A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of 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 supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the 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 both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine

Development of an agglutination-dependent E. coli-based biosensor for diagnostic applications

Whole-cell biosensors are powerful diagnostic tools for detection of various analytes, including proteins which have difficulty in transporting across the cell membrane. Here, we developed a novel biosensor based on the agglutination reaction by using an E. coli chassis displaying nanobodies (VHHs) as the detection element for selectively binding to a target of interest. As a proof-of-concept, we demonstrated that this design was able to detect a model protein having two antigenic determinants in a semi-quantitative manner with a detectable change in the output, which was easily visualised using the naked eye. Subsequently, the designer cells were re-engineered to develop tests for clinically relevant biomarkers by the display of specific VHHs against human fibrinogen (Fib) and 3-phenoxybenzoic acid (3-PBA). Fib is a bivalent protein so detection was achieved in the same manner as the prototype by observing cell agglutination. 3-PBA is a small molecule, therefore, detection was achieved by modifying the platform based on a competitive ELISA technique. Binding with a 3-PBA-protein conjugate, allowed cell crosslinking, which was disrupted by the presence of free 3-PBA, thus resulting in cell pellets. The optimised cell biosensors detected both analytes at nanomolar concentrations and exhibited robust function in complex sample backgrounds such as human plasma and synthetic urine. Furthermore, we studied the feasibility of using the bacterial LuxIR quorum sensing as a quantitative output design for differentiating cell agglutination versus pellets by monitoring the production of the fluorescent reporter. Despite the verified functional constructs, there was no signal difference between the two states, possibly due to a similar spatial cell distribution in a 96-well plate. This agglutination-dependent E. coli biosensor was shown to be a promising platform for identifying both protein and chemical analytes. Therefore this simple, affordable design could be developed as a potential diagnostic tool for field use.Open Acces

BeQuIK (Biosensor Engineered Quorum Induced Killing): designer bacteria for destroying recalcitrant biofilms.

This opinion piece describes a new design for the remediation of recalcitrant biofilms. It builds on previous work to develop engineered E. coli that recognize quorum sensing signals from pathogens in a biofilm and secrete toxins in response. To solve the challenge of dilute signalling molecules, we propose to use nanobodies and enzymes displayed on the surface of the cells to localize them to the biofilm and degrade the extracellular polymeric substances, thus creating a solution with better 'seek and destroy' capabilities

A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure

There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments

A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure

There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments

Financiamento de rodovias por meio da cobrança de pedágio : uma avaliação da Empresa Gaúcha de Rodovias

O presente trabalho discorre a respeito dos métodos de financiamento empregados em rodovias, com maior enfoque na cobrança de pedágio, e faz uma análise da eficiência econômica e operacional da Empresa Gaúcha de Rodovias (EGR), uma empresa estatal que atua na cobrança de pedágio em rodovias do estado do Rio Grande do Sul. Após uma revisão da literatura sobre os métodos de financiamento de rodovias e de um breve panorama da situação atual das rodovias no Brasil, foram elencadas empresas privadas concessionárias de rodovias para servirem de base para comparações e futura análise do desempenho da EGR. Foram estudados, além da EGR, os grupos Triunfo e EcoRodovias, representados pelos seus setores de concessão de rodovias, nos anos de 2013 e 2014. As comparações e análises foram baseadas em indicadores de desempenho desenvolvidos ao longo do trabalho, os quais abrangeram elementos como número de acidentes, número de atendimentos, extensão dos trechos administrados, volume de tráfego, custo dos serviços prestados, despesas operacionais das empresas e volume total arrecadado. A definição dos indicadores foi limitada pela disponibilidade de dados, os quais foram coletados a partir de pesquisa documental realizada junto às empresas e à Agência Nacional de Transportes Terrestres (ANTT). Os indicadores apontaram que as rodovias administradas pela EGR, quando comparadas com as das demais empresas estudadas, apresentam bom desempenho no quesito segurança. Entretanto, todos os demais indicadores foram desfavoráveis à empresa, concluindo-se, por fim, que a Empresa Gaúcha de Rodovias apresenta, de um modo geral, desempenhos econômico e operacional inferiores quando comparada com empresas privadas concessionárias de rodovias de porte semelhante

Whole-cell biosensor with tuneable limit of detection enables low-cost agglutination assays for medical diagnostic applications

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 can 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 straight-forward 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 nanbodies 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

Whole-cell biosensor with tunable limit of detection enables low-cost agglutination assays for medical diagnostic applications

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.1