Role of the Probe Sequence/Structure in Developing an Ultra-Efficient Label-Free COVID-19 Detection Method Based on Competitive Dual-Emission Ratiometric DNA-Templated Silver Nanoclusters as Single Fluorescent Probes

Abstract

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3′ end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

Cancer-Associated Fibroblast Mimetic AIE Probe for Precision Imaging-Guided Full-Cycle Management of Ovarian Cancer Surgery

“…27 To avoid this drawback, CAF-probe uses AIE molecules to provide the fluorescence signal, which has the advantage that the higher the molecular aggregation, the stronger the fluorescence brightness. 28,29 P3-PPh 3 is a typical AIE molecule that we reported previously (Figure S1), 30 and the fluorescence brightness gradually increases as the aggregation degree of the molecule increases (Figure 1A). CAFs from different tumor tissue sources are specific for the corresponding cancer cells.…”

“…Cell membrane mimicry probes require the encapsulation of fluorescent dyes, which tend to accumulate in narrow spaces to induce aggregation-caused quenching and reduce the fluorescence brightness of the probes . To avoid this drawback, CAF-probe uses AIE molecules to provide the fluorescence signal, which has the advantage that the higher the molecular aggregation, the stronger the fluorescence brightness. , P3-PPh 3 is a typical AIE molecule that we reported previously (Figure S1), and the fluorescence brightness gradually increases as the aggregation degree of the molecule increases (Figure A). CAFs from different tumor tissue sources are specific for the corresponding cancer cells. , To ensure the specific targeting of the CAF-probe to ovarian cancer cells, we isolated and obtained CAFs from ovarian cancer tissues (Figure S2).…”

Ratiometric Fluorescence Biosensing of Tandem Biemissive Ag Clusters Boosted by Confined Catalytic DNA Assembly

“…The change of the DNA template component and length determines the clusters’ number and geometry and therefore electronic structure and adjusts their color and brightness. , The clustering scaffolds and recognizable elements can be readily designed and programmed in diverse DNA-based probes, structures, or devices. − Once presenting a specific target, the molecular binding event is activated to guide their conformation switches; thus, the template sequences are liberated or locked at precise positions to develop or dissociate these fluorescent adducts, further modulating their emission spectra for tailored signaling modes. For example, Miao’s group reported chameleon Ag clusters through the directional disassembly of hairpin structures to construct logic operations or ratiometric sensing platforms. , Owing to the intrinsic built-in correction capable of minimizing false positive backgrounds, dual-color DNA/AgCs as ratiometric signal reporters are more desirable for accurately sensing a univariate target than a single cluster emitter. − By encoding the host templates in a shared DNA tweezer or three-way junction, we have explored the ratiometric fluorescence of bicolor Ag clusters under a target-stimulated conformation switch. − The ability of two clusters with configuration responsiveness from molecule-recognizing events enables accurate fluorescent assay of analytes at the ultralow level. Even greater gains in sensitivity are achievable through combining a recognition process with isothermal amplification tools, such as RCA, CDA, and/or target recycling. ,, Predictably, the logic integration of the three events would provide great opportunities and accessibilities to encode the cluster-hosting templates and thus achieve a more rapid and efficient signal conversion and amplification.…”

Dual-emissive phenylalanine dehydrogenase-templated gold nanoclusters as a new highly sensitive label-free ratiometric fluorescent probe: heavy metal ions and thiols measurement with live-cell imaging

“…In the ratiometric uorescence system, the built-in self-calibration provided by calculating the intensity ratio of two uorescence signals can minimize the inuence of environmental factors and cause more accurate and sensitive detection. 41,42 However, most of the previously reported ratiometric uorescence sensors experience some practical disadvantages including interference between two uorescence peaks, the need for external uorophores, low biocompatibility, cytotoxicity, small Stokes shis and the need for surface modication. 41,43 It is interesting to note that only few researchers have reported ratiometric uorescence assays using enzyme-protected nanoclusters.…”

“…41,42 However, most of the previously reported ratiometric uorescence sensors experience some practical disadvantages including interference between two uorescence peaks, the need for external uorophores, low biocompatibility, cytotoxicity, small Stokes shis and the need for surface modication. 41,43 It is interesting to note that only few researchers have reported ratiometric uorescence assays using enzyme-protected nanoclusters. [43][44][45] Among all the analytes, detection of heavy metals such as Hg 2+ and Cu 2+ have received increasing attention due to their high toxicity to the natural environment and human health.…”