Recent Advances in Developing Cell-Free Protein Synthesis Biosensors for Medical Diagnostics and Environmental Monitoring
Abstract
1. Introduction
2. Cell-Free Biosensors for Environmental and Medical Applications
2.1. Environmental Detection
2.2. Medical Diagnostics
3. Technological Advances and Optimization Strategies in Cell-Free Protein Synthesis for Biosensor Development
3.1. Optimization of Yield and Sensitivity
3.2. Preservation and Field Deployment Strategies
4. Integration of Synthetic Biology with Cell-Free Systems for Advanced Biosensor Applications
4.1. Complex Signal Processing and Multiplexed Systems
4.2. Novel Sensor Design Strategies
5. Emerging Materials and Future Directions
5.1. Advanced Materials for Cell-Free Biosensors
5.2. Future Challenges and Opportunities
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CFPS | Cell-free protein synthesis |
LCMS | Liquid chromatography–mass spectrometry |
aTF | Allosteric transcription factor |
RAPID | Rapid adaptable portable in vitro detection |
TLISA | T7 RNA polymerase-linked immunosensing assay |
GABA | γ-aminobutyric acid |
SLP | Supported lipid bilayer |
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Target Analyte | Detection Method/System | Limit of Detection | Selectivity/Specificity | Sample Matrix |
---|---|---|---|---|
Mercury [22] | Paper-based, dual- filter, smartphone readout | 6 μg/L | Selective for mercury (activation ratio >8–14 for Hg, <2 for others) | Water |
Mercury [14] | merR gene, plasmid DNA, firefly luciferase/eGFP | 1 ppb | Selective for Hg2+; pH optimization and chelating agents enhance specificity | Water |
Mercury [24] | Allosteric transcription factors (aTFs) | 0.5 nM | High selectivity for target metals; validated in real water samples with 91–123% recovery rates | Water |
Lead [24] | aTFs | 0.1 nM | High selectivity for target metals; validated in real water samples with 91–123% recovery rates | Water |
Lead [25] | Engineered PbrR mutants | 50 nM | Selective for lead | Water |
Arsenic and mercury [26] | Optimized transcription factors | Arsenic ≤10 μg/L, Mercury ≤6 μg/L | Minimal response to nontoxic ions | Water |
Tetracyclines [30] | Riboswitch-based, RNA aptamers | 0.4 μM | Broad-spectrum for tetracycline family | Milk samples |
Biological warfare agents [32] | 16S rRNA targeting, retroreflective particles | 1 to 11 fM | Specific for B. anthracis, F. tularensis, Y. pestis, B. pseudomallei, B. abortus | Laboratory samples |
Atrazine [28] | Metabolic pathway and cyanuric acid biosensor | 50 μM | Specific for atrazine via metabolic conversion | Laboratory samples |
Target Analyte | Detection Method/System | Limit of Detection | Selectivity/Specificity | Sample Matrix |
---|---|---|---|---|
Thyroid receptor ligands [37] | Allosterically activated fusion protein | 48 to 75 nM | β-specific endocrine disruptors | Laboratory samples |
Estrogenic compounds [38] | RAPID platform | 9 to 330 nM | selective for estrogenic activity | Human blood/urine |
3-oxo-C12-HSL [41] | Quorum sensing detection | 4.9 nM | Highly specific for 3-oxo-C12-HSL; results comparable to LC-MS | P. aeruginosa-infected sputum |
Glutamine [7] | Metabolically engineered system | 10 μM | Inhibitor-based approach ensures glutamine specificity | Human serum |
Bile acids [42] | Transcription factor-based | 0.61 μM | Selective for deoxycholic acid | Fecal water, wastewater, serum |
Homocysteine [16] | Colorimetric detection | 1 μM | Selective detection at clinically relevant thresholds | Plasma |
Progesterone [20] | CRISPR-Cas14a + aTFs | 67 pM to 0.33 μM | Single-nucleotide discrimination | 2 μL sample |
Pentachlorophenol [45] | aTF NalC + NASBA | 0.002 μM | high specificity for pentachlorophenol; validated with 101–114% recovery | Environmental samples |
Protein biomarkers [46] | Split T7 RNA polymerase (TLISA) | 50 to 200 nM | Various protein targets | Serum/saliva |
SARS-CoV-2 RNA [15] | Paper-based toehold switches | 60 nM | High specificity for target RNA | Human saliva |
System Type | Detection Method | Limit of Detection | Key Innovation | Sample Compatibility |
---|---|---|---|---|
Signal amplification [60] | Ribozyme cleavage circuits | 0.045 μM | Eliminates translation steps | Small molecules |
Amplification circuit [76] | Polymerase strand recycling | 5 nm to 1 μM | T7 RNA polymerase recycling | Laboratory samples |
Electrochemical multiplexing [77] | Gene-circuit-based sensors | 65 nM | Electrochemical readouts | Laboratory samples |
Multiplexed pathogen detection [78] | Multi-arm RNA junctions | 20 aM | Molecular logic operations | Diagnostic samples |
Quantum dot multiplexing [79] | Hybrid nucleic acid-QD assemblies | 1 enzyme unit 1 | Multiple enzymatic monitoring | Laboratory samples |
CRISPR-based detection [20] | CRISPR-Cas systems integration | 4.2 pM | Signal processing enhancement | Complex biological matrices |
Pathogen multiplexing [32] | 16S rRNA targeting | 2.4 nM | Simultaneous multi-pathogen ID | Laboratory samples |
Metabolic transducers [33] | Plug-and-play cascades | 1 to 10 μM | Synthetic metabolic networks | Complex media/urine |
System Type | Detection Method | Limit of Detection | Key Innovation | Sample Compatibility |
---|---|---|---|---|
Split reporter system [86] | Split T7 promoter | 10 pM | Three-way junction structure | Nucleic acids |
Split protein system [87] | Split mNeonGreen | Not reported | Reduced synthesis workload | Laboratory samples |
Metabolic biosensing [88] | GABA transaminase + CFPS | 47 μM | Enzymatic conversion approach | Laboratory samples |
Dopamine detection [84] | Synthetic riboswitches | 0.48 μM | Engineered aptamer riboswitches | Human urine |
Fluoride detection [85] | Natural riboswitches | 0.2 mM | B. cereus crcB riboswitch | Field conditions |
Mechanosensitive systems [82] | MscL + biosensing | Not reported | Osmotic pressure integration | Synthetic liposomes |
Adaptive synthetic cells [83] | Inducible genetic circuits | Not reported | Mechanosensitivity + gene expression | Synthetic systems |
Colorimetric multiplexing [89] | Multi-enzyme systems | Not reported | Visual detection without instruments | Laboratory samples |
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Green, T.P.; Talley, J.P.; Bundy, B.C. Recent Advances in Developing Cell-Free Protein Synthesis Biosensors for Medical Diagnostics and Environmental Monitoring. Biosensors 2025, 15, 499. https://doi.org/10.3390/bios15080499
Green TP, Talley JP, Bundy BC. Recent Advances in Developing Cell-Free Protein Synthesis Biosensors for Medical Diagnostics and Environmental Monitoring. Biosensors. 2025; 15(8):499. https://doi.org/10.3390/bios15080499
Chicago/Turabian StyleGreen, Tyler P., Joseph P. Talley, and Bradley C. Bundy. 2025. "Recent Advances in Developing Cell-Free Protein Synthesis Biosensors for Medical Diagnostics and Environmental Monitoring" Biosensors 15, no. 8: 499. https://doi.org/10.3390/bios15080499
APA StyleGreen, T. P., Talley, J. P., & Bundy, B. C. (2025). Recent Advances in Developing Cell-Free Protein Synthesis Biosensors for Medical Diagnostics and Environmental Monitoring. Biosensors, 15(8), 499. https://doi.org/10.3390/bios15080499