Editing Biosensor (section)

===Reagentless fluorescent biosensor===
A reagentless biosensor can monitor a target analyte in a complex biological mixture without additional reagent. Therefore, it can function continuously if immobilized on a solid support. A fluorescent biosensor reacts to the interaction with its target analyte by a change of its fluorescence properties. A Reagentless Fluorescent biosensor (RF biosensor) can be obtained by integrating a biological receptor, which is directed against the target analyte, and a [[solvatochromism|solvatochromic]] fluorophore, whose emission properties are sensitive to the nature of its local environment, in a single macromolecule. The fluorophore transduces the recognition event into a measurable optical signal. The use of extrinsic fluorophores, whose emission properties differ widely from those of the intrinsic fluorophores of proteins, tryptophan and tyrosine, enables one to immediately detect and quantify the analyte in complex biological mixtures. The integration of the fluorophore must be done in a site where it is sensitive to the binding of the analyte without perturbing the affinity of the receptor.

Antibodies and artificial families of Antigen Binding Proteins (AgBP) are well suited to provide the recognition module of RF biosensors since they can be directed against any antigen (see the paragraph on bioreceptors). A general approach to integrate a solvatochromic fluorophore in an AgBP when the atomic structure of the complex with its antigen is known, and thus transform it into a RF biosensor, has been described.<ref name="pmid19945965"/> A residue of the AgBP is identified in the neighborhood of the antigen in their complex. This residue is changed into a cysteine by site-directed mutagenesis. The fluorophore is chemically coupled to the mutant cysteine. When the design is successful, the coupled fluorophore does not prevent the binding of the antigen, this binding shields the fluorophore from the solvent, and it can be detected by a change of fluorescence. This strategy is also valid for antibody fragments.<ref>{{cite journal|last1=Renard|first1=M|last2=Belkadi|first2=L|last3=Hugo|first3=N|last4=England|first4=P|last5=Altschuh|first5=D|last6=Bedouelle|first6=H|title=Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies|journal=J Mol Biol|date=Apr 2002|volume=318|issue=2|pages=429–442|doi=10.1016/S0022-2836(02)00023-2|pmid=12051849}}</ref><ref>{{cite journal|last1=Renard|first1=M|last2=Bedouelle|first2=H|title=Improving the sensitivity and dynamic range of reagentless fluorescent immunosensors by knowledge-based design|journal=Biochemistry|date=Dec 2004|volume=43|issue=49|pages=15453–15462|doi=10.1021/bi048922s|pmid=15581357|citeseerx=10.1.1.622.3557|s2cid=25795463}}</ref>

However, in the absence of specific structural data, other strategies must be applied. Antibodies and artificial families of AgBPs are constituted by a set of hypervariable (or randomized) residue positions, located in a unique sub-region of the protein, and supported by a constant polypeptide scaffold. The residues that form the binding site for a given antigen, are selected among the hypervariable residues. It is possible to transform any AgBP of these families into a RF biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. More specifically, the strategy consists in individually changing the residues of the hypervariable positions into cysteine at the genetic level, in chemically coupling a solvatochromic fluorophore with the mutant cysteine, and then in keeping the resulting conjugates that have the highest sensitivity (a parameter that involves both affinity and variation of fluorescence signal).<ref name="pmid21565483"/> This approach is also valid for families of antibody fragments.<ref>{{cite journal|last1=Renard|first1=M|last2=Belkadi|first2=L|last3=Bedouelle|first3=H|title=Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors|journal=J. Mol. Biol.|date=Feb 2003|volume=326|issue=1|pages=167–175|doi=10.1016/S0022-2836(02)01334-7|pmid=12547199}}</ref>

A posteriori studies have shown that the best reagentless fluorescent biosensors are obtained when the fluorophore does not make non-covalent interactions with the surface of the bioreceptor, which would increase the background signal, and when it interacts with a binding pocket at the surface of the target antigen.<ref>{{cite journal|last1=de Picciotto|first1=S|last2=Dickson|first2=PM|last3=Traxlmayr|first3=MW|last4=Marques|first4=BS|last5=Socher|first5=E|last6=Zhao|first6=S|last7=Cheung|first7=S|last8=Kiefer|first8=JD|last9=Wand|first9=AJ|last10=Griffith|first10=LG|last11=Imperiali|first11=B|last12=Wittrup|first12=KD|title=Design Principles for {{not a typo|SuCESsFul}} Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions|journal=J Mol Biol|date=Jul 2016|doi=10.1016/j.jmb.2016.07.004|pmid=27448945|pmc=5048519|volume=428|issue=20|pages=4228–4241}}</ref> The RF biosensors that are obtained by the above methods, can function and detect target analytes inside living cells.<ref>{{cite journal|last1=Kummer|first1=L|last2=Hsu|first2=CW|last3=Dagliyan|first3=O|last4=MacNevin|first4=C|last5=Kaufholz|first5=M|last6=Zimmermann|first6=B|last7=Dokholyan|first7=NV|last8=Hahn|first8=KM|last9=Plückthun|first9=A|title=Knowledge-based design of a biosensor to quantify localized ERK activation in living cells|journal=Chem Biol|date=Jun 2013|volume=20|issue=6|pages=847–856|doi=10.1016/j.chembiol.2013.04.016|pmid=23790495|pmc=4154710}}</ref>