Engineering Biomimetic Biosensor Using Dual-Targeting Multivalent Aptamer Regulated 3D DNA Walker Enables High-Performance Detection of Heterogeneous Circulating Tumor Cells.

The phenotypic heterogeneity of circulating tumor cells (CTCs) and the nonspecific adsorption of background cells impede the effective and sensitive detection of rare CTCs. Although leukocyte membrane coating approach has a good antileukocyte adhesion ability and holds great promise for addressing the challenge of capture purity, its limited specificity and sensitivity prevent its use in the detection of heterogeneous CTCs. To overcome these obstacles, a biomimetic biosensor that integrated dual-targeting multivalent aptamer/walker duplex functionalized biomimetic magnetic beads and an enzyme-powered DNA walker signal amplification strategy is designed. As compared to conventional leukocyte membrane coating, the biomimetic biosensor achieves efficient and high purity enrichment of heterogeneous CTCs with different epithelial cell adhesion molecule (EpCAM) expression while minimizing the interference of leukocytes. Meanwhile, the capture of target cells can trigger the release of walker strands to activate an enzyme-powered DNA walker, resulting in cascade signal amplification and the ultrasensitive and accurate detection of rare heterogeneous CTCs. Importantly, the captured CTCs remained viable and can be recultured in vitro with success. Overall, this work provides a new perspective for the efficient detection of heterogeneous CTCs by biomimetic membrane coating and paves the way for early cancer diagnosis.

Allosteric Probe Recognition Initiated Cascade Transcription Amplification for Sensitive Analysis of Exosomes.

Sensitive and accurate analysis of exosomes is important for many biological processes including as a biomarker for cerebral venous thrombosis (CVT) diagnosis and therapy. Herein, we established a sensitive and specific exosome detection approach based on target recognition initiated cascade signal amplification. In this method, an allosteric probe was designed with a hairpin structure for specific recognition of the exosome followed by signal amplification. After the cascade signal amplification process, spinach RNA sequences bind to DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) to produce enhanced fluorescence signal (approximate 2000 fold than that of inactive DFHBI). Compared with former proposed exosome detection techniques, this method exhibited a comparable detection range, but with an easy-to-design toolbox. Therefore, we believe that the proposed approach holds great potential for exosome based early diagnosis and prognosis of disease.

A single nucleotide polymorphism electrochemical sensor based on DNA-functionalized Cd-MOFs-74 as cascade signal amplification probes.

An ultrasensitive electrochemical sensor has been constructed for the detection of single nucleotide polymorphisms (SNPs) based on DNA-functionalized Cd-MOFs-74 as cascade signal amplification probe under enzyme-free conditions. Interestingly, the introduction of an auxiliary probe did not disturb the detection of SNP targets, but could bind more Cd-MOFs-74 signal elements to enhance the different pulse voltammetry electrochemical signal 2~3 times as compared to sensing system without auxiliary probe, which obviously improves the sensitivity of the proposed sensor. Experimental results taking p53 tumor suppressor gene as SNP model demonstrated that the proposed method can be employed to sensitively and selectively detect target p53 gene fragment with a linear response ranging from 0.01 to 30 pmol/L (detection limit of 6.3 fmol/L) under enzyme-free conditions. Utilizing this strategy, the ultrasensitive SNP electrochemical sensor is a promising tool for the determination  of SNPs in biomedicine. Graphical Abstract.

Highly sensitive SERS assay of DENV gene via a cascade signal amplification strategy of localized catalytic hairpin assembly and hybridization chain reaction.

Pathogenic viruses with worldwide distribution, high incidence and great harm are significantly and increasingly threatening human health. However, there is still lack of sufficient, highly sensitive and specific detection methods for on-time and early diagnosis of virus infection. In this work, taking dengue virus (DENV) as an example, a highly sensitive SERS assay of DENV gene was proposed via a cascade signal amplification strategy of localized catalytic hairpin assembly (LCHA) and hybridization chain reaction (HCR). The SERS assay was performed by two steps, i.e., the operation of cascade signal amplification strategy and the following SERS measurements by transferring the products on SERS-active AgNRs arrays. The sensitivity of the cascade signal amplification strategy is significantly amplified, which is 4.5 times that of individual CHA, and the signal-to-noise ratio is also improved to 5.4 relative to 1.8 of the CHA. The SERS sensing possesses a linear calibration curve from 1 fM to 10 nM with the limit of detection low to 0.49 fM, and has good specificity, uniformity and recovery, which indicates that the highly sensitive SERS assay provides an attractive tool for reliable, early diagnosis of DENV gene and is worth to be popularized in a wide detection of other viruses.

"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.

Enhanced luminol electrochemiluminescence biosensing system based on highly dispersed bimetallic nanozyme coreaction accelerator and 3D DNA walker signal amplifier.

In this work, a platinum-nickel based nanozyme is prepared and used as a coreaction accelerator in the luminol-H2O2 electrochemiluminescence (ECL) system to construct an ECL biosensor for dimethyl phthalate (DMP) detection. The PtNi/NC nanozyme possesses dispersed metal active sites, and the synergistic effect of Pt and Ni endows it with excellent catalytic performance, which effectively converts H2O2 into more superoxide anions, and then significantly enhances the ECL intensity of the luminol system. The ECL mechanism is investigated by combining cyclic voltammetry and ECL with different types of free radical scavengers. Simultaneously, an "off-on" biosensor is constructed by integrating 3D DNA walker with enzyme-free recycling amplification for ultrasensitive detection of DMP. The biosensor based on PtNi/NC nanozyme mediated luminol-H2O2 system and 3D DNA walker exhibits a linear range of 1 × 10-16 to 1 × 10-6 M with a detection limit of 4.3 × 10-17 M (S/N = 3), and displays good stability and specificity. This study demonstrates the advantages of PtNi/NC nanozyme in enhancing the luminol-H2O2 ECL system, providing new strategy for designing efficient ECL emitter and offering a new method for detecting phthalate esters.

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.

Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella.

Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL-1. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.

Rapid and accurate SERS assay of disease-related nucleic acids based on isothermal cascade signal amplifications of CRISPR/Cas13a system and catalytic hairpin assembly.

Developing rapid, accurate and convenient nucleic acid diagnostic techniques is essential for the prevention and control of contagious diseases that are prone to gene mutations and may have homologous sequences, especially emerging infectious diseases such as the SARS-CoV-2 pandemic. Herein, a one-pot SERS assay integrating isothermal cascade signal amplification strategy (i.e., CRISPR/Cas13a system (Cas13a) and catalytic hairpin assembly (CHA), Cas13a-CHA) and SERS-active silver nanorods (AgNRs) sensing chips was proposed for rapid and accurate detection of disease-related nucleic acids. Taking SARS-CoV-2 RNA assay as a model, the Cas13a-CHA based SERS sensing strategy can achieve ultra-high sensitivity low to 5.18 × 102 copies·mL-1 within 60 min, and excellent specificity, i.e., not only the ability to identify SARS-CoV-2 RNA from gene mutations, but also incompatibility with coronaviruses such as severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and other respiratory viruses. The proposed Cas13a-CHA based SERS assay for SARS-CoV-2 RNA has satisfactory sensitivity, specificity, uniformity, and repeatability, and can be easily expanded and universalized for screening different viruses, which is expected to promise as a crucial role for diagnosis of disease-related nucleic acids in various medical application scenarios.

Cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction: Toward an ultrasensitive split-type fluorescent immunoassay.

An ultrasensitive split-type fluorescent immunobiosensor has been reported based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. In this strategy, Cu2+ could oxidize chemically o-phenylenediamine (OPD) to generate photosensitive 2, 3-diaminophenazine (DAP) and Cu+/Cu0. On one hand, the generated Cu0 in turn catalyzed the oxidation of OPD. On the other hand, the introduced H2O2 reacted with Cu + ion to produce hydroxyl radicals (·OH) and Cu2+ ion through a Cu + -mediated Fenton-like reaction. The produced ·OH and recycled Cu2+ ion could take turns oxidizing OPD to generate more photoactive DAP, which triggering a self-sustaining chemical redox-cycling reaction and leading to a remarkable fluorescent improvement. It was worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Based on the H2O2-triggered cascade signal amplification, the strategy was exploited for the construction of split-type fluorescent immunoassay by taking interleukin-6 (IL-6) as the model target. It was realized for the ultrasensitive determination of IL-6 in a linear ranging from 20 fg/mL to 10 pg/mL with a limit of detection of 5 fg/mL. The study validated the practicability of the cascade signal amplification on the fluorescent bioanalysis and the superior performance in fluorescent immunoassay. It is expected that the strategy would offer new opportunities to develop ultrasensitive fluorescent methods for biosensor and bioanalysis.

Analyte-triggered cascade signal amplification strategy for highly sensitive detection of iodate in table salt with dual-readout signals.

A novel and highly sensitive upconversion fluorescence and colorimetric dual readout iodate (IO3-) nanosensor system was constructed by using both the outstanding optical performance of NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and the analyte-triggered cascade signal amplification (CSA) technique. The construction of the sensing system consisted of three processes. First, IO3- oxidized o-phenylenediamine (OPD) to diaminophenazine (OPDox), while IO3- was reduced to I2. Second, the generated I2 can further oxidize OPD to OPDox. This mechanism has been verified by 1H NMR spectra titration analysis and HRMS measurement, which effectively improves the selectivity and sensitivity of the measurement of IO3-. Third, the generated OPDox can effectively quench the fluorescence of UCNPs via the inner filter effect (IFE), realize analyte-triggered CSA, and allow quantitative determination of IO3-. Under the optimized conditions, the fluorescence quenching efficiency showed a good linear relationship to IO3- concentration in the range of 0.06-100 µM, and the detection limit reached 0.026 µM (3RSD/slope). Moreover, this method was applied to detect IO3- in table salt samples, yielding satisfactory determination results with excellent recoveries (95.5-105%) and high precision (RSD <5.5%). These results suggest that the dual-readout sensing strategy with well-defined response mechanisms has promising application prospects in physiological and pathological studies.

Holographically Activatable Nanoprobe via Glutathione/Albumin-Mediated Exponential Signal Amplification for High-Contrast Tumor Imaging.

Glutathione (GSH)-activatable probes hold great promise for in vivo cancer imaging, but are restricted by their dependence on non-selective intracellular GSH enrichment and uncontrollable background noise. Here, a holographically activatable nanoprobe caging manganese tetraoxide is shown for tumor-selective contrast enhancement in magnetic resonance imaging (MRI) through cooperative GSH/albumin-mediated cascade signal amplification in tumors and rapid elimination in normal tissues. Once targeting tumors, the endocytosed nanoprobe effectively senses the lysosomal microenvironment to undergo instantaneous decomposition into Mn2+ with threshold GSH concentration of ≈ 0.12 mm for brightening MRI signals, thus achieving high contrast tumor imaging and flexible monitoring of GSH-relevant cisplatin resistance during chemotherapy. Upon efficient up-regulation of extracellular GSH in tumor via exogenous injection, the relaxivity-silent interstitial nanoprobe remarkably evolves into Mn2+ that are further captured/retained and re-activated into ultrahigh-relaxivity-capable complex by stromal albumin in the tumor, and simultaneously allows the renal clearance of off-targeted nanoprobe in the form of Mn2+ via lymphatic vessels for suppressing background noise to distinguish tiny liver metastasis. These findings demonstrate the concept of holographic tumor activation via both tumor GSH/albumin-mediated cascade signal amplification and simultaneous background suppression for precise tumor malignancy detection, surveillance, and surgical guidance.

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification.

A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.

A novel cascade signal amplification strategy integrating CRISPR/Cas13a and branched hybridization chain reaction for ultra-sensitive and specific SERS detection of disease-related nucleic acids.

The molecular diagnosis of disease by high-sensitively and specifically detecting extremely trace amounts of nucleic acid biomarkers in biological samples is still a great challenge, and the powerful sensing strategy has become an urgent need for basic researches and clinical applications. Herein, a novel one-pot cascade signal amplification strategy (Cas13a-bHCR) integrating CRISPR/Cas13a system (Cas13a) and branched hybridization chain reaction (bHCR) was proposed for ultra-highly sensitive and specific SERS assay of disease-related nucleic acids on SERS-active silver nanorods sensing chips. The Cas13a-bHCR based SERS assay of gastric cancer-related miRNA-106a (miR-106a) can be achieved within 60 min and output significantly enhanced SERS signal due to the multiple signal amplification, which possesses a good linear calibration curve from 10 aM to 1 nM with the limit of detection (LOD) low to 8.55 aM for detecting gastric cancer-related miR-106a in human serum. The Cas13a-bHCR based SERS sensing also shows good specificity, uniformity, repeatability and reliability, and has good practicability for detection of miR-106a in clinical samples, which can provide a potential powerful tool for SERS detection of disease-related nucleic acids and promise brighter prospects in the field of clinical diagnosis of early disease.

A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly.

Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5'-phosphorylated terminus to form dsDNA with a blunt 5'-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.

Ultrafast synthesized monometallic nanohybrids as an efficient quencher and recognition antenna of upconversion nanoparticles for the detection of xanthine with enhanced sensitivity and selectivity.

Upconversion nanoparticles (UCNPs) have shown great promise in bioanalytical applications owing to their excellent optical properties. Generally, most analytical applications are based on the fluorescence resonance energy transfer (FRET) principle to quench the fluorescence of UCNPs. However, each UCNP contains thousands of emission center ions, and most of them exceed the FRET critical distance, which hinders FRET efficiency and leads to a low signal-to-background ratio (SBR). Herein, a novel nanoprobe for the detection of Xanthine (XA) based on inner filter effects (IFE) and cascade signal amplification strategy was constructed by decorating UCNP with trypsin-chymotrypsin-stabilized gold nanoparticles-gold nanoclusters (Try-chy-AuNPs-AuNCs) monometallic nanohybrids. The Try-chy-AuNPs-AuNCs prepared by ultrafast (3 min) and green synthesis method have efficient upconversion fluorescence quenching ability (the quenching efficiency up to 90.9%), which can effectively improve the SBR of the probe, so as to improve the sensitivity. In addition, the Try-chy-AuNPs-AuNCs have a unique spatial structure, which can effectively prevent the interaction between large-size biothiol (glutathione) and the probe, thus improving its selectivity. Besides, combined with the excellent optical performance of UCNPs and cascaded signal amplification strategy, the sensitivity of the probe can be further improved. Under the optimized conditions, the linear response range of the probe was obtained from 0.05 to 50 µM, 0.06-80 µM and with the low detection limit of 22.6 nM and 26.3 nM for H2O2 and XA, respectively. Meanwhile, the developed method has been further applied to the detection of XA in human serum with satisfactory results.

DNAzyme-based cascade signal amplification strategy for highly sensitive detection of lead ions in the environment.

Lead ions are one of many common environmental pollutants, that can cause posing a serious threat to people's health, thus, their efficient and sensitive detection is important. We propose a cascade signal amplification sensor using a DNAzyme-based strand displacement amplification (SDA) and hybridization chain reaction (HCR) for the high-sensitivity detection of Pb2+. In the demonstrated sensor system, the target metal ion can activate DNAzyme to cause a strand displacement reaction. Under the synergistic action of polymerase and nickase, large numbers of DNA strands are generated that can initiate HCR. The subsequent HCR can restore the fluorescence intensity of the FAM quenched for the fluorescence resonance energy transfer effect, which exhibits a strong fluorescence signal. The DNAzyme-based sensor allowed the detection of Pb2+ down to 16.8 pM and resulted in a good dynamic line relationship ranging from 50 pM to 500 nM, and the biosensor also showed good selectivity. Furthermore, we confirmed that the proposed sensor can still detect lead ions in complex environments such as lake water, milk, and serum. We believe these findings will provide new ideas for the detection of toxic elements ions in the environment and food.

Target-triggered cascade signal amplification for sensitive electrochemical detection of SARS-CoV-2 with clinical application.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the outbreak of the 2019 coronavirus (COVID-19) disease, which greatly challenges the global economy and health. Simple and sensitive diagnosis of COVID-19 at the early stage is important to prevent the spread of pandemics. Herein, we have proposed a target-triggered cascade signal amplification in this work for sensitive analysis of SARS-CoV-2 RNA. Specifically, the presence of SARS-CoV-2 RNA can trigger the catalytic hairpin assembly to generate plenty of DNA duplexes with free 3'-OH termini, which can be recognized and catalyzed by the terminal deoxynucleotidyl transferase (TdT) to generate long strand DNA. The prolonged DNA can absorb substantial Ru(NH3)63+ molecules via electrostatic interaction and produce an enhanced current response. The incorporation of catalytic hairpin assembly and TdT-mediated polymerization effectively lowers the detection limit to 45 fM, with a wide linear range from 0.1 pM to 3000 pM. Moreover, the proposed strategy possesses excellent selectivity to distinguish target RNA with single-base mismatched, three-base mismatched, and random sequences. Notably, the proposed electrochemical biosensor can be applied to analyze targets in complex circumstances containing 10% saliva, which implies its high stability and anti-interference. Moreover, the proposed strategy has been successfully applied to SARS CoV-2 RNA detection in clinical samples and may have the potential to be cultivated as an effective tool for COVID-19 diagnosis.

Smartphone-assisted bioenzyme-nanozyme-chromogen all-in-one test strip with enhanced cascade signal amplification for convenient paraoxon sensing.

Monitoring of pesticide residues in food and environmental matrices is undoubtedly crucial to guarantee food safety and ecological health, yet how to realize their sensitive and convenient detection is still challenging. Herein, we propose an all-in-one test strip that elaborately integrates bioenzyme, nanozyme and chromogen together, and achieve the highly sensitive and convenient sensing of pesticide residues assisted by a smartphone. A sequential self-assembly strategy was first explored to acquire an integrative bioenzyme-nanozyme-chromogen assembly, and then the assembly was confined in a biocompatible hydrogel to construct the test strip. Thanks to both the proximity and confinement effects, a ∼1.2-fold improvement of the cascade catalytic efficiency was gained to benefit high-sensitivity detection. More importantly, since all the sensing elements, including target recognition units and signal amplification modules, were rationally integrated in the test strip, detection operation was significantly simplified, making it possible for in-field rapid analysis. Besides, the microenvironment provided by the alginate hydrogel carrier endowed the test strip with an excellent sensing stability. By taking paraoxon as a typical pesticide, high-performance detection of the target was accomplished via the smartphone-assisted all-in-one test strip. Moreover, the test strip was successfully applied for paraoxon detection in various real samples and exhibited good correlations with commercial kits, demonstrating its great prospect for practical applications. Our work not only offers a new tool for the high-sensitivity and convenient monitoring of pesticide residues, but will also inspire the development of efficient multi-enzyme sensing platforms.

The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity.

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10-5 U mL-1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.