Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications

Abstract

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.

Biosensors for the detection of respiratory viruses: A review

“…Nevertheless, organisms used to develop whole-cell biosensors are generally experimentally modified to incorporate transducer capacity or increase their sensitivity [71] , [72] , [73] , [74] , [75] . The whole cell-based biosensor is increasingly being reported in the literature, and these reports have shown high selectivity, sensitivity, and great potential for their use in biomedical diagnostics [76] , [77] , [78] , [79] .…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The bacteria can be directly spotted on a surface and be used as immunocapture elements of biosensors or as both the capture and detection regents of an ELISA test [87,93]. As an alternative, their capacity of agglutinating in solution can be exploited to quantify the antigens present in liquid samples [94]. The system is therefore faster, less expensive and simpler to implement than the conventional protocol that foresees protein purification and functionalization before immobilization.…”

“…The system is therefore faster, less expensive and simpler to implement than the conventional protocol that foresees protein purification and functionalization before immobilization. Despite the impossibility to quantify exactly the nanobody concentration in an E. coli sample, the binder amount can be tuned by controlling the level of VHH expression [94]. Nanobody displaying bacteria can co-express cytoplasmic fluorescent proteins for obtaining colored living immunoreagents suitable for light microscopy and flow cytometry applications [87].…”

Camelid‐derived single‐chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications

“…Incubation of plasma samples containing fibrinogen with these sdAb-expressing bacteria results in a dose-dependent agglutination. 78 Given the high affinity and specificity, this type of approaches could be used for the detection of other hemostatic proteins as well.…”