A Split Enzyme-based Self Amplification System for Ultrasensitive Detection of Proteins and Small Molecules at the Point of Care

Introduction:: Rapid, inexpensive detection of biomarkers at the point of care is vital for many clinical purposes. While the lateral flow assay (LFA) has allowed rapid diagnostic information to be gathered without equipment for many conditions, limitations in the LFA platform have prevented its application when detecting biomarkers at low concentrations, forcing clinicians to rely either on potentially inaccurate empirical diagnosis or expensive, slow lab tests to make treatment decisions. Sensitive detection of nucleic acid targets has been readily achieved by amplifying signals, but there has been a lack of innovation for detection of low concentration proteins and small molecules at the point of care. Biology, on the other hand, has evolved intricate mechanisms for rapidly amplifying protein signals in vivo via post-translational modification and protein based signaling networks. We have developed a novel sensing platform that leverages split enzyme engineering and a novel autocatalytic feedback loop made up of engineered protein components that results in rapid, bistable, ultrasensitive in vitro sensing of biomarkers.

Our detection platform uses split adenylate cyclase (sAC) to detect the presence of an analyte of interest in vitro. The analyte mediates interactions of the two enzyme component halves fused to binding domains (i.e., a sandwich assay in solution), resulting in cAMP accumulation. Because of the rapid turnover of cAMP from a single reconstituted sAC, detection in the system is not limited by 1-to-1 analyte-to-color change agent as in LFAs, but rather by the binding kinetics with amplification through enzyme turnover.


Materials and Methods:: Experimental Methods: Proof of concept demonstration has been performed by detecting rapamycin, a small molecule, via the well characterized FKBP12-rapamycin-FRB complex. We have engineered protein constructs with N- and C- termini of sAC fused to the FRB and FKBP12 protein domains, such that in the presence of rapamycin, sAC is reconstituted, generating cAMP. Detection of cAMP is achieved via a FRET-based cAMP biosensor that shifts fluorescence upon cAMP binding.

Theoretical Introduction of Bistability: We have further designed a theoretical autocatalytic feedback loop that when incorporated with the sAC sensing fusions and the cAMP biosensor gives rise to bistable response dynamics: a system that exists in only the ON or OFF states. We designed a feed-forward system composed of sAC fused to cAMP receptor protein (CRP) and small DNA fragments that act as binding scaffolds. When cAMP is generated by the sAC sensor, cAMP cooperatively binds CRP-sAC fusions, driving the fusions to bind to short DNA fragments containing multiple repeats of the CRP promotor sequence. As multiple CRP-sAC fusions bind to the scaffold DNA, more sAC is reconstituted, driving increased production of cAMP and further reconstituting CRP-sAC fragments: an autocatalytic positive feedback loop. Activation of the system can be tuned to trigger predetermined analyte concentrations by the inclusion of phosphodiesterase (PDE), degrading cAMP to inactive AMP which does not bind CRP. Therefore, the system triggers only when the rate of cAMP generation reliably exceeds that of degradation by PDE.


Results, Conclusions, and Discussions:: Experimental Results: When the sAC-rapamycin sensing fusions were mixed with a serial dilution of rapamycin, the system transitioned from an OFF state to an ON state between the concentrations of 10 and 100nM (Fig. 1). The OFF state had comparable activity levels to the sAC negative controls (sAC fragments with no binding domains), and the ON state had comparable activity to the positive controls (sAC fragments fused to self-assembling coiled-coil domains). The biosensor has also been experimentally validated and shows an immediate shift in fluorescence in the presence of cAMP (Fig. 2).

Computational Results: We have developed an ordinary differential equation-based model to simulate the dynamics of the protein-protein interactions in this assay using available kinetic parameters of PDE and in vivo expression of sAC. This integrated model combines the initial rapamycin sensing and FRET-based sensing with the autocatalytic feedback loop. ON/OFF behavior across a range of the rapamycin concentrations was achieved by tuning PDE concentration (Fig. 3). Importantly, the modeled system also showed bistability, as illustrated by the hysteresis in the modeled dose response curve of activated biosensor across rapamycin concentrations (Fig. 4); once the system is activated at the “trigger” concentration, the antigen concentration would need to decrease by an order of magnitude for the system to revert to the OFF state.

Conclusions and Discussion: Here, we have demonstrated proof-of-concept for a novel sensing platform for proteins and small molecules that integrates signal amplification, allowing for more sensitive detection than current point-of-care diagnostic technologies. This work is significant because our system bypasses many of the time and infrastructure barriers preventing the implementation of molecular diagnostics at the point of care, while providing sensitive detection of proteins and small molecules in less than a minute in a simple to measure fluorometric output. Further, this system will be broadly applicable for low-concentration antigen detection and can be used to detect a wide range of target analytes with known antibody binding domains. As such, this system could be used as platform for the detection of many analytes currently unable to be rapidly detected at the point of care.


Acknowledgements (Optional): : Research reported in this abstract was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number F32EB031608. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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