A Peptide-Based, Ratiometric Biosensor Construct for Direct Fluorescence Detection of a Protein Analyte

Abstract

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.

Molecular-Size Fluorescence Emitters

“…In this work the 3HC dye 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC) was attached covalently to a sole exposed SH-group in one of subunits of this protein. The same derivative when attached to antigenic peptide allowed detecting its specific interaction with antibody (Enander et al 2008) and being attached to enzyme inhibitor reported on interaction with enzyme elastase (Boudier et al 2009 Synthetic amino acid analogs containing fluorophore as the side group can be incorporated at desired sites of synthetic peptides. Based on different photophysical mechanisms, they can provide responsive functions (Krueger and Imperiali 2013).…”

Investigation of the effects of design parameters on sensitivity of surface plasmon resonance biosensors

“…They developed the Au/SiO x surfaces where the SPR was employed to calculate layer thickness and amount of grafted oligonucleotides. Various biosensing techniques, such as impedance spectroscopy [44], microcantilever [45], carbon nanotube FET [46] and fluorescence [47], are currently available for protein detection. Unlike these detection platforms, the SPR approach does not involve any filtering scheme or complex arrangement.…”

A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays

“…Fab57P was expressed and affinity purified as described elsewhere (Enander et al, 2008). The peptides P1, corresponding to amino acids 134-151 of the tobacco mosaic virus coat protein with an additional biotinylated N-terminal glutamic acid residue (sequence: CH 3 CO-E Bio RGTGSYNRSSFESSSGLV-CONH 2 ), P2 (CH 3 -CO-E Bio KSYNRSSFETNSGLT-CONH 2 ), P3 (CH 3 CO-E Bio NKTSFSPP-PLSI-CONH 2 ), and P4 (CH 3 CO-E Bio RGSGTRSYSNEGFSSLSV-CONH 2 ) were synthesized on the solid phase using standard fluorenylmethoxycarbonyl (Fmoc) chemistry.…”

“…The kinetic parameters describing the interaction between the TMVP-derived peptide (here denoted P1) and Fab57P have been determined, giving K d = 1.8 and 1.6 nM. (Choulier et al, 2001;Enander et al, 2008) P1 was employed as the highest affinity recognition molecule in the array. The second peptide, P2, was rationally designed by deleting three N-terminal amino acids in P1 and exchanging Ser146 for Thr, Ser147 for Asn, and Val151 for Thr.…”