CRISPR/Cas12a-Responsive Smart DNA Hydrogel for Sensitive Electrochemiluminescence Detection of the Huanglongbing Outer Membrane Protein Gene.

Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 - I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.

H2O2 self-supplying and GSH-depleting nanosystem for amplified NIR mediated-chemodynamic therapy of MRSA biofilm-associated infections.

Reactive oxygen species (ROS) has emerged as potent therapeutic agents for biofilm-associated bacterial infections. Chemodynamic therapy (CDT), involving the generation of high-energy ROS, displays great potential in the therapy of bacterial infections. However, challenges such as insufficient hydrogen peroxide (H2O2) and over-expressed glutathione (GSH) levels within the microenvironment of bacterial biofilms severely limit the antibacterial efficacy of CDT. Herein, we have developed a multifunctional nanoplatform (CuS@CaO2@Dex) by integrating copper sulfide (CuS) and calcium peroxide (CaO2) into dextran (Dex)-coated nanoparticles. This innovative platform enhanced ROS generation for highly efficient biofilm elimination by simultaneously supplying H2O2 and depleting GSH. The Dex-coating facilitated the penetrability of CuS@CaO2@Dex into biofilms, while CaO2 generated a substantial amount of H2O2 in the acidic biofilm microenvironment. CuS, through a Fenton-like reaction, catalyzed the conversion of self-supplied H2O2 into hydroxyl radicals (•OH) and consumed the overexpressed GSH. Additionally, the incorporation of near-infrared II (NIR II) laser irradiation enhanced the photothermal properties of CuS, improving the catalytic efficiency of the Fenton-like reaction for enhanced antibacterial effects. In vivo experiments have demonstrated that CuS@CaO2@Dex exhibited remarkable antibacterial and antibiofilm efficacy, exceptional wound healing capabilities, and notable biosafety. In summary, the Dex-coated nanoplatform proposed in this study, with its self-sterilization capability through ROS, holds significant potential for future biomedical applications.

Graphene Oxide Nanoparticles Combined with CRISPR/Cas9 System Enable Efficient Inhibition of Pseudorabies Virus.

We describe an application where graphene oxide nanoparticles (GONs) enable combined inhibition of Pseudorabies Virus (PRV) through delivery of a CRISPR/Cas9 system for targeted cleaving of a PRV genome and direct interaction with viral particles. The sheeted GONs could load CRISPR plasmid DNA (pDNA) to form a small sized, near-spheroidal GONs-CRISPR complex, which enables CRISPR pDNA efficient intracellular delivery and transient expression under serum conditions. Cell studies showed that GONs-CRISPR could allow rapid cellular uptake, endolysosomes escape, and nucleus transport within 3 h. Virus studies demonstrated that the pure GONs have antiviral activity and GONs-CRISPR could significantly inhibit PRV replication and result in progeny PRV decreasing by approximately 4000 times in infected host cells. Transmission electron microscopy (TEM) imaging showed that GONs-CRISPR could destroy the PRV structures by directly interacting with viral particles. This GONs-based strategy may extend the advanced application of the CRISPR system for antiviral action.

Dual-Mode Immunosensor for Electrochemiluminescence Resonance Energy Transfer and Electrochemical Detection of Rabies Virus Glycoprotein Based on Ru(bpy)32+-Loaded Dendritic Mesoporous Silica Nanoparticles.

Rabies is a serious zoonotic disease in almost all warm-blooded animals and causes fatal encephalitis. The detection of rabies virus (RABV) is critical and remains a significant challenge. Herein, an electrochemiluminescence resonance energy transfer (ECL-RET) and electrochemical (EC) dual-mode immunosensor was developed for highly sensitive detection of RABV glycoprotein. Dendritic mesoporous silica nanoparticles (DMSNs) were employed to load Ru(bpy)32+ and to obtain ECL probes (Ru@DMSNs). Ru@DMSNs were decorated on the electrode surface, followed by the modification of the RABV antibody (Ab1). RABV was specifically recognized and captured by Ab1, causing the decline of the ECL signal due to the obstruction of electron transfer. Additionally, manganese oxide nanoparticles (MnOx) modified with Ab2 can further quench the ECL signal of Ru@DMSNs via the RET between Ru@DMSNs and MnOx. Meanwhile, MnOx can catalyze the oxidation of o-phenylenediamine (o-PD), generating a significant differential pulse voltammetry (DPV) signal as a second signal to monitor RABV glycoprotein concentration. Consequently, an immunosensor was developed to achieve dual-signal detection of RABV and improve reliability. Under the optimal conditions, detection ranges of 0.10 pg·mL-1 to 10 ng·mL-1 for ECL (with an 88 fg·mL-1 detection limit) and 1 pg·mL-1 to 2 ng·mL-1 for EC (with a 0.1 pg·mL-1 detection limit) were obtained for RABV detection. The reliability of this immunoassay was validated by eight brain tissue samples. The results were found to be compatible with the results of the real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, indicating the potential applicability of this method for RABV diagnosis.

Biomimetic Nanoplatelets to Target Delivery Hirudin for Site-Specific Photothermal/Photodynamic Thrombolysis and Preventing Venous Thrombus Formation.

Due to the high recurrence rate and mortality of venous thrombosis, there is an urgent need for research on antithrombotic strategies. Because of the short half-life, poor targeting capabilities, bleeding complications, and neurotoxic effects of conventional pharmacological thrombolysis methods, it is essential to develop an alternative strategy to noninvasive thrombolysis and decrease the recurrence rate of venous thrombosis. A platelet-mimetic porphyrin-based covalent organic framework-engineered melanin nanoplatform, to target delivery of hirudin to the vein thrombus site for noninvasive thrombolysis and effective anticoagulation, is first proposed. Owing to the thrombus-hosting properties of platelet membranes, the nanoplatform can target the thrombus site and then activate hyperthermia and reactive oxygen species for thrombolysis under near-infrared light irradiation. The photothermal therapy/photodynamic therapy combo can substantially improve the effectiveness (85.7%) of thrombolysis and prevent secondary embolism of larger fragments. Afterward, the highly loaded (97%) and slow-release hirudin (14 days) are effective in preventing the recurrence of blood clots without the danger of thrombocytopenia. The described biomimetic nanostructures offer a promising option for improving the efficacy of thrombolytic therapy and reducing the risk of bleeding complications in thrombus associated diseases.

Genome-wide association study reveals the genetic architecture of 27 agronomic traits in tomato.

Tomato (Solanum lycopersicum) is a highly valuable fruit crop, and yield is one of the most important agronomic traits. However, the genetic architecture underlying tomato yield-related traits has not been fully addressed. Based on ∼4.4 million single nucleotide polymorphisms obtained from 605 diverse accessions, we performed a comprehensive genome-wide association study for 27 agronomic traits in tomato. A total of 239 significant associations corresponding to 129 loci, harboring many previously reported and additional genes related to vegetative and reproductive development, were identified, and these loci explained an average of ∼8.8% of the phenotypic variance. A total of 51 loci associated with 25 traits have been under selection during tomato domestication and improvement. Furthermore, a candidate gene, Sl-ACTIVATED MALATE TRANSPORTER15, that encodes an aluminum-activated malate transporter was functionally characterized and shown to act as a pivotal regulator of leaf stomata formation, thereby affecting photosynthesis and drought resistance. This study provides valuable information for tomato genetic research and breeding.

Novel approach to enhance Bradyrhizobium diazoefficiens nodulation through continuous induction of ROS by manganese ferrite nanomaterials in soybean.

BACKGROUND: The study of symbiotic nitrogen fixation between (SNF) legumes and rhizobia has always been a hot frontier in scientific research. Nanotechnology provides a new strategy for biological nitrogen fixation research. However, how to construct abiotic nano-structure-biological system, using the special properties of nanomaterials, to realize the self-enhancement of biological nitrogen fixation capacity is important. RESULTS: In order to construct a more efficient SNF system, in this study, we applied manganese ferrite nanoparticles (MF-NPs) with sustainable diatomic catalysis to produce reactive oxygen species (ROS), thus regulating the nodulation pathway and increasing the number of nodules in soybean (Glycine max), eventually enhancing symbiotic nitrogen fixation. Symbiosis cultivation of MF-NPs and soybean plants resulted in 50.85% and 61.4% increase in nodule weight and number, respectively, thus inducing a 151.36% nitrogen fixation efficiency increase, finally leading to a 25.70% biomass accumulation increase despite no substantial effect on the nitrogenase activity per unit. Transcriptome sequencing analysis showed that of 36 differentially expressed genes (DEGs), 31 DEGs related to soybean nodulation were upregulated in late rhizobium inoculation stage (12 d), indicating that the increase of nodules was derived from nodule-related genes (Nod-R) continuous inductions by MF-NPs. CONCLUSIONS: Our results indicated that the nodule number could be effectively increased by extending the nodulation period without threatening the vegetative growth of plants or triggering the autoregulation of nodulation (AON) pathway. This study provides an effective strategy for induction of super-conventional nodulation.

Efficient Gene Silencing in Intact Plant Cells Using siRNA Delivered By Functional Graphene Oxide Nanoparticles.

Delivery of small interfering RNA (siRNA) to intact plants for gene silencing mainly relies on viral vectors and Agrobacterium-mediated transformation due to the barrier of intact plant cell wall. Here, we reported that polymer functionalized graphene oxide nanoparticles (GONs) enable siRNA transfer into intact plant cells and bring about efficient gene silencing. We found that sheeted GONs could efficiently load siRNA to form small sized, near-spheroidal GONs-siRNA complex, which could be across the cell wall and internalize in the plant cell. The GONs-siRNA exhibited transient and strong silencing (97.2 % efficiency) in plant tissues at 24 h after treatment and returned to normal level at 5 days after treatment. This method has the obvious advantages of efficient, transient, simple, stability and well biocompatibility, which should greatly stimulate the application of nanomaterials as gene-engineering tools in plant research.

Novel Porphyrin Zr Metal-Organic Framework (PCN-224)-Based Ultrastable Electrochemiluminescence System for PEDV Sensing.

The sensitive detection of coronavirus is of vital importance for the prevention of its rapid spread. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets. In this work, a novel PCN-224-based electrochemiluminescence (ECL) system was constructed for PEDV detection with high sensitivity. We found that PCN-224 can be employed as an ECL reporter with a strong signal because of its zirconium-based organic porous frame nanomaterial with a large specific surface area and stable structure. TiO2 nanoparticles were used as an accelerator for the first time to promote the reduction of coreactant potassium peroxydisulfate on the cathode; thus, the initial ECL signal of PCN-224 was significantly amplified. In the presence of PEDV, the ECL signal decreased due to the block effect to electron transfer. As a result, the novel "signal off" biosensor achieved a sensitive detection of PEDV ranging from 1 pg/mL to 10 ng/mL, with a detection limit of 0.4 pg/mL (S/N = 3). Importantly, the PCN-224 nanomaterial enriched the ECL system in biological analysis, and the proposed strategy provided a new route for coronavirus detection.

Gastric Acid Powered Nanomotors Release Antibiotics for In Vivo Treatment of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) infection has ≈75% probability of causing gastric cancer, so it is considered to be the strongest single risk factor for gastric malignancies. However, the harsh gastric acid environment has created obstacles to medical treatment. This work reports a nanomotor with a bottle-shaped container that can be loaded with small molecules of clarithromycin, nano calcium peroxide (CaO2 ), and Pt nanoparticles (Pt NPs) by ultrasound. Nanomotors can quickly consume gastric acid through the chemical reaction of CaO2 to temporarily neutralize gastric acid. The product hydrogen peroxide (H2 O2 ) is catalytically decomposed into a large amount of oxygen (O2 ) by Pt NPs. The local concentration gradient of O2 bubbles causes it to be expelled from the nanobottles through a narrow opening, and then push the nanobottles forward to provide maximum release and prodrug efficacy. Experiments in animal models show that 15 mg nanomotors can safely and quickly neutralize gastric acid in the stomach and simultaneously release prodrugs to achieve good therapeutic effects without causing acute toxicity. H. pylori burden in mice was 2.6 orders of magnitude lower than that in the control group. The stomach returns to normal pH within 1 d after administration.

A portable SERS reader coupled with catalytic hairpin assembly for sensitive microRNA-21 lateral flow sensing.

Nucleic acid lateral flow sensing has drawn great research attention since it has the advantages of being simple, rapid, and cost-effective. However, considering the trace amounts of the nucleic acid targets, its sensitivity is still limited. Although enormous efforts have been devoted to enhancing its sensitivity, developing a simple lateral flow sensing platform with high sensitivity remains challenging. We report a novel lateral flow microRNA-21 biosensing platform based on a portable surface enhanced Raman scattering (SERS) reader coupled with a catalytic hairpin assembly signal amplification strategy. Hairpin DNA probes were anchored on Au@Ag nanotags, and the presence of microRNA-21 triggered the formation of numerous double-stranded DNAs along with the exposure of the biotin groups. By this means, the target was recycled and signal amplification was achieved. The Au@Ag nanoprobes with exposed biotin can be captured on the test line via its interaction with streptavidin. By scanning the strip with a portable SERS reader, the sensitive quantification of microRNA-21 was realized with a detection limit as low as 84 fM. The proposed strategy was employed to detect the target in a serum sample, demonstrating its great potential in amplified point-of-care biosensing for clinical diagnosis.

A novel signal amplified electrochemiluminescence biosensor based on MIL-53(Al)@CdS QDs and SiO2@AuNPs for trichlorfon detection.

An ultrasensitive electrochemiluminescence (ECL) biosensor was developed based on MIL-53(Al)@CdS QDs and SiO2@AuNPs for trichlorfon detection. Metal-organic frameworks (MOFs) were used as a loading platform that provided a large surface area to load targets and modified materials onto the electrode. At the same time, SiO2@AuNPs loaded plenty of AuNPs which effectively increased the ECL resonance energy transfer between the CdS QDs, so that the ECL signal was strongly quenched and resulted in an amplified response. In the range of 10-11-10-4 M, the ECL response showed a linear relationship with the concentration (logarithm) of trichlorfon, and the detection limit was 5.1 × 10-12 M (S/N = 3). When the biosensor was applied to detect trichlorfon in lettuce, broccoli, cucumber, and chives, the recoveries obtained from the spiked samples were 97%-105%, 102%-104%, 100%-104%, and 98%-104%, respectively. Thus, this novel ECL biosensor has potential applications for the analysis of trichlorfon in food samples.

Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model.

Rabies, which is caused by rabies virus (RABV), poses an ever-present threat to public health in most countries of the world. Once clinical signs appear, the mortality of rabies approaches 100%. To date, no effective method for early rabies diagnosis has been developed. In this study, an RPA-CRISPR nucleic-acid-based assay was developed for early rabies diagnosis by detecting viral RNA shedding in the cerebrospinal fluid (CSF) of rats. This method can detect a single copy of RABV genomic RNA in 1 µL of liquid. RABV genomic RNA released from viral particles in the CSF could be detected via RPA-CRISPR as early as 3 days postinfection in a rat model. This study provides an RPA-CRISPR technique for early detection of RABV with potential application in the clinical diagnosis of human rabies.

Programmable DNA Tweezer-Actuated SERS Probe for the Sensitive Detection of AFB1.

A DNA tweezer is a dynamic DNA nanomachine that can reversibly switch its state between open and closed. Here, we employed a DNA tweezer for the first time to dynamically control the distance between plasmonic silver nanoparticles (Ag NPs) for a surface enhanced Raman scattering (SERS) biosensing application. Two DNA and 4-nitrothiophenol (4-NTP) modified Ag NPs were linked to the arms of the DNA tweezer (DNA tweezer-Ag NPs probe) by complementary base pairing. Activation of the Raman intensity was achieved by the state transformation of the DNA tweezer-Ag NPs probe from open to closed. The distances between two Ag NPs in open and closed state were 8.1 ± 2.7 nm and 3.2 ± 0.8 nm, respectively. Furthermore, the two Ag NPs were spatially separated in the open state with a low Raman signal, whereas in the closed state, Raman intensity was enhanced because of the proximity of two Ag NPs. The developed biosensing system exhibited a good linear relationship when the concentration of aflatoxin B1 (AFB1) ranged from 1 ng/mL to 0.01 pg/mL, and the limit of detection (LOD) was 5.07 fg/mL. In addition, spike recovery and certificated real foodstuffs were used to examine the feasibility in a real situation. This protocol provides a potential candidate for SERS detection and can be used as a promising technology for biological and chemical sensors.

Pomegranate-Inspired Silica Nanotags Enable Sensitive Dual-Modal Detection of Rabies Virus Nucleoprotein.

The outbreak of rabies virus (RABV) in Asia and Africa has attracted widespread concern due to its 100% mortality rate, and RABV detection is crucial to its diagnosis and treatment. Herein, we report a sensitive and reliable strategy for the dual-modal RABV detection using pomegranate-shaped dendritic silica nanospheres fabricated with densely incorporated quantum dots (QDs) and horseradish peroxidase (HRP)-labeled antibody. The immunoassay involves the specific interaction between virus and nanospheres-conjugated antibody coupled with robust fluorescence signal originating from QDs and naked-eye discernible colorimetric signal on the oxTMB. The ultrahigh loading capacity of QDs enables the detection limit down to 8 pg/mL via fluorescence modality, a 348-fold improvement as compared with conventional enzyme-linked immunosorbent assay (ELISA). In addition, the detection range was from 1.20 × 102 to 2.34 × 104 pg/mL by plotting the absorbance at 652 nm with RABV concentrations with a detection limit of 91 pg/mL, which is nearly 2 order of magnitude lower than that of the conventional ELISA. Validated with 12 brain tissue samples, our immunoassay results are completely consistent with polymerase chain reaction (PCR) results. Compared with the PCR assay, our approach requires no complex sample pretreatments or expensive instruments. This is the first report on RABV diagnosis using nanomaterials for colorimetry-based prescreening and fluorescence-based quantitative detection, which may pave the way for virus-related disease diagnosis and clinical analysis.

Miniature Hollow Gold Nanorods with Enhanced Effect for In Vivo Photoacoustic Imaging in the NIR-II Window.

The miniaturization of gold nanorods exhibits a bright prospect for intravital photoacoustic imaging (PAI) and the hollow structure possesses a better plasmonic property. Herein, miniature hollow gold nanorods (M-AuHNRs) (≈46 nm in length) possessing strong plasmonic absorbance in the second near-infrared (NIR-II) window (1000-1350 nm) are developed, which are considered as the most suitable range for the intravital PAI. The as-prepared M-AuHNRs exhibit 3.5 times stronger photoacoustic signal intensity than the large hollow Au nanorods (≈105 nm in length) at 0.2 optical density under 1064 nm laser irradiation. The in vivo biodistribution measurement shows that the accumulation in tumor of miniature nanorods is twofold as high as that of the large counterpart. After modifying with a tumor-targeting molecule and fluorochrome, in living tumor-bearing mice, the M-AuHNRs group gives a high fluorescence intensity in tumors, which is 3.6-fold that of the large ones with the same functionalization. Moreover, in the intravital PAI of living tumor-bearing mice, the M-AuHNRs generate longer-lasting and stronger photoacoustic signal than the large counterpart in the NIR-II window. Overall, this study presents the fabrication of M-AuHNRs as a promising contrast agent for intravital PAI.

Kanamycin Adsorption on Gold Nanoparticles Dominates Its Label-Free Colorimetric Sensing with Its Aptamer.

A short kanamycin-binding aptamer has been widely used for detecting kanamycin. One of the popular signaling methods is based on the color change of gold nanoparticles (AuNPs) to develop label-free colorimetric biosensors. The general perception was that aptamer binding to its target would inhibit aptamer adsorption by the AuNPs. This inhibited adsorption results in the aggregation of the AuNPs and a color change upon addition of salt. However, the potential adsorption of kanamycin was ignored. Herein, we carefully studied the adsorption of kanamycin on AuNPs and performed a comprehensive analysis using two mutated aptamers and a randomly sequenced DNA which were not supposed to bind kanamycin. In addition, a total of six antibiotics were studied over a wide concentration range. As low as 90 nM kanamycin can induce the aggregation of 3 nM citrate-capped AuNPs, indicating very strong adsorption of kanamycin. The color change was independent of DNA sequence, and all the tested sequences showed a similar color response, regardless of aptamer. Among the different antibiotics, kanamycin and streptomycin induced a color change but not the other four. Our results support an alternative mechanism that kanamycin and streptomycin adsorption by the AuNPs was the main reason for the color change instead of aptamer binding.

Assembling PVP-Au NPs as portable chip for sensitive detection of cyanide with surface-enhanced Raman spectroscopy.

Cyanide (C≡N) can lead to blood, cardiovascular system, and nervous system disorders owing to the acute and chronic toxicity; thus, aiming at the group or individual poisoning incidents, it is necessary to develop the sensitive and credible method for rapid on-site detection of poisons cyanide. Surface-enhanced Raman spectroscopy (SERS) with the advantages of providing fingerprint information of target molecules and single-molecules sensitivity has been widely used in on-site analysis; however, the SERS measurements always suffer from the problem of the stability of substrates. Here, the polyvinylpyrrolidone (PVP)-stabilized Au NPs (PVP-Au NPs) have been assembled through the simple, convenient evaporation-induced strategy with the large-scale hotspots substrates. The presence of PVP can not only facilitate the assembly of Au NPs but also prevent the corrosion of CN- towards the Au NPs with the formation of [Au (CN)2]-1, providing high stable and reproducible SERS signals. Moreover, the PVP-Au NPs have been assembled on the Si wafer to fabricate the portable SERS chip for rapid on-site detection of CN- with an RSD of 5.8% and limitation of 100 ppb. Furthermore, by coupling a portable Raman spectrometer, the SERS spectra of CN- spiked into different specimens to simulate the poison samples have been collected and analyzed on SERS chips with the recovery of 89-103% and RSD not higher than 11.3%. Consequently, the fabricated SERS chip with assembled PVP-Au NPs can provide sensitive and credible detection for CN- in different specimens, and then would satisfy the rapid on-site evaluation of CN- in poisoning incidents with the portable Raman spectrometer. Graphical Abstract.

Bioapplications of DNA nanotechnology at the solid-liquid interface.

DNA nanotechnology engineered at the solid-liquid interface has advanced our fundamental understanding of DNA hybridization kinetics and facilitated the design of improved biosensing, bioimaging and therapeutic platforms. Three research branches of DNA nanotechnology exist: (i) structural DNA nanotechnology for the construction of various nanoscale patterns; (ii) dynamic DNA nanotechnology for the operation of nanodevices; and (iii) functional DNA nanotechnology for the exploration of new DNA functions. Although the initial stages of DNA nanotechnology research began in aqueous solution, current research efforts have shifted to solid-liquid interfaces. Based on shape and component features, these interfaces can be classified as flat interfaces, nanoparticle interfaces, and soft interfaces of DNA origami and cell membranes. This review briefly discusses the development of DNA nanotechnology. We then highlight the important roles of structural DNA nanotechnology in tailoring the properties of flat interfaces and modifications of nanoparticle interfaces, and extensively review their successful bioapplications. In addition, engineering advances in DNA nanodevices at interfaces for improved biosensing both in vitro and in vivo are presented. The use of DNA nanotechnology as a tool to engineer cell membranes to reveal protein levels and cell behavior is also discussed. Finally, we present challenges and an outlook for this emerging field.

Inhibition of Porcine Epidemic Diarrhea Virus Replication and Viral 3C-Like Protease by Quercetin.

For the last decade, porcine epidemic diarrhea virus (PEDV) variant strains have caused severe damage to the global pig industry. Until now, no effective antivirals have been developed for the therapeutic treatment of PEDV infection. In the present study, we found that quercetin significantly suppressed PEDV infection at noncytotoxic concentrations. A molecular docking study indicated that quercetin might bind the active site and binding pocket of PEDV 3C-like protease (3CLpro). Surface plasmon resonance (SPR) analysis revealed that quercetin exhibited a binding affinity to PEDV 3CLpro. Based on the results of the fluorescence resonance energy transfer (FRET) assay, quercetin was proven to exert an inhibitory effect on PEDV 3CLpro. Since coronavirus 3CLpro is an important drug target and participates in the viral replication process, quercetin should be developed as a novel drug in the control of PEDV infection.