Point-of-care (POC) antigen detection plays a crucial role in curbing the spread of viruses. Paper-based fluorescence aptasensors are expected to offer a low-cost tool to meet the needs of decentralized POC diagnosis. Herein, we report on a fluorescent paper-based sensing system for detecting the SARS-CoV-2 spike protein. The sensing system was constructed by loading multi-layer Nb₂C MXene nano-quenchers and carbon-dot-labeled aptamer (G-CDs@Apt) probes onto a mixed cellulose ester (MCE) paper substrate. On the Nb₂C MXene/G-CDs@Apt sensing paper, abundant G-CDs@Apt probes were attached to the multilayer MXene nano-quenchers and kept in a fluorescence-off state, while recognition of the target detached the G-CDs@Apt probes formed the nano--quenchers, resulting in fluorescence recovery of the sensing paper. The developed paper-based sensor performed well in the one-step detection of the SARS-CoV-2 S1 protein with a detection limit of 0.067 ng/mL (0.335 pg/test). The assay exhibited good selectivity and anti-interference in the detection of the SARS-CoV-2 S1 protein in artificial saliva. Moreover, the paper-based aptasensor was successfully used to detect the SARS-CoV-2 S1 protein in actual environmental samples with recoveries of 90.87–100.55% and relative standard deviations of 1.52–3.41%. The proposed technology provides a cost-effective alternative to traditional antibody test strips for a wide range of POC diagnostic applications.

Ochratoxin A (OTA) is a mycotoxin widely found in foodstuffs such as cereal grains. It greatly threatens human health owing to its strong toxicity and high stability. Aptasensors have emerged as promising tools for the analysis of small molecule contaminants. Nucleic-acid-based signal amplification enables detectable signals to be obtained from aptasensors. However, this strategy often requires the use of complex primers or multiple enzymes, entailing problems such as complex system instability. Herein, we propose a fluorescent aptasensor for the ultrasensitive detection of OTA in cereal products, with signal amplification through RecJ_f exonuclease-assisted target recycling. The aptamer/fluorescein-labeled complementary DNA (cDNA-FAM) duplex was effectively used as the target-recognition unit as well as the potential substrate for RecJ_f exonuclease cleavage. When the target invaded the aptamer-cDNA-FAM duplex to release cDNA-FAM, RecJ_f exonuclease could cleave the aptamer bonded with the target and release the target. Thus, the target-triggered cleavage cycling would continuously generate cDNA-FAM as a signaling group, specifically amplifying the response signal. The proposed exonuclease-assisted fluorescent aptasensor exhibited a good linear relationship with OTA concentration in the range from 1 pg/mL to 10 ng/mL with an ultralow limit of detection (6.2 ng/kg of cereal). The analytical method showed that recoveries of the cereal samples ranged from 83.7 to 109.3% with a repeatability relative standard deviation below 8%. Importantly, the proposed strategy is expected to become a common detection model because it can be adapted for other targets by replacing the aptamer. Thus, this model can guide the development of facile approaches for point-of-care testing applications.

Antigen detection provides rapid and convenient diagnosis of respiratory infections. This study develops an innovative dual-fluorescence aptasensing method based on polydopamine-functionalized MXene (PDA-MXene) for the simultaneous detection of spike protein and hemagglutinin protein. The method employs green- and red-emitting quantum dot (QD) probes as fluorescence reporters, and the PDA-MXene as an effective adsorption and separation substrate. Coupled with a centrifugation-assisted separation strategy, this design method reduces background interference and enhances detection reliability. The method demonstrates good analytical performance, with detection limits of 0.82 ng/mL for spike protein and 2.11 ng/mL for hemagglutinin protein in single-channel mode. The dual-channel mode enables reliable and simultaneous quantification of both target proteins with minimal spectral cross-talk. Furthermore, this method exhibits high specificity against interferents including ions, proteins, and toxins. Artificial saliva, chosen as real sample, is spiked with target proteins to investigate the practical applicability of the method, showing recovery rates for both target proteins between 100 and 114 sensing strategy is simple to operate and allows the detection of new targets by simply replacing the azide-modified aptamer lyophilized powder. It therefore holds promising application for the simultaneous detection of multiple proteins in point-of-care testing and health monitoring fields.