Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permiQed BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

DNAzyme Sensor Uses Chemiluminescence Resonance Energy Transfer for Rapid, Portable, and Ratiometric Detection of Metal Ions.

DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.

Cysteamine-coated gold nanoparticles for bimodal colorimetric detection with inverse sensitivity: a proof-of-concept with lysozyme.

Cysteamine-coated gold nanoparticles (cysAuNPs) are positively charged as-synthesised and hence can interact with negatively charged DNA with ease. We have investigated the dependency of the particles' dispersion stage on different concentrations of lysozyme-binding aptamer (LBA). On top of the commonly reported phenomenon where cysAuNPs aggregate as the concentration of LBA increases, we observed that cysAuNPs redispersed after the amount of LBA achieved a certain threshold, dubbed as the critical redispersion concentration (CRC). By harnessing the aggregation and dispersion behaviour of cysAuNPs at LBA below and above the CRC, respectively, we have demonstrated a bimodal colorimetric aptasensor to detect lysozyme as a proof-of-concept study. Apart from being able to quantify the lysozyme in different ranges of concentrations with a visual change in colour, this aptasensor also demonstrated a novel concept of inverse sensitivity (i.e. higher signal with less analyte), leading to a 24-fold higher of signal-to-noise ratio (SNR), in comparison to the conventional sensors. The aptasensor can also selectively distinguish lysozyme and eliminate false results from other control proteins via both modes. The generalisability, as well as potential of cysAuNPs for bimodal colorimetric detection and inverse sensitivity behaviour have made this material an interesting alternative to citrate-coated AuNPs.