Mechanistic discoveries and simulation‐guided assay optimization of portable hormone biosensors with cell‐free protein synthesis

Abstract

Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.

Electrochemical Cell‐Free Biosensors for Antibody Detection

“…[2][3][4][5][6] Cell-free biosensors consist of rationally designed responsive gene circuits that, upon binding to a specific target, trigger the transcription/translation of a reporter molecule. Cell-free biosensors for the detection of specific nucleic acid sequences, [5][6][7][8][9][10][11][12][13] small molecules, [3,[13][14][15][16][17][18][19] metal ions, [19][20][21][22] and biomolecules [23] including antibodies [24] have been reported to date demonstrating the possibility of using similar systems as analytical tools that are not only sensitive and specific but also rapid, low-cost and easy to use. The majority of these sensors employs as reporter species optically-active proteins (such as green fluorescent protein, GFP) or light-up RNA aptamers (such as spinach) that are respectively expressed or transcribed upon target recognition by the responsive gene circuit.…”

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

“…Here, we describe a cell-free platform for producing recombinant allergens that overcomes the shortcomings of traditional allergen extract preparations and endows several favorable features beyond the capabilities of cell-based recombinant methods. Cell-free protein synthesis (CFPS) − is a technique that has been applied to protein biologic discovery, , glycoprotein synthesis, − point-of-care manufacturing, − distribution of medicines to resource-limited settings, , small molecules, − diagnostics, − and education. − …”

Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors

“…Recently we reported the utility of a chimeric fusion protein containing a human thyroid receptor domain (hTRβ) fused to a beta lactamase (β-lac) reporter [9]. This fusion protein only detects ligands during protein folding and thus must be translated in the presence of the ligand or sample [10], as depicted in Figure 1. Therefore, this fusion protein was expressed using a BL21 E. coli-based cell-free protein synthesis (CFPS) system that enabled the rapid detection of human thyroid receptor ligands with a β-lac/nitrocefin colorimetric enzymatic readout [9][10][11].…”

“…This fusion protein only detects ligands during protein folding and thus must be translated in the presence of the ligand or sample [10], as depicted in Figure 1. Therefore, this fusion protein was expressed using a BL21 E. coli-based cell-free protein synthesis (CFPS) system that enabled the rapid detection of human thyroid receptor ligands with a β-lac/nitrocefin colorimetric enzymatic readout [9][10][11]. However, the relatively high cost and poor stability of nitrocefin [12][13][14] motivate efforts to reengineer the fusion protein with alternative reporter enzyme systems.…”