Apart from the conventional design strategy, a unique modular strategy for tailoring fluorescence selleckbio biosensors by a simple combination of a receptor and a signal transducer has recently been proposed in the field of signaling aptamers [16,17]. We will discuss the advantage of the strategy and will refer to the perspective of fluorescent biosensors based on macromolecular receptors.2.?Auto-Fluorescent Protein (AFP) Based BiosensorsAuto-fluorescent proteins (AFPs) such as green fluorescent protein (GFP) from the jellyfish [18] are well-established and versatile reporter proteins for monitoring gene expression profiles [19] and protein localizations [20] in a variety of systems. It is noteworthy that AFPs exhibit spontaneous fluorescence emission in cells by the autocatalytic formation of the chromophore after translation [21,22].

Therefore, AFPs can be endogenously expressed in cells or tissues just by transfection of the plasmid DNA without interfering with their fluorescence properties and damaging the cells. In addition to the application of AFPs as a reporter tag, various kinds of AFP-based biosensors have recently been developed by fusion of receptor proteins or mutation of AFPs. There are practically two strategies for the construction of AFP-based biosensors; (a) analyte-sensitive sensors and, (b) conformation-sensitive sensors [23].The design of analyte-sensitive sensors was based on AFP variants, whose fluorescent properties were directly affected by the interaction between a target molecule and a chromophore moiety in AFP.

Initially, pH and halide-sensitive AFP variants have been developed exploiting the intrinsic pH sensitivity of GFP mutants [24�C26] and the high pKas of YFP mutants [27�C29]. Mutations in close proximity to the GFP chromophore or the barrel structure of BFP lead to the specific biosensor for Hg2+ [30] or Zn2+ [31], respectively. In this type of sensor, the receptor function was directly integrated into the chromophore itself by the alteration of the chemical nature around the chromophore.The conformation-sensitive sensors are designed so that the conformational change of the receptor associated with the ligand-binding event transduces to a significant fluorescence response of AFPs. This design strategy is more versatile than that for the analyte-sensitive sensor, because this type of sensor could be applied to a variety of native receptor proteins. Actually, biosensors for Ca2+ [32�C43] as well as for small organic molecules such as ATP [44], cAMP [45�C48], cGMP [49�C51], tryptophan [52], glutamate [53,54], and inositol phosphates [55,56] have been reported based on this strategy.