Surface Of Magnetic Beads Research Articles

A Novel DNAzyme Walker Biosensor for the Determination of Chromium(III)

A new DNAzyme walker biosensor has been constructed to sensitively determine chromium (III). The DNAzyme walker consisted of enzyme strands (ES) as the walking strand and carboxyfluorescein-labeled substrate strands (SS) carrying a nicking cleavage site on the surface of streptavidin-modified magnetic beads (MB@ES/SS). Cr3+ activates the nicking endonuclease activity of DNAzyme and its corresponding cleavage on SS to drive ES to move autonomously along the MB surface. As a result, FAM-labeled fragments from SS are released, producing the fluorescence signal. The MB@ES/SS walker biosensor provides rapid analysis and a low limit of detection (3.12 nM) within a linear range from 0.01 to 20 µM. This work offers a new method for Cr3+ determination and broadens its application for metal ion analysis.

Sensitive and reliable miRNA analysis based on cyclic reverse transcription and CRISPR-Cas12a-assisted signal cycle

MicroRNAs (miRNAs), a category of small molecules that possess significant regulatory capabilities, have been extensively employed as biomarkers in the domain of biosensing to facilitate the early detection of diverse ailments. However, sensitive and accurate miRNA detection remains a huge challenge due to the high similarity between the homologous sequences and low abundance. Therefore, it is essential to develop methods with high sensitivity and specificity for miRNA detection. In this study, we present the development of a signal cycle-based platform that utilizes cyclic reverse transcription (CRT) and CRISPR-Cas12a to enable the precise and sensitive detection of microRNAs. The CRT mechanism facilitates precise target recognition in the presence of target miRNA, thereby converting miRNA signals to DNA signals. The trans-cleavage activity of the Cas12a protein is triggered by the formation of complete hairpin-shaped CRT products; this results in the cleavage of the DNA section contained in the H probe, while the RNA section (“4”@MBs) remains loaded onto the surface of magnetic beads (MB). By binding with the “reporter” sensor, the “4” sequences create an RNA/DNA duplex that the duplex-specific nuclease (DSN) can recognize. The “reporter” probe is thus metabolized, leading to the reappearance of the fluorescence signal. By capitalizing on the exceptional fidelity and selectivity of CRISPR/Cas12a, as well as the substantial impact of triggered enzymatic cycle amplification, this approach demonstrated remarkable sensitivity and specificity in miRNA detection, even in a complex environment containing 10% fetal bovine serum (FBS) and a serum sample. In contrast, a detection limit of 3.2 fM is conceivable. Furthermore, this approach maintained a notable degree of stability, which was anticipated to result in the detection of miRNAs in an effective and sensitive manner.

Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni2+

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and NC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.

BSA‐Gold Nanoclusters Enhanced Clusterization‐Triggered–Emission of Nonconventional Luminophores‐Hydrogels in Dilute Aqueous Solution

AbstractThe application of nonconventional nonaromatic luminophores‐based hydrogels (NLHs) in bioanalysis is severely limited due to their luminescence in concentrated solution or solid state. Herein, a nonconventional NLH (B‐Au@PDMAA) with high signal‐to‐noise ratio and stability in dilute solutions is designed by enhancing nonaromatic luminophores intermolecular aggregation through bovine serum albumin‐gold nanoclusters (B‐Au) confined space interaction. Importantly, the established NLHs could be rapidly polymerized in situ on magnetic beads (MB) surface in dilute solutions, and the fluorescence intensity after polymerization is 5.63‐fold higher than that of direct polymerization. Moreover, proposed B‐Au@PDMAA@MB constructed homogeneous immunoassay platform is demonstrated to be highly sensitive for detecting hepatitis B surface antigen with a sensitivity of 3.39 ng mL−1, which is 28.6‐fold lower than that of B‐Au@PDMAA based immunoassay and comparable to the sensitivity of the chemiluminescence immunoassay. This work presents a new strategy for the synthesis of NLHs with high brightness and stability in dilute solutions, and opens new avenues for the construction of novel homogeneous immunoassay platforms.

Studying the small extracellular vesicle capture efficiency of magnetic beads coated with tannic acid.

The isolation of small extracellular vesicles (sEVs), including those secreted by pathological cells, with high efficiency and purity is highly demanded for research studies and practical applications. Conventional sEV isolation methods suffer from low yield, presence of contaminants, long-term operation and high costs. Bead-assisted platforms are considered to be effective for trapping sEVs with high recovery yield and sufficient purity for further molecular profiling. In this study, magnetically responsive beads made of calcium carbonate (CaCO3) particles impregnated with iron oxide (Fe3O4) nanoparticles are fabricated using a freezing-induced loading (FIL) method. The developed magnetic beads demonstrate sufficient magnetization and can be collected by a permanent magnet, ensuring their rapid and gentle capture from an aqueous solution. The tannic acid on the surface of magnetic beads is formed by a layer-by-layer (LbL) method and is used to induce coupling of sEVs with the surface of magnetic beads. These tannic acid coated magnetic beads (TAMB) were applied to capture sEVs derived from MCF7 and HCT116 cell lines. Quantitative data derived from nanoparticle tracking analysis (NTA) and BCA methods revealed the capture efficiency and recovery yield of about 60%. High-resolution transmission electron microscopy (HRTEM) imaging of sEVs on the surface of TAMBs indicated their structural integrity. Compared with the size exclusion chromatography (SEC) method, the proposed approach demonstrated comparable efficiency in terms of recovery yield and purity, while offering a relatively short operation time. These results highlight the high potential of the TAMB approach for the enrichment of sEVs from biological fluids, such as cell culture media.

A controllable Y-shaped DNA structure assisted aptasensor for the simultaneous detection of AFB1 and OTA based on ARGET ATRP.

The development of a simple, rapid, and sensitive technology for the simultaneous detection of mycotoxins is of great significance in ensuring the safety of foods and drugs. Herein, a fluorescence aptasensor with high sensitivity and reproducibility for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was developed. In this sensing system, AFB1 and OTA aptamers were co-immobilized on the surface of magnetic beads (MBs) to form a Y-shaped structure through the principle of complementary base pairing, and were used as recognition probes to specifically capture the target. Activators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP) was used as a signal amplification strategy to improve the sensitivity. The initiator modified at the end of an antibody initiates the ARGET ATRP reaction. Different fluorescence signals were designed to achieve the simultaneous detection of OTA and AFB1 with limits of 426.18 and 79.55 fg mL-1 for AFB1 and OTA, respectively. In addition, experiments were conducted on three types of samples, and the recoveries of the two mycotoxins ranged from 87.30% to 109.50%, with relative standard deviations ranging from 0.50% to 4.92% under reproducible conditions. The results suggest that the developed aptasensor is sufficient to meet the different regulatory requirements of the two mycotoxins in food and drug safety and shows great potential.

Label-free ratiometric fluorescence detection of Pb2+via structure-specific fluorescent dyes and dual signal amplification.

Lead ions (Pb2+) are a widely distributed and highly toxic heavy metal pollutant, which seriously threatens the environment, economy and human safety. Here, a label-free ratiometric fluorescent biosensor was constructed for Pb2+ detection using DNAzyme-driven target cycling and exonuclease III (Exo III)-mediated DNA cycling as a dual signal amplification strategy. The SYBR Green I (SGI) and N-methyl mesoporphyrin IX (NMM) used in this study are characterized by low cost, storage resistance, and short preparation time compared with conventional signaling probes labeled with fluorescent groups. Unlike the single-emission fluorescence strategy, monitoring the fluorescence intensity ratio of SGI and NMM can effectively reduce external interference to achieve accurate detection of Pb2+. DNAzyme structures on the surface of magnetic beads (MBs) can recognize Pb2+ and activate the target circulatory system to cleave single-stranded DNA (ssDNA). The ssDNA further initiated the Exo III-assisted DNA circulatory system to digest double-stranded DNA (dsDNA) and release guanine-rich G1. Finally, the fluorescence signals of SGI and NMM were weakened and enhanced, respectively. The sensing strategy achieved a wide linear range from 0.5 to 500 nM and a low limit of detection (LOD) of 26.4 pM. Furthermore, its anti-interference ability and potential applicability for Pb2+ detection in actual samples were verified. This work ingeniously combines the dual signal amplification strategy with the ratiometric sensing strategy constructed by structure-specific fluorescent dyes, which provides a promising method for constructing sensitive and accurate fluorescent biosensors.

Ultrasensitive detection of the H5N1 nucleic acid fragment by ICP-MS using DNA dendrimer-carried silver nanoparticle labeling.

The importance of avian influenza virus (AIV) detection in clinical diagnosis and prognosis has been deeply recognized. In this study, the ultrasensitive detection of AIV subtype H5N1 was achieved by ICP-MS combined with DNA dendrimer-carried silver nanoparticle (AgNP) labeling. First, a magnetic control system was constructed by anchoring double-strand DNAs (dsDNAs) which contained a complementary sequence of H5N1 and two locked triggers on the surface of magnetic beads (MBs). When H5N1 was present, the two triggers were released and initiated dendrimer hybridization chain reactions which led to the generation of DNA dendrimer-carried AgNPs on the surface of the MBs. Finally, the AgNPs were collected via magnetic separation, digested by nitric acid, and tested using ICP-MS. The signal intensities of 107Ag were positively correlated with the concentrations of H5N1. Notably, the DNA dendrimer assembly contributed to significant signal amplification and good sensitivity with the limit of detection as low as 2.0 × 10-11 mol L-1. Moreover, the method displayed favorable selectivity against mismatched H5N1 and good recoveries in human serum samples. It is a promising analytical tool for the H5N1 virus and other subtypes of AIV, and has potential value in clinical diagnosis and prognosis of infectious diseases.

Ultra-sensitive detection of tumor necrosis factor alpha based on silver-coated gold core shell and magnetically separated recognition of SERS aptamer sensors.

A highly sensitive tumor necrosis factor α (TNF-α) detection method based on a surface-enhanced Raman scattering (SERS) magnetic patch sensor is reported. Magnetic beads (MNPs) and core shells were used as the capture matrix and signaling probe, respectively. For this purpose, antibodies were immobilized on the surface of magnetic beads, and then Au@4-MBN@Ag core-shell structures coupled with aptamers and TNF-α antigen were added sequentially to form a sandwich immune complex. Quantitative analysis was performed by monitoring changes in the characteristic SERS signal intensity of the Raman reporter molecule 4-MBN. The results showed that the limit of detection (LOD) of the proposed method was 4.37 × 10-15 mg·mL-1 with good linearity (R2 = 0.9918) over the concentration range 10-12 to 10-5 mg·mL-1. Excellent assay accuracy was also demonstrated, with recoveries in the range 102% to 114%. Since all reactions occur in solution and are separated by magnetic adsorption of magnetic beads, this SERS-based immunoassay technique solves the kinetic problems of limited diffusion and difficult separation on solid substrates. The method is therefore expected to be a good clinical tool for the diagnosis of the inflammatory biomarker THF-α and in vivo inflammation screening.

CRISPR/Cas12a controlled chemiluminescence resonance energy transfer sensor for detection of carcinoembryonic antigen

A CRISPR-Cas12a controlled chemiluminescence resonance energy transfer (CRET) biosensor was constructed for the detection of cancer biomarker carcinoembryonic antigen (CEA). The CRET probe, a single-strand DNA (ssDNA) with hemin/G-quadruplex on one end and CdSe/ZnS quantum dots (QDs) nanosphere on the other end, was used as the substrate for the trans-cleavage of CRISPR/Cas12a system. The hemin/G-quadruplexes catalyzed the oxidation of luminol in the presence of H2O2. The generating chemiluminescence could excite the CdSe/ZnS QDs encapsulated in the nanosphere, producing CRET. When target CEA specifically identified dual aptamers on the surface of magnetic beads (MBs), the terminal sequence of Aptamer 2 triggered the hybridization chain reaction (HCR), whose product initiated the trans-cleavage activity of CRISPR/Cas12a and destroyed the CRET system. As a result, the luminescence of QDs nanosphere was declined. With HCR, CRISPR-Cas12a trans-cleavage, and mimic DNAzyme catalytic amplification, the biosensor gave a detection limit down to 5.6 pg/mL. Due to the application of CRISPR/Cas12a, the CRET sensor with the characteristics of easy operation and no external light source required was more suitable for point-of-care testing (POCT) detection of various targets.

ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction

The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen.

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.

Aptamer-functionalized polydiacetylene biosensor for the detection of three foodborne pathogens.

Rapid, simple and sensitive screening of foodborne pathogens is of great significance to ensure food safety. In this study, an aptamer-functionalized polydiacetylene (Apta-PDA) biosensor was developed for the detection of E. coli O157:H7, S. typhimurium or V. parahaemolyticus. First, aptamers responding to the target bacteria were modified on the surface of magnetic beads by covalent binding to form MBs-oligonucleotide conjugates for bacterial enrichment. Then, an Apta-PDA biosensor was obtained by connecting the aptamers to the PDA nanovesicles using the carbodiimide method. Molecular recognition occurred in the presence of the target bacteria, whereby the aptamer folded into a sequence-defined unique structure, resulting in an MBs-Apta/bacteria/Apta-PDA sandwich structure. Due to the optical properties of PDA, the blue-red transition of the detection system could be observed by the naked eye and quantified by the colorimetric response percentage (CR%). Under optimized conditions, the detection limits of E. coli O157:H7, S. typhimurium and V. parahaemolyticus were 39, 60 and 60CFU/ml, respectively, with a selectivity of 100% and a reaction time of 30min. Compared with the gold standard method, the accuracy of the three target bacteria detection reached 98%, 97.5% and 97%, respectively, and the sensitivity and specificity were both greater than 90%. The entire detection process was rapid and easy to execute without any special equipment, making this technology particularly suitable for resource-poor laboratories or regions.

B-259 Exocas-2 Rapid and Pure Isolation of Exosomes by Anionic Exchange using Magnetic Beads

Abstract Background Extracellular vesicles (EVs) are considered essential biomarkers in liquid biopsies. Despite intensive efforts aimed at employing EVs in a clinical setting, workable approaches are currently limited owing to the fact that EV-isolation technologies are still in a nascent stage. This study introduces a magnetic bead-based ion exchange platform for isolating EVs called ExoCAS-2 (exosome clustering and scattering). Owing to their negative charge, exosomes can easily adhere to magnetic beads coated with a polycationic polymer. Owing to the features of magnetic beads, exosomes can be easily processed via washing and elution steps and isolated with high purity and yield within 40 min. Methods Cationic salt is coated on the surface of magnetic beads with carboxyl group surface residues. Inject the cation coated magnetic bead (ExoCAS) into the plasma solution and incubate in 4°C for 30 minutes. By charge interaction, negative charged EVs are attached to the cationic beads. And then, pH 6 washing buffer and 1M NaCl elution buffer were used for EV isolation. Results The present results confirmed the isolation of exosomes through analyses of size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared with the commercially available methods, the proposed method showed superior performance in terms of key aspects, including operation time, purity, and recovery rate. Conclusion This highlights the potential of this magnetic bead-based ion exchange platform for isolating exosomes present in blood plasma.

First biosensing platform for detecting traces of almonds in processed foods by electrochemical determination of the allergenic protein Pru du 6

A novel electrochemical bioplatform for Pru du 6determination, one of the most abundant and allergenic almond proteins, was developed to detect traces of almonds in processed foods. The bioplatform combines the application of a sandwich immunoassay on the surface of magnetic beads using specific antibodies unmodified or conjugated to horseradish peroxidase as capture and detection antibodies. The resulting magnetic bioconjugates are trapped on the surface of disposable carbon electrodes, and amperometry in the presence of hydrogen peroxide and hydroquinone is employed to measure the change in the cathodic current, which is proportional to Pru du 6 concentration. The developed bioplatform exhibited good selectivity and sensitivity, offering a detection limit of 0.12 ng mL−1 of Pru du 6. It could determine Pru du 6 in raw dough and baked cookies incurred with 0.2 µg g−1 of almond protein with acceptable precision, providing results comparable to those obtained using an ELISA technique.

A CRISPR/Cas12a-SERS platform for amplification-free detection of African swine fever virus genes

A surface-enhanced Raman scattering (SERS) strategy combined with a CRISPR/Cas12a system is designed for the amplification-free gene detection of African swine fever virus (ASFV). A SERS sensing probe was fabricated by conjugating plasmonic SERS tags on the magnetic bead (MB) surface with an single-stranded DNA (ssDNA) as a linker. The target ASFV gene-activated Cas12a protein starts the trans-cleavage function on the linker ssDNA, which causes the release of SERS tags, leading to a decrease of the SERS signal detected above the collective MBs. Two signal enhancement strategies were adopted to improve the liquid-phase detection sensitivity arriving at the fM level. One is the unlimited trans-cleavage function of the Cas12a protein, and the other is the magnetic-induced collection of probes that can significantly gather the analytes from the solution to the laser spot and provide SERS hotspots during SERS measurement. Detection range is from 100 nM to 10 fM without the gene amplification steps. This sensing method achieved the SERS detection of ASFV gene in the serum system and the extracted nucleic acids in viral samples with high sensitivity and selectivity at a relative standard deviation of <8%. This sensing platform is mainly in use for site inspection and quick testing of gene samples.

SERS sensor combined with the dual DNA cycling amplification assay for the sensitive detection of antibiotic resistance gene in environmental samples

Antibiotic resistance genes (ARGs) have emerged as new environmental pollutants, and even minute quantities of them can seriously affect the environment. Thus, it is urgently required to test trace ARGs. Herein, a surface-enhanced Raman scattering (SERS) sensor based on the dual cycling amplification has been developed for the ultrasensitive detection of bla-TEM. First, the capture DNA on the surface of magnetic beads and the Hg2+ aptamer-based capture strand could achieve the capture, separation, and enrichment of the bla-TEM from the relatively complex samples. Then, the dual amplification process was finished under the catalysis of the enzymes, and the entire process was operated in one step under the isothermal condition. Finally, the enriched SERS nanoprobes were obtained via the feasible magnetic separation and provided Raman signals for the detected bla-TEM under the laser irradiation. Under the optimum experimental conditions, the sensor exhibited excellent sensitivity and good selectivity, obtaining a linear range of 0.1 zM-300 aM and a limit of detection (LOD) of 0.02 zM. It can be successfully used to measure bla-TEM in actual environmental samples, which was in good accordance with the conventional polymerase chain reaction (PCR) analysis, showing great promise for detecting trace ARGs in the environmental samples.

Construction of a dual-signal sensing platform based on DNA enhanced peroxidase-activity of iron cobalt oxide nanosheets for thrombin detection

Thrombin played important roles for the diagnosis of neurodegenerative and cardiovascular diseases. Herein, we successfully constructed a rapid and sensitive fluorescence and colorimetric dual-signal sensing strategy for the detection of thrombin by taking advantage of the DNA-enhanced peroxidase like activity FeCo-ONSs and magnetic separation. In this work, single-stranded DNA S1 with thrombin aptamer was used to enhance the catalytic activity of FeCo-ONSs, and single-stranded DNA S2 with different thrombin aptamer was modified on the surface of magnetic beads (MBs) to construct MBs-S2 for subsequent magnetic separation. In the presence of thrombin, S1 and MBs-S2 quickly combined with thrombin and the amount of S1 decreased in the solution after magnetic separation. The enhancement of peroxidase like activity of S1/FeCo-ONSs was weakened, resulting in the inhibition of the catalytic oxidation process of substrate TMB. The formation of oxTMB and the fluorescence quenching efficiency of MoS2 QDs were further affected. Therefore, the quantitative detection of thrombin can be achieved by measuring the absorption signal of oxTMB and the fluorescence signal of MoS2 QDs. The limits of detection for thrombin were 0.67 pM and 2.36 pM by the outputting fluorometric and colorimetric dual signals, respectively. This sensing strategy not only has high specificity, but also can be expanded as a more universal platform for sensitive detection of different proteins by changing the aptamer probe pairs.

Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform.

A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant isvery important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developedmethod is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked withdifferent percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λex/λem of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.

Dual-Toehold-Probe-Mediated Exonuclease-III-Assisted Signal Recycles Integrated with CHA for Detection of mecA Gene Using a Personal Glucose Meter in Skin and Soft Tissue Infection.

Staphylococcus aureus integrated with mecA gene, which codes for penicillin-binding protein 2a, is resistant to all penicillins and other beta-lactam antibiotics, resulting in poor treatment expectations in skin and soft tissue infections. The development of a simple, sensitive and portable biosensor for mecA gene analysis in S. aureus is urgently needed. Herein, we propose a dual-toehold-probe (sensing probe)-mediated exonuclease-III (Exo-III)-assisted signal recycling for portable detection of the mecA gene in S. aureus. When the target mecA gene is present, it hybridizes with the sensing probe, initiating Exo III-assisted dual signal recycles, which in turn release numerous "3" sequences. The released "3" sequences initiate catalytic hairpin amplification, resulting in the fixation of a sucrase-labeled H2 probe on the surface of magnetic beads (MBs). After magnet-based enrichment of an MB-H1-H2-sucrase complex and removal of a liquid supernatant containing free sucrase, the complex is then used to catalyze sucrose to glucose, which can be quantitatively detected by a personal glucose meter. With a limit of detection of 4.36 fM for mecA gene, the developed strategy exhibits high sensitivity. In addition, good selectivity and anti-interference capability were also attained with this method, making it promising for antibiotic tolerance analysis at the point-of-care.