Assessment of Colorimetric Reporter Enzymes in the PURE System

Abstract

Colorimetric reporter enzymes are useful for generating eye-readable biosensor readouts that do not require a device to interpret, an attractive property for applications in remote or developing parts of the world. The use of cell-free gene expression further facilitates such applications via amenability to lyophilization and incorporation into materials like paper. Currently, detection of multiple analytes simultaneously with these systems requires multiple reactions or a device. Here we evaluate seven enzymes and 15 corresponding substrates for functionality in a particular cell-free expression system known as PURE. We report eight enzyme/substrate pairs spanning four enzymes that are compatible with PURE. Of the four enzymes, three pairings exhibit no cross-reactivity. We finally show that at least one pairing can be used to create a third color when both are present, highlighting the potential use of these reporters for multiplex sensing.

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors

“…Similar to Sharpes's finding of incompatibility between some enzyme−substrate systems when mixed together, we also find that our NcoI system was not functional in the presence of the two other reporters when all were coexpressed in the same reaction. 13 We note that when the NcoI system was expressed by itself in the reaction mix individually, it did work albeit far more slowly. Further analysis is required to clearly understand the reason for NcoI reporter's inefficacy, but we speculate possible incompatibility between QD 625 (due to its large surface area) and some PURExpress enzyme(s)'s coordination on the QD surface or our use of a sub-optimized NcoI gene sequence, whereas the commercially obtained pure enzyme may in actuality be a vastly improved mutationally selected version that is proprietary.…”

“…In a similarly focused study, Sharpes et al identified seven candidate colorimetric reporter enzymes from the literature with each enzyme having one to three substrates that were all commercially available for screening in cell-free assays as output signals. 13 After intensive screening of several different combinations, they showed that at least one pair of these reporter/substrate combinations can produce four distinct colors from two enzymes, suggesting utility for multiplexing of sensors in a single reaction. In this report, we focused on developing multiplexed optical outputs from reporter enzymes expressed as part of biosensing in cell-free reactions.…”

“…Cell-free reaction systems are an emergent technology with broad applications in sensor development and point-of-contact manufacturing. − Cell-free systems typically seek to mimic biological activities, specifically transcription and translation (TX-TL), with minimized components while functioning in the absence of encapsulating cellular membranes. − Reduced to this simplicity and less restrained by potential biological toxicity issues, cell-free systems are conducive to bioproduction of a wide array of biomolecules at the point of need including pharmaceuticals and enzymes. The latter type of products also enables sensor development since they can be used as coupled assay reporter outputs. − Cell-free sensors are biosensors by definition and typically demonstrate the same functionality, namely, that the presence of some target analyte is “sensed” by a component of the reaction mixture resulting in the coupled TX-TL of an output reporter, which can range from an enzyme to a fluorescent protein. − The lack of a living component here imparts two distinct advantages compared to cell-based biosensors; first, cell-free systems do not need to proliferate (i.e., reproduce or grow and expand) to function in field-based detection, and second, as a non-living system, they can be integrated into materials and/or lyophilized for prolonged storage over a range of temperatures and conditions without significant loss of activity. − Most cell-free sensors relying on enzyme-based or fluorescent protein reporters have shown success in assay development, but developing multiplexed capabilities for a single reaction mix (as opposed to multiple parallel individual yet differentially targeted reaction mixes) has not been fully achieved to date. Either multiplexing is achieved through colorimetric assays or signals from multiple sensors triggering the same reporter output. ,, Most fluorescence-based reporters typically have excitation/emission profiles that give rise to complex and overlapping spectral windows that are difficult to deconvolve when combined, while enzymatic systems generally exploit single colorimetric readouts. , Here, we introduce a design for an optically multiplexed cell-free biosensor reporter platform that utilizes a collection of highly sensitive and minimally overlapping spectrally distinct photoluminescent (PL) outputs that are all simultaneously excited in a broad range of UV–visible wavelengths.…”

“…Either multiplexing is achieved through colorimetric assays or signals from multiple sensors triggering the same reporter output. 3,13,20 give rise to complex and overlapping spectral windows that are difficult to deconvolve when combined, while enzymatic systems generally exploit single colorimetric readouts. 12,21 Here, we introduce a design for an optically multiplexed cellfree biosensor reporter platform that utilizes a collection of highly sensitive and minimally overlapping spectrally distinct photoluminescent (PL) outputs that are all simultaneously excited in a broad range of UV−visible wavelengths.…”

Biotechnology Applications of Cell-Free Expression Systems

“…Cell-free biosensors that can operate free of any device are also attractive for applications where factors such as weight and portability are especially important. One such example of this is the utilization of the PURE system towards biosensing, which demonstrated compatibility with colorimetric reporter enzymes that would allow simple visual detection of multiple target analytes [63]. Technological innovations in the integration of cell-free systems demonstrate the advantage they present for the biosensing of target analytes, especially those that are toxic or present challenges for cell-based systems.…”