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Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine. ; We would like to thank the following funding sources: EPSRC EP/K038648/1 (K.M.P. and P.S.F.); BBSRC CASE Studentship (N.K.); Royal Thai Government Scholarship,6 MICIU/ AEI/FEDER, EU BIO2017-89081R (L.A.F.); EPSRC EP/ P009352/1 and EP/M002187/1 (G.-B.V.S.); Imperial College President's PhD Scholarship.18

Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine. ; We would like to thank the following funding sources: EPSRC EP/K038648/1 (K.M.P. and P.S.F.); BBSRC CASE Studentship (N.K.); Royal Thai Government Scholarship,6 MICIU/ AEI/FEDER, EU BIO2017-89081R (L.A.F.); EPSRC EP/ P009352/1 and EP/M002187/1 (G.-B.V.S.); Imperial College President's PhD Scholarship.18

H.E.L. was supported by an Imperial College President's Ph.D. Scholarship. We thank UKRI EPSRC (EP/K038648/1, EP/L011573/1 to P.S.F.) and the European Union's Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 614779 GenoChemetics to R.J.M.G.) for funding. A.M.C.O. receives funding from EPSRC CRITICAT, EP/L016419/1. ; Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds. ; Publisher PDF ; Peer reviewed