Triggered Signal Amplification Research Articles

Fluorometric determination of mecA gene in MRSA with a graphene-oxide based bioassay using flap endonuclease 1-assisted target recycling and Klenow fragment-triggered signal amplification

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes a wide range of illnesses, necessitating the development of new technologies for its detection. Herein, we propose a graphene oxide (GO)-based sensing platform for the detection of mecA gene in MRSA using flap endonuclease 1 (FEN1)-assisted target recycling and Klenow fragment (KF)-triggered signal amplification. Without the target, all the DNA probes were adsorbed onto GO, resulting in fluorescence quenching of the dye. Upon the addition of the target, a triple complex was formed that triggered FEN1-assisted target recycling and initiated two polymerization reactions with the assistance of KF polymerase, generating numerous dsDNA that were repelled by GO. These dsDNAs triggered fluorescence enhancement when SYBR Green I was added. Therefore, the target DNA was quantified by measuring the fluorescence at excitation and emission wavelengths of 480/526 nm. This mecA gene assay showed a good linear range from 1 to 50 nM with a lower limit of detection of 0.26 nM, and displayed good applicability to the analysis of real samples. Thus, a new method for monitoring MRSA has been developed that has great potential for early clinical diagnosis and treatment.

Fluorescent immunoassay for chloramphenicol based on the label-free polyadenine-mediated spherical nucleic acids triggered signal amplification.

A fluorescent immunosorbent assay incorporating signal amplification away from the surface of spherical nucleic acid (SNA) was developed for the detection of chloramphenicol (CAP). Through the conjugation of antibodies and poly-adenine (polyA) DNA onto the surface of gold nanoparticles (AuNPs), the fabrication of the nano-immunoprobe was achieved in a more straightforward and cost-effective manner. Moreover, a strategy utilizing the hybridization chain reaction (HCR) in the amplification step was devised, with particular attention given to the enzyme inhibition associated with SNA. The results demonstrated good performance on CAP detection with a linear range of 0.01-5ng/L with a detection limit of 0.005ng/L. The significance of this work mainly lies in the polyA-SNA-based immunoprobe and the thoughtful design to prevent enzyme inhibition.

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.

In situ Hg2+ improved the peroxidase-like activity and triggered "ON" the oxidase-like activity of yolk-shell Co3S4 microspheres for the detection of Hg2.

Exploring highly active nanozymes is an important task to realize the real-time detection of some heavy metal ions in water. In this work, yolk-shell Co3S4 microspheres have been verified to possess excellent peroxidase-like activity, which can be further improved by adding Hg2+. Very interestingly, Hg2+ can trigger "ON" the oxidase-like activity of Co3S4 microspheres. The dual peroxidase-/oxidase-like activity of the yolk-shell Co3S4 microspheres is evaluated by using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). Furthermore, comprehensive studies verify that the enhanced peroxidase-like activity, together with the "ON" oxidase-like activity of the yolk-shell Co3S4 microspheres, is attributed to the in situ generation of HgS on the surface of Co3S4 microspheres and then the release of more active sites. Importantly, the in situ generated HgS on the surface of Co3S4 microspheres can form a heterojunction, which also accelerates the catalytic process. During the catalytic reaction, some active species (O2- and h+) can be detected by ESR. Thus, a colorimetric sensing platform based on Hg2+-triggered signal amplification has been successfully constructed, which can be validated by the detection of Hg2+ residue in environmental water.

Quantitative SERS detection of multiple breast cancer miRNAs based on duplex specific nuclease-mediated signal amplification.

Simultaneous and ultrasensitive detection of multiple microRNA (miRNA) biomarkers is an essential precondition for early cancer diagnosis and treatment. Here we developed a sandwich surface-enhanced Raman scattering (SERS) sensor based on Au@Ag core-shell nanorods combined with duplex specific nuclease-mediated signal amplification (DSNSA) for quantitative detection of multiple breast cancer miRNA biomarkers. The DSNSA strategy enables quantitative detection of target miRNA through rehybridizing the capture probe DNA-SERSnanotag conjugates to trigger signal amplification. The Au@Ag core-shell nanorods coated with an Ag shell exhibit excellent SERS performance, implying that molecules can be concentrated by the Ag shell at the hot spots. By monitoring the Raman signal attenuation of hot spots in the presence of target miRNAs, three breast cancer associated miRNAs (miR-21, miR-155, and let 7b) were simultaneously determined using the sandwich SERS sensor, and their detection limits (LODs) were 0.05 fM, 0.063 fM and 0.037 fM, respectively. These results indicated that our sandwich SERS sensor combined with the DSNSA strategy holds remarkable promise for multiplex detection of cancer biomarkers and contributes to early diagnosis of cancer.

Construction of simple and sensitive pancreatitis related microRNA detection strategy via self-priming triggered cascade signal amplification.

Pancreatic diseases, such as pancreatitis and pancreatic cancer, remain the most threatening gastrointestinal diseases with a high mortality due to atypical symptoms. MicroRNA plays crucial roles in regulating metastasis and cell proliferation of pancreatic cancer, constituting important biomarkers for the early diagnosis of pancreatic cancers. Herein, we develop a sensitive and simple exosomal miRNA detection method with only a dual-hairpin-probe. In detail, the dual-hairpin-probe is constructed through combination of two functional sections for both target miRNA identification and signal amplification. With only one probe, the method possesses the capability to avoid interferences from concentration changes of other probes, and exhibits a higher stability which is demonstrated through the obtained low coefficients of variation (CV) of 6.73%. With let-7a as detection target, the LOD of the established method is determined to be 243 aM, while maintaining a high discriminating capability towards let-7a homogenous miRNAs.

Ultrasensitive detection of tumor-specific exosomal proteins by a Single Microbead-based Aptasensor coupled with Terminal deoxynucleotidyl transferase-initiated DNA amplification (SMAT)

Tumor-derived exosomes carry lots of tumor-specific proteins, attracting more attention as ideal biomarkers for non-invasive diagnosis. However, due to the low concentration of exosomes in biological samples, the development of an ultrasensitive method for tumor-specific exosomal protein detection is highly desired. Here, a sensitive Single Microbead (MB)-based Aptasensor coupled with Terminal deoxynucleotidyl transferase (TdT)-triggered signal amplification (SMAT) is reported to detect tumor-specific exosomal proteins. In the SMAT detection system, a single MB is used as the reaction carrier and firstly modified by the antibodies of generic membrane biomarkers (anti-CD63 and anti-CD81) of exosomes for efficient enrichment of exosomes in samples. Then, the DNA aptamers immobilized on gold nanoparticles (AuNPs) are applied to specifically bind to the tumor-specific proteins on exosome surfaces. Subsequently, the DNA aptamers can be efficiently extended by TdT to generate a long poly(dT) tail. By hybridization with lots of fluorescein-labeled poly(dA)25 (FAM-poly(dA)25) probes, large amounts of fluorescence molecules will be highly enriched on one MB and in situ monitored with fluorescence imaging. With this strategy, exosomal EpCAM protein (Exo-EpCAM) has been sensitively detected from the samples containing 55 particles/μL tumor-derived exosomes. Furthermore, the proposed method could be applied for detection of Exo-EpCAM from plasma microsamples, indicating great application prospect in future for clinical diagnosis.

Imaging and Quantification of mRNA Molecules at Single-Cell Resolution in the Human Fungal Pathogen Candida albicans.

ABSTRACTThe study of gene expression in fungi has typically relied on measuring transcripts in populations of cells. A major disadvantage of this approach is that the transcripts’ spatial distribution and stochastic variation among individual cells within a clonal population is lost. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. mRNA imaging also revealed the subcellular localization of specific transcripts. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. albicans cell. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. albicans genes in infected organs at single-cell resolution.IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. Widespread phenomena such as stochastic variation in transcript levels among cells—even within a clonal population—seem to play important roles in the biology of many microorganisms. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution.

Detection of locus-specific N6-methyladenosine modification based on Ag+-assisted ligation and supersandwich signal amplification.

Emerging evidence reveals that the epitranscriptomic mark N6-methyladenosine (m6A) plays vital roles in organisms, including gene regulation and disease progression. However, developing sensitive methods to detect m6A modification, especially the identification of m6A marks at the single-site level, remains a challenge. Therefore, based on target-specific triggered signal amplification, we developed a highly sensitive electrochemical method to detect site-specific m6A modifications in DNA. In this work, the m6A site in DNA can restrict the ligation assisted by Ag+, and this restriction effect can activate the subsequent strand displacement reaction and hybridization chain reaction (HCR), thus achieving signal amplification from the m6A site, and finally realizing high sensitivity analysis of m6A methylation. Benefiting from the high specificity of base pairs and the extremely weak binding affinity between Ag+ and m6A, the proposed method was used for not only detecting the target DNA with a putative m6A site, but also identifying m6A marks at the single-site level in DNA. In addition, this study does not rely on antibodies and radiolabeling, so it has the advantage of cost-effectiveness. Therefore, we believe that the proposed strategy may provide a new perspective for methylation research, which can be used to test more clinical samples in further research.

Hg2+ Significantly Enhancing the Peroxidase-Like Activity of H2TCPP/ZnS/CoS Nanoperoxidases by Inducing the Formation of Surface-Cation Defects and Application for the Sensitive and Selective Detection of Hg2+ in the Environment

Exploring excellent peroxidase mimics with enhanced peroxidase-like activity is important to the construction of a fast, low-cost, and convenient colorimetric sensing platform for heavy ions. In this work, 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (H2TCPP) was first used to modify ZnS/CoS and make it show better peroxidase-like activity. The metal-cation vacancies generated by Hg2+ contacting H2TCPP/ZnS/CoS further stimulate the catalytic activity. It is reported that the addition of Hg2+ usually causes a decrease of the peroxidase-like activity of metal sulfides. Oppositely, in our work, Hg2+ can trigger the colorimetric signal amplification because of lots of metal-cation vacancies generated on the surface of the nanocomposites (bimetallic sulfides). The peroxidase-like activity of ZnS/CoS was evaluated by virtue of the chromogenic substrate 3,3,5,5-tetramethylbenzidine (TMB) from colorless to blue in 3 min. The enhanced catalytic activity of H2TCPP/ZnS/CoS was attributed to lots of active sites from the metal-cation defects on the surface of H2TCPP/ZnS/CoS as well as the synergistic effect of porphyrin molecules and ZnS/CoS. The adsorption behavior of H2O2 on the H2TCPP/ZnS/CoS surface with defects was studied by density functional theory calculation. Thus, a colorimetric sensing platform based on Hg2+ trigger signal amplification has been successfully constructed, which can be used to sensitively and selectively determine Hg2+ in environmental samples.

Self-Assembly Trigger Signal Amplification for MicroRNA Sensing in Living Cells with GSH-Cleavable Nanoprobes

Accurate sensing of endogenous microRNAs remains a great challenge for unsatisfactory signal-to-background ratios of traditional techniques. Meanwhile, low sensitivity and insufficient specificity ...

Photoelectrochemical immunosensor for methylated RNA detection based on WS2 and poly(U) polymerase–triggered signal amplification

A novel photoelectrochemical immunosensor has beenconstructed for the determination of methylated RNA. MoS2 nanosheets with large specific area were employed as photoactive material, gold nanoparticles were used as signal amplification unit and immobilization matrix of 4-mercaptophenylboronic acid, anti-m6A antibody was adopted as methylated RNA recognition reagent, and poly(U) polymerase-mediated RNA chain extension and Ru(NH3)63+ were used as assisted signal amplification unit. With the sensitization effect of Ru(NH3)63+, the photoactivity of WS2 nanosheets was improved greatly, which also improved the sensitivity. Using visible-light excitation and ascorbic acid as electron donor, the sensitive determination of methylated RNA was achieved by monitoring the photocurrent change with different concentrations of methylated RNA. This photoelectrochemical immunosensor hasa wide linear relationship with methylated RNA concentration from 0.05 to 35nM under optimal experimental conditions. The low detection limit of 14.5pM was realized based on 3σcriterion. In addition tothe good selectivity, this sensor also presents highreproducibility with arelative standard deviation of 1.4% for the photocurrent of seven electrodes. The applicability of the developed method was also investigated by detecting the level of methylated RNA in corn seedling leaves with and without sulfadiazine treatment. Graphical abstract A novel photoelectrochemical immunosensor was developed for methylated RNA detection using the photoactive material of MoS2 and poly(U) polymerase-mediated RNA chain extension.

Near-Infrared Light Activated Nucleic Acid Cascade Recycling Amplification for Spatiotemporally Controllable Signal Amplified mRNA Imaging.

The expression level and subcellular distribution of mRNA dynamically changed during the different cell circles. Spatiotemporally controllable signal amplification methods capable of controlling the when and where of the amplification process could allow the sensitive mRNA imaging of selected living cells at dictated time-intervals of the cell life-cycle. However, the present methods for amplified mRNA imaging are hard to control the where and when of the signal amplification due to the lack of an effective strategy to precisely trigger and control the signal amplification process. Herein, we present a conceptual study termed as photocontrollable nucleic acid cascade recycling amplification which uses near-infrared (NIR) light to precisely control and trigger the whole process. This strategy is achieved by integrating photocontrollable nucleic acid displacement reaction with exonuclease III (EXO III) assisted nucleic acid cascade recycling amplification and combination with upconversion nanoparticles (UCNPs), thus resulting in a NIR light activatable signal amplification. As a proof of concept, we demonstrate this developed NIR light triggered signal amplification process in selected living cancer cells for spatiotemporally controllable signal amplified mRNA imaging.

A TiO2 mesocrystals triggered signal amplification strategy of photoelectrochemical immunoassay for α-fetoprotein detection based on indirect competitive reaction

A versatile TiO2 mesocrystals trigged signal amplification photoelectrochemical (PEC) sensing platform was designed for sensitive and accurate determination of α-fetoprotein (AFP). To construct such a biosensor, Bi2S3 nanosheets with notable visible light photoactivity and biocompatibilities were employed as electron transfer mediator as well as a substrate for AFP antigen immobilization. Meanwhile, the Polyethylenimine (PEI) functionalized TiO2 mesocrystals was used to anchor AFP antibody and the resulting composite Ab-TiO2-PEI was utilized as trace tag for signal amplification. The trace tag anchored onto the electrode interface via indirect competitive recognition between free target AFP and immobilized AFP to combine Ab-TiO2-PEI. As a result, a cascade structure of band-edge levels formed between TiO2 mesocrystals and Bi2S3 nanosheets, which notably promoted the photo-to-current conversion efficiency and enhance photocurrent response significantly. By merit of these, the concentration of AFP could be accurately determined through an indirect competitive reaction immunoassay. Upon the optimal condition, this PEC biosensor performed favorable sensitivity, selectivity and reproducibility. Hence, the designed indirect competitive PEC immunoassay possesses a potential application for clinical test and will inspire more development of simple and practical PEC sensor toward different biomolecules.

Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification.

A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525nm and the DNA concentration in the range from 0.2 pM to 20nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA. Graphical abstract Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.

Multifunctionalized ZIFs nanoprobe-initiated tandem reaction for signal amplified electrochemical immunoassay of carbohydrate antigen 24-2

Based on multifunctionalized zeolitic imidazolate frameworks (ZIFs)-initiated cascade reaction triggered signal amplification strategy, a novel sandwich-type amperometric immunosensor was constructed for ultrasensitive detection of carbohydrate antigen 24-2 (CA 242). The methylene blue-glucose oxidase-ZIF-8/reduced graphene oxide-Au (MB-GOx-ZIF-8/Au-rGO) was synthesized by a facile coprecipitation method for enzyme immobilization and redox species loading. The multifunctionalized ZIFs conjugated with labeling antibodies were employed as immunoprobe. In the presence of glucose, tandem reaction was driven by the immunoprobe to catalyze the glucose oxidation to yield H2O2. Simultaneously, the generated H2O2 induced the decomposition of poly(anilineboronicacid) (PABA)/poly(vinyl alcohol) (PVA) films on substrate, which made the PVA chains breaking away from PABA polymer. Due to the poor conductivity of PVA chains, this decomposition reaction can amplify the current signal. The current difference (ΔI) caused by per unit concentration target with tandem reaction amplification was elevated prominently, resulting in ultrasensitive analytical performance of the immunosensor. Under optimal conditions, the proposed immunosensor displayed wide linear range from 0.001 to 1000 U mL−1 with an ultralow limit of detection 69.34 μU mL−1 (S/N = 3). This method successfully implemented functionalized ZIFs in immunoprobe construction for sensitivity amplification, providing a promising strategy to construct ultrasensitive immunosensing platform for analysis of other tumor marker.

How Wnt ligands achieve specificity

Developmental Signaling Wnt signaling is essential for development, tissue homeostasis, and disease. The 19 members of the Wnt family interact promiscuously with the 10 Frizzled receptors, raising the question of how ligand-specific discrimination is achieved in a biological context. Eubelen et al. used experiments in zebrafish to show that cells are equipped with decoding modules that bind Wnt with high specificity and trigger signal amplification via their recruitment into higher-order Frizzled signalosomes (see the Perspective by Kim and Goentoro). Thus, distinct Wnt ligand-receptor pairs can be targeted specifically for therapeutic purposes. Science , this issue p. [eaat1178][1]; see also p. [643][2] [1]: /lookup/doi/10.1126/science.aat1178 [2]: /lookup/doi/10.1126/science.aau6457

Sensitive colorimetric detection of K(I) using catalytically active gold nanoparticles triggered signal amplification

In this work, we report a simple, ultrasensitive, and feasible colorimetric assay for metal ion (K+, used as a model) via inherent peroxidase-like enzymatic amplification strategy of gold nanoparticles (AuNPs). It is shown that peroxidase-like activity of AuNPs can be improved dramatically by its surface activation with target-specific aptamer molecules. Whereas when the target exists, the aptamers leave the surface of AuNPs in a target concentration-dependent manner, resulting in a decrease of the nanoenzymatic catalytic ability of AuNPs. Thus, K+ can be quantified in the presence of AuNPs by using a colorimetric sensing probe (3,3′,5,5′-tetramethylbenzidine). The color change of the solution is relevant to the dose of the target, and this can be achieved with the naked eyes and monitored by UV–vis spectrometry. A linear dependence between the absorbance and target K+ concentration is obtained under optimal conditions in the range from 0. 1nM to 1μM with a detection limit (LOD) of 0.06nM estimated at the 3Sblank level. The sensitivity displays to be 2–9 orders of magnitude better than those of other K+ detection methods. This sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interest.

Dynamic Monitoring of MicroRNA-DNA Hybridization Using DNAase-Triggered Signal Amplification.

Dynamically monitoring microRNA (miRNA)-DNA reactions is critical for elucidating various biological processes. However, traditional strategies fail to capture this dynamic event because the original targets are preamplified. In the present study, we developed an amplification-free strategy for real-time monitoring of miRNA-DNA hybridization that integrates the advantages of both duplex-specific nuclease (DSN)-triggered signal amplification and single-stranded DNA probe coating facilitated by reduced graphene oxide. DSN-mediated miRNA recognition was found to consist of two phases: hybridization and hybridization cleavage. In the presence of miRNA and DSN, hybridization of a 22-mer miRNA-DNA could be completed within 7 min by observing the angle increase in a surface plasmon resonance (SPR) biosensor. The subsequent hybridization-cleavage process could be visualized as a gradual SPR angle decrease that occurred until all coated probes were hydrolyzed. In addition, for miRNA-21 detection, the proposed linear signal amplification assay demonstrated a sensitivity of 3 fM over a dynamic range of 5 orders of magnitude.

Hyperbranched Hybridization Chain Reaction for Triggered Signal Amplification and Concatenated Logic Circuits

A hyper-branched hybridization chain reaction (HB-HCR) is presented herein, which consists of only six species that can metastably coexist until the introduction of an initiator DNA to trigger a cascade of hybridization events, leading to the self-sustained assembly of hyper-branched and nicked double-stranded DNA structures. The system can readily achieve ultrasensitive detection of target DNA. Moreover, the HB-HCR principle is successfully applied to construct three-input concatenated logic circuits with excellent specificity and extended to design a security-mimicking keypad lock system. Significantly, the HB-HCR-based keypad lock can alarm immediately if the "password" is incorrect. Overall, the proposed HB-HCR with high amplification efficiency is simple, homogeneous, fast, robust, and low-cost, and holds great promise in the development of biosensing, in the programmable assembly of DNA architectures, and in molecular logic operations.