Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

Abstract

Cell-free protein synthesis-based biosensors have been developed as highly accurate, low-cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor’s dynamic range must be increased in the system to achieve low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein, mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder green fluorescent protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce the false-positive proportion.

Toehold switch plus signal amplification enables rapid detection

“…We did a preliminary test comparing the intensity of mNeonGreen to enhanced green fluorescent protein (eGFP) [17] generated in the CFPS and after 1 h of synthesis at 37 • C, the mNeonGreen sample was ca. 5 times more intense (Figure S1), an observation reported recently by Copeland et al [18] Using this construct, toehold switches were identified that bound to SARS-CoV-2 genomic trigger RNA, allowing subsequent stem loop relaxation and translation of the reporter gene. Once toehold switches that successfully produced a positive signal using the mNeonGreen reporter were identified, a new downstream amplification system based on TEVp was engineered and tested for increased sensitivity.…”

Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging

“…However, the emission wavelengths of these modulated FP chromophore analogs fall in the yellow or green spectral region, which causes photodamage to biological samples 10,11 and limits the depth of penetration in biological tissues. [12][13][14][15] Furthermore, the reported works on FP chromophore analogs mainly focus on applications such as viscosity probes, [16][17][18] DNA sensors, [19][20][21] and fluorescence imaging. 22 There has been less exploration in the construction of FP photosensitizers and photodynamic therapy (PDT).…”

Expanding the Cell-Free Reporter Protein Toolbox by Employing a Split mNeonGreen System to Reduce Protein Synthesis Workload

“…The full-length sfGFP and mNG have previously been characterized in the CFS as bright reporter protein candidates. 9 For the split mNG used in this study, we particularly utilized the complementation-enhanced version of the split mNG to maintain complementation capability even at low expression levels. 7 In this study, we elucidated the feasibility of the split protein interaction compared to the whole-cell system and explored the complementation efficiencies and brightness between sfGFP and mNG split protein systems in the CFS.…”