Abstract
The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Instead, the signal from affinity-based measurements of these biomolecules depends linearly on the probe concentration. Substituting antibody-based probes tagged for fluorescent quantification with lasing detection probes would create a new platform for biomarker quantification based on optical rather than enzymatic amplification. Here, we construct a virus laser which bridges synthetic biology and laser physics, and demonstrate virus-lasing probes for biosensing. Our virus-lasing probes display an unprecedented > 10,000 times increase in signal from only a 50% increase in probe concentration, using fluorimeter-compatible optics, and can detect biomolecules at sub-100 fmol mL−1 concentrations.
Highlights
The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells
Antibody-based probes tagged for fluorescent quantification can bind a wide range of biomolecular targets with high specificity, but generate a weak signal, which can be difficult to distinguish from background noise.[1]
The structural order and repeating chemical landscape on the surface of M13 provide a versatile and amenable model system for modulating the spectrum and threshold dynamics of the laser using the principles of synthetic biology
Summary
The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Coli28,29—has been used effectively as a substitute for antibody probes in cell imaging, flow cytometry and enzyme-linked immunosorbent assays (ELISA)[30,31] and as the key component in nanosystems such as virus-based lithium-ion batteries and piezoelectric generators.[32,33,34] Using phage display, M13 can be routinely programmed to bind specific target biomolecules either by display of known antibody domains or binding proteins fused to either the gene 3 or 8 coat proteins, or by selection from phage-displayed combinatorial libraries to isolate those with the required targetbinding specificity and affinity.[30,32,33,35,36,37,38] Phage surfaces can be functionalised by coupling amine-reactive dyes to α-amines at Ala[1] and ε-amines at Lys[8] on the 2700 50 amino-acid alphahelical gene 8 coat proteins, or to solvent-exposed primary amines on the five gene 3 coat proteins.[28,29] The gene 8 coat proteins form an overlapping quasicrystalline lattice with a 5-fold rotation axis and a two-fold screw axis,[39] which brings the dyes into close proximity—much smaller than the wavelength of visible light—enabling electronic interactions and leading to resonant energy transfer between dye molecules as well as fluorescence quenching (Fig. 1a).[40,41,42]. In a proof-of-concept study, we optically engineer a mix-andmeasure ligand-binding assay sensitive to 90 fmol mL−1 monoclonal antibody, suggesting that clinically relevant concentrations of biomolecules can be detected without immobilization of the ligand or probe on surfaces and without invasive wash steps
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