Quantum Dot and Nucleic Acid Optical Readout for Cell-Free Biosensing

Cell-free biosensors combine in vitro bacterial transcription-translation systems with operons to detect analytes, such as heavy-metal ions. These sensors are highly desirable due to their easy portability and long shelf life. Typically, the expression of a fluorescent RNA aptamer or protein tied to the presence of an analyte is used as an optical readout for detection in such biosensors. While these readouts have demonstrated tremendous success in testing water potability, the readout is limited by how many different RNA aptamers and proteins can be used simultaneously. The quantum yield of these biological fluorescent molecules is low as well. Recently, we demonstrated a semiconductor quantum dot (QD)-based reporter system that is fully compatible with cell-free transcription-translation systems. Our reporter, abbreviated as QD-PDD (Peptide-PNA DNA Dye), uses nucleic acid specificity to trigger a change in Förster resonance energy transfer (FRET) between the QD and its acceptor fluorophore (Cy3) when a restriction enzyme (<i>Bam</i>HI) is expressed. Given the high specificity of nucleic acids and the quantum yield of QDs, the question remained whether QD-PDD reporters could be plugged downstream of heavy-metal cell-free biosensors. Herein, we connected an operon sensitive to cadmium ions to the cell-free expression of <i>Bam</i>HI, which triggered a FRET change in the QD-PDD reporter. The operon system can successfully detect cadmium in water-based cadmium chloride solutions. This system serves as a proof of concept showing that QD-PDD can enable the departure of fluorescent biomolecules (aptamers and proteins) in cell-free biosensors.