Fluorescence Signal Amplification Research Articles

Sensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.

GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.

Fluorescent biosensor based on magnetic separation platform and spore-like breakable organosilica nanocapsules controlled-release carbon dots for the detection of Escherichia coli O157:H7

Ultrasensitive and rapid detection of low concentration of Escherichia coli O157: H7 (E. coli O157:H7) in food is essential for food safety and public health. In this study, A novel fluorescence signal amplification biosensor based on magnetic separation platform and red fluorescent carbon dots (R-CDs)-encapsulated breakable organosilica nanocapsules (BONs) for ultrasensitive detection of E. coli O157:H7 was established. Wulff-type boronic acid functionalized magnetic nanoparticles (MNPs@B–N/APBA) with broad-spectrum bacterial recognition ability were synthesized for the first time to recognize and capture E. coli O157: H7 in food samples. R-CDs@BONs labeled with anti-E. coli O157:H7 monoclonal antibody (mAb@R-CDs@BONs-NH2) were used as the second recognition element to ensure the specificity for E. coli O157:H7 and form MNPs@B–N/APBA∼ E. coli O157:H7∼mAb@R-CDs@BONs-NH2 sandwich complexes, followed by releasing R-CDs to generate amplified fluorescence response signals for quantitative detection of E. coli O157:H7. The proposed method had a limit of detection with 25 CFU/mL in pure culture and contaminated lettuce samples, which the whole detection process took about 120 min. This fluorescence signal amplification biosensor has the potential to detect other pathogens in food by altering specific antibodies.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

A novel entropy-driven dual-output mode integrated with DNAzyme for enhanced microRNA detection

Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg2+-dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application.

Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Multiply Guaranteed Catalytic DNA Circuit for Cancer-Cell-Selective Imaging of miRNA and Robust Evaluation of Drug Resistance.

Catalytic DNA circuits are desirable for sensitive bioimaging in living cells; yet, it remains a challenge to monitor these intricate signal communications because of the uncontrolled circuitry leakage and insufficient cell selectivity. Herein, a simple yet powerful DNA-repairing enzyme (APE1) activation strategy is introduced to achieve the site-specific exposure of a catalytic DNA circuit for realizing the selectively amplified imaging of intracellular microRNA and robust evaluation of the APE1-involved drug resistance. Specifically, the circuitry reactants are firmly blocked by the enzyme recognition/cleavage site to prevent undesirable off-site circuitry leakage. The caged DNA circuit has no target-sensing activity until its circuitry components are activated via the enzyme-mediated structural reconstitution and finally transduces the amplified fluorescence signal within the miRNA stimulation. The designed DNA circuit demonstrates an enhanced signal-to-background ratio of miRNA assay as compared with the conventional DNA circuit and enables the cancer-cell-selective imaging of miRNA. In addition, it shows robust sensing performance in visualizing the APE1-mediated chemoresistance in living cells, which is anticipated to achieve in-depth clinical diagnosis and chemotherapy research.

Activity-Based Fluorescence Diagnostics for Cancer.

Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.

“Two in one”: A novel DNA cascade amplification strategy for trace detection of dual targets

According to the characteristics of DNA programming, the cascaded nucleic acid amplification technology with larger output can overcome the problem of insufficient sensitivity of single nucleic acid amplification technology, and it combines the advantages of two or even multiple nucleic acid amplification technologies at the same time. In this work, a novel cascade signal amplification strategy with strand displacement amplification (SDA) and cascade hybridization chain reaction (HCR) was proposed for trace detection of hAAG and VEGF165. HAAG-induced SDA produced a large amount of S2 to open H2 on Polystyrene (PS) nanospheres, thereby triggering cascade HCR to form DNA dendritic nanostructures with rich fluorescence (FL) signal probes (565 nm). It could realize the amplification of FL signals for the detection of hAAG. Moreover, many doxorubicin (Dox) were loaded into the GC bases of DNA dendritic nanostructures, and its FL signal was effectively shielded. VEGF165 specifically bound to its aptamer to form G-quadruplex structures, which released Dox to produce a high FL signal (590 nm) for detection of VEGF165. This work developed a unique multifunctional DNA dendritic nanostructure fluorescence probe, and cleverly designed a new “On-off” switch strategy for sensitive trace detection of cancer markers.

Self-Feedback DNAzyme Motor for Cascade-Amplified Imaging of mRNA in Live Cells and In Vivo.

DNA motors have attracted extensive interest in biosensing and bioimaging. However, the amplification capacity of the existing DNA motor systems is limited since the products from the walking process are unable to feedback into the original DNA motor systems. As a result, the sensitivities of such systems are limited in the contexts of biosensing and bioimaging. In this study, we report a novel self-feedback DNAzyme motor for the sensitive imaging of tumor-related mRNA in live cells and in vivo with cascade signal amplification capacity. Gold nanoparticles (AuNPs) are modified with hairpin-locked DNAzyme walker and track strands formed by hybridizing Cy5-labeled DNA trigger-incorporated substrate strands with assistant strands. Hybridization of the target mRNA with the hairpin strands activates DNAzyme and promotes the autonomous walking of DNAzyme on AuNPs through DNAzyme-catalyzed substrate cleavage, resulting in the release of many Cy5-labeled substrate segments containing DNA triggers and the generation of an amplified fluorescence signal. Moreover, each released DNA trigger can also bind with the hairpin strand to activate and operate the original motor system, which induces further signal amplification via a feedback mechanism. This motor exhibits a 102-fold improvement in detection sensitivity over conventional DNAzyme motors and high selectivity for target mRNA. It has been successfully applied to distinguish cancer cells from normal cells and diagnose tumors in vivo based on mRNA imaging. The proposed DNAzyme motor provides a promising paradigm for the amplified detection and sensitive imaging of low-abundance biomolecules in vivo.

DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Host-guest synergistic hydrogen bond triggered signal amplification for visualizing the plant hormone salicylic acid

Salicylic acid (SA), the crucial phytohormone, is biosynthesized by plants in response to fight against multifarious phytopathogens and is a central mediator for regulating plant physiology and pathology processes including biotic and abiotic stress. However, current research still lacks of effective and simple tactics to track SA for understanding the underlying mechanisms in plants. Herein, we constructed a supramolecular host–guest nanoprobe (RAD&CB[7]) based on cucurbit[7]uril (CB[7]) and adamantane (AD)-modified rhodamine derivative, the nanoprobe selectivity and sensitivity for SA were very satisfactory. The nanoprobe RAD&CB[7] has been employed to (1) improve the fluorescent signal for detecting SA about 330-fold at lower concentration of SA (20 μM) in vitro, which is very difficult to realize by previous SA fluorescent probes. The density functional theory (DFT) calculations demonstrated the signal amplification is significantly higher than other fluorescent probes reported in literature due to the synergetic effects of host–guest and hydrogen-bond interaction. (2) realize the detection of SA in living Nicotiana glutinosa L. callus, Arabidopsis thaliana, and tomato seedlings even at ultra-low concentrations of RAD&CB[7] (minimum 1 μM) and SA (minimum 5 μM), compared with previous probes (minimum 10 μM) for detecting SA (minimum 25 μM) in vivo, thus realized quantitatively detected SA in callus and Arabidopsis thaliana roots. (3) verify SA-induced stomatal closure in the leaves of Arabidopsis thaliana by host–guest fluorescent probe technology for the first time. The remarkable in vitro and in vivo performance make this fluorescence signal amplification strategy a powerful tool for studying SA-related physiology and pathology in biological processes and discovering novel immune activators for crop protection.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs featureimproved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are providedby a double-stranded DNA fragment,a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and applicationin nucleic acid detection assays.

Development of a Catalytic Hairpin Assembly-Based Fluorescent Assay for the Rapid Detection of SARS-CoV-2 Target RNA

To develop a catalytic hairpin assembly (CHA)-based fluorescent assay for the detection of the target RNA of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), so as to realize the rapid nucleic acid testing of SARS-CoV-2. A 24-nt segment of the SARS-CoV-2 nucleocapsid protein gene (N gene, NC_045512.2) was chosen as the target RNA and the hairpin motif 1 (H1) and hairpin motif 2 (H2) were designed based on the principle of CHA reaction. The H1 motif was labelled with a fluorophore group as well as a quencher group. When the target RNA was added to the hairpin motifs, CHA reaction was triggered at room temperature (25 ℃), which led to the amplification of fluorescence signal, thereby enabling the rapid detection of the target RNA. After the optimization of the hairpin motifs and the experimental conditions, the sensitivity and the specificity of the testing method were measured to evaluate its performance. We successfully constructed a CHA-based fluorescent assay specifically for the target RNA of SARS-CoV-2. With this method, testing could be completed at room temperature within 30 min. This testing method exhibited excellent specificity and could be used to accurately distinguish the perfectly-matched target RNA from the target RNA with single-base mutations. In addition, the testing method demonstrated good sensitivity, with a detection limit of 50 pmol/L. The proposed assay enables the simple and rapid detection of the SARS-CoV-2 target RNA with excellent sensitivity and specificity, showing great promise for further optimization and subsequent clinical application for the rapid detection of SARS-CoV-2 nucleic acid.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy

BackgroundDNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. ResultsWe designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1–10,000 pg mL −1 and 10–100,000 pg mL −1, respectively, with corresponding detection limits of 0.933 pg mL −1 and 1.07 pg mL −1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SignificanceThe constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

An enzyme-free and label-free ratiometric fluorescence signal amplification biosensor based on DNA-silver nanoclusters and catalytic hairpin assembly for tetracycline detection

Sensitive and accurate in antibiotic detection is crucial for food security, environmental conservation, public health, and environmental protection. In this study, we developed an enzyme-free and label-free ratiometric fluorescence biosensor for highly sensitive detection of tetracycline (TET). This biosensor utilized a combination of target-triggered catalytic hairpin assembly (CHA) and DNA-silver nanocluster (DNA-AgNCs) dual-color fluorescent signal probes. Upon TET binding to the aptamer, it initiated the leakage of CHA initiator, resulting in the formation of numerous double-stranded DNA complexes. This process exposed the foothold sequence of hairpin-structured DNA-AgNCs (H3-AgNCs) that exhibit yellow fluorescence. Subsequently, the conformational changes in H3-AgNCs significantly reduced the yellow fluorescence intensity (FI) and reveal the auxiliary DNA-AgNCs (a-DNA-AgNCs) linkage sequence. The binding of a-DNA-AgNCs brought two dark AgNCs close, forming nanocluster dimers emitting red fluorescence. The results demonstrate that the variation in ratiometric signal is linear with TET concentration between 0.1 and 50 ng/mL, with a detection limit as low as 0.086 ng/mL. Additionally, this approach exhibited good recovery results with spiked milk samples. This enzyme-free and label-free combined signal amplification method with proportional signal output based on DNA-AgNCs provides valuable empirical reference for creating low-cost, stable, and highly-sensitive biosensors.

A target-triggered mimic nucleic acid enzyme enables versatile and sequence-independent biosensing

Simultaneous quantification of multiple microRNAs (miRNAs) is very important because lots of pathologies are associated with abnormal expressions of several miRNAs. In this work, we proposed an enzyme-assisted cleavage of nucleic acids signal amplification (TMNAE enzyme sensing) based method enable simple, highly sensitive and selective multiple miRNAs detection. Two unmodified probes and analyte were assembled into a Y-shaped structure that contains a nicking endonuclease recognition site in the complementary region of the unmodified probe and two side arms complementary to reporter probe. Thus, it enables the loading of a Nicking endonuclease (NEase) on the complementary region of the unmodified probe, localization to a specific locus through hybridization of the side arms with reporter probe, and cleavage thereof. NEase is employed to recycle the process of Y-shaped structure assisted digestion of reporter probe, thus, resulting in a significant fluorescence signal amplification through which one analyte molecule cleaves thousands of reporter probe molecules. We demonstrate the efficiency of this TMNAE enzyme sensing strategy for rapid direct quantification of multiple sequence specific miRNAs in homogeneous solution with the detection limit in the femtomolar range, nearly 4 orders of magnitude lower than that of conventional hybridization assay methods. This assay also has the ability to discriminate the target from other miRNAs or even single-base mismatched sequences. Moreover, the TMNAE enzyme sensing strategy can be used to detect other molecules apart from nucleic acids. The detection of thrombin in biological samples showed a LOD of as low as 1 nM. Overall, our proposed TMNAE enzyme sensing strategy provides a versatile, reliable, simple, highly sensitive, and selective detection method and shows promising application in molecular disease diagnosis and biomedicine.

Primer exchange reaction-coupled transcription isothermal amplification as a sensitive biomolecular assay.

We devised a novel strategy that relies on a combination of the primer exchange reaction (PER) with transcription isothermal amplification, termed PER-Trap, for a sensitive biomolecular assay. Its design allowed light-up RNA aptamers to be produced as the final product, leading to the generation of an amplified fluorescence signal. The utility of PER-Trap was successfully demonstrated by the detection of exosomes.

Homogeneous fluorescence immunoassay based on AuNPs quenching dendritic silica assembled with multicolor QDs for the simultaneous determination of four mycotoxins in cereals

In this work, a novel homogeneous fluorescence immunoassay for the simultaneous detection of ochratoxin A (OTA), aflatoxin B1 (AFB1), fumonisins B1 (FB1), and zearalenone (ZEN) in cereal samples is established. Here, amino groups and thiol groups are grafted onto the surface of dendritic mesoporous silica nanoparticles (DMSNs) to improve loading rate and fluorescence preservation rate of quantum dots (QDs), enabling enrichment and amplification of fluorescence signals. In this test system, the fluorescence of the signal probe (DMSNs@QDs@Ab) is quenched by AuNPs, and then the presence of the targets makes the fluorescence of the signal probes recover. The correlation between the recovered fluorescence value and mycotoxin concentration is applied to achieve quantitative detecting mycotoxins. This assay displays good linear correlation in range of 0.01–100 μg L-1, with the limit of detection (LOD) of 0.0001 μg L-1 for OTA, 0.0008 μg L-1 for AFB1, 0.001 μg L-1 for FB1, and 0.0006 μg L-1 for ZEN, respectively. The proposed assay is applied to detect multiple mycotoxins in contaminated cereal samples and the test results is confirmed by liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Avail of silica with high loading capacity for assembling multicolor QDs to achieve simultaneous multi-signal output, a simple and efficient homogeneous fluorescence immunoassay is proposed for the first time. This multiplex test strategy provides a good idea for simultaneous, rapid, and sensitive detection of multiple hazardous substances in food and environment samples.