recognition element (an analog of the primary analyte) that interacts with the receptor protein. These two entities are self-assembled on the surface of a microtiter well and their close proximity, when the biorecognition elements bind the analog on the modular arm, results in FRET between the dyes.
Detection of the targeted analyte is achieved when the analyte displaces the analog on the arm and alters FRET in a quantifiable manner. The useful sensing range is easily altered and extended through the use of different protein mutants and the addition of a DNA complement to the DNA flexible linker.
The FRET-based surface-bound biosensor overcomes the single-use limitation of previously known homogenous bioanalytical systems. The invention provides for fully reversible, reagentless, self-assembling biosensors. The modularity of different portions of the biosensor allows functional flexibility. The biosensor is adaptable to measure many different analytes or targets, and operates without additional development reagents, requiring only the presence of analyte or target for function.