Distinguishable Magnetic Reporter Coordination with Buoyancy-Magnetism Separation for Immobilization-Free Dual-Target Electrochemical Immunosensing.

Simultaneous sensitive and precise determination of multibiomarkers is of great significance for improving detection efficiency, reducing diagnosis and treatment expenses, and elevating survival rates. However, the development of simple and portable biosensors for simultaneous determination of multiplexed targets in biological fluids still faces challenges. Herein, a unique and versatile immobilization-free dual-target electrochemical biosensing platform, which combines distinguishable magnetic signal reporters with buoyancy-magnetism separation, was designed and constructed for simultaneous detection of carcinoembryonic (CEA) and α-fetoprotein (AFP) in intricate biological fluids. To construct such distinguishable magnetic signal reporters with signal transduction, amplification, and output, secondary antibodies of CEA and AFP were respectively functionalized on methylene blue (MB) and 6-(ferrocenyl)hexanethiol (FeC) modified Fe3O4@Au magnetic nanocomposites. Meanwhile, a multifunctional flotation probe with dual target recognition, capture, and isolation capability was prepared by conjugating primary antibodies (Ab1-CEA, Ab1-AFP) to hollow buoyant microspheres. The target antigens of CEA and AFP can trigger a flotation-mediated sandwich-type immunoreaction and capture a certain amount of the distinguishable magnetic signal reporter, which enables the conversion of the target CEA and AFP quantities to the signal of the potential-resolved MB and FeC. Thus, the MB and FeC currents of magnetically adsorbed distinguishable magnetic reporters can be used to determine the CEA and AFP targets simultaneously and precisely. Accordingly, the proposed strategy exhibited a delightful linear response for CEA and AFP in the range of 100 fg·mL-1-100 ng·mL-1 with detection limits of 33.34 and 17.02 fg·mL-1 (S/N = 3), respectively. Meanwhile, no significant nonspecific adsorption and cross-talk were observed. The biosensing platform has shown satisfactory performance in the determination of real clinical samples. More importantly, the proposed approach can be conveniently extended to universal detection just by simply substituting biorecognition events. Thus, this work opens up a new promising perspective for dual and even multiple targets and offers promising potential applications in clinical diagnosis.

Chemical Constituents, Hypolipidemic, and Hypoglycemic Activities of Edgeworthia gardneri Flowers.

The flowers of Edgeworthia gardneri are used as herbal tea and medicine to treat various metabolic diseases including hyperglycemia, hypertension, and hyperlipidemia. This paper investigate the chemical constituents and biological activities of ethanolic extract and its different fractions from E. gardneri flowers. Firstly, the E. gardneri flowers was extracted by ethanol-aqueous solution to obtain crude extract (CE), which was subsequently fractionated by different polar organic solution to yield precipitated crystal (PC), dichloromethane (DCF), ethyl acetate (EAF), n-butanol (n-BuF), and residue water (RWF) fractions. UHPLC-ESI-HRMS/MS analysis resulted in the identification of 25 compounds, and the main compounds were flavonoids and coumarins. The precipitated crystal fraction showed the highest phenolic and flavonoid contents with 344.4 ± 3.38 mg GAE/g extract and 305.86 ± 0.87 mg RE/g extract. The EAF had the strongest antioxidant capacity and inhibitory effect on α-glucosidase and pancreatic lipase with IC50 values of 126.459 ± 7.82 and 23.16 ± 0.79 µg/mL. Besides, both PC and EAF significantly regulated the glucose and lipid metabolism disorders by increasing glucose consumption and reducing TG levels in HepG2 cells. Molecular docking results suggested that kaempferol-3-O-glucoside and tiliroside had good binding ability with enzymes, indicating that they may be potential α-glucosidase and pancreatic lipase inhibitors. Therefore, the E. gardneri flowers could be served as a bioactive agent for the regulation of metabolic disorders.

Fascinating Immobilization-Free Electrochemical Immunosensing Strategy Based on the Cooperation of Buoyancy and Magnetism.

Rapid and accurate detection of biomolecules is of vital importance for the diagnosis of disease and for performing timely treatments. The point-of-care analysis of cancer biomarkers in the blood with low cost and easy processing is still challenging. Herein, an advanced and robust strategy, which integrates the buoyant recognition probe with the magnetic reporter probe in one solution, was first proposed for immobilization-free electrochemical immunosensing. The tumor marker of alpha fetoprotein (AFP) can be captured immune-buoyantly, and then a multifunctional magnetic reporter probe in pseudo-homogeneous solution was further captured to fulfill a sandwich-type immunoreaction. The residual magnetic reporter probe can be firmly and efficiently attracted on a magnetic glassy carbon electrode to fulfill the conversion of the target AFP amount into the residual magnetic electrochemical signal indicator. As a result, the electrochemical signal of methylene blue can accurately reflect the original level of target antigen AFP concentration. By integrating buoyancy-driven quasi-homogenous biorecognition with magnetism-mediated amplification and signal output, the proposed immobilization-free electrochemical immunosensing strategy displayed a wide range of linear response (100 fg mL-1 to 10 ng mL-1), low detection limit (14.52 fg mL-1), and good reproducibility, selectivity, and stability. The designed strategy manifests remarkable advantages including assay simplicity, rapidness, and high sensitivity owing to the in-solution instead of on-electrode biorecognition that could accelerate and improve the biorecognition efficiency. To the best of our knowledge, this is the first cooperation of buoyancy-driven biorecognition with magnetism-mediated signal output in bioanalysis, which would be attractive for rapid clinic biomedical application. Thus, this work provides a fresh perspective for convenient and favorable immobilization-free electrochemical biosensing of universal biomolecules.

N/S-Co-doped carbon dot-based FRET ratiometric fluorescence aptasensing platform modulated with entropy-driven DNA amplifier for ochratoxin A detection.

This study proposes a nitrogen and sulfur co-doped carbon dot (N/S-CD)-based FRET ratiometric fluorescence aptasensing strategy modulated with entropy-driven DNA amplifier for sensitive and accurate detection of ochratoxin A (OTA). In the strategy, a duplex DNA probe containing OTA aptamer and complementary DNA (cDNA) is designed as a recognition and transformation element. Upon sensing of target OTA, the cDNA was liberated, and triggered a three-chain DNA composite-based entropy-driven DNA circuit amplification, making CuO probes anchor on a magnetic bead (MB). The CuO-encoded MB complex probe is finally turned into abundant Cu2+, which oxidizes o-phenylenediamine (oPD) to generate 2,3-diaminophenazine (DAP) with yellow fluorescence and further triggers FRET between the blue fluorescent N/S-CDs and DAP. The changes in ratiometric fluorescence are related to the OTA concentration. Originating from the synergistic amplifications from the entropy-driven DNA circuits and Cu2+ amplification, the strategy dramatically enhanced detection performance. A limit of detection as low as 0.006 pg/mL of OTA was achieved. Significantly, the aptasensor can visually evaluate the OTA via on-site visual screening. Moreover, the high-confidence quantification of the OTA in real samples with results consistent with that of the LC-MS method indicated that the proposed strategy has practical application prospects for sensitive and accurate quantification in food safety.

Engineering an Ag/Au bimetallic nanoparticle-based acetylcholinesterase SERS biosensor for in situ sensitive detection of organophosphorus pesticide residues in food.

Developing simple, efficient, and inexpensive method for trace amount organophosphorus pesticides' (OPs) detection with high sensitivity and specificity is of significant importance for guaranteeing food safety. Herein, an Ag/Au bimetallic nanoparticle-based acetylcholinesterase (AChE) surface-enhanced Raman scattering (SERS) biosensor was constructed for in situ simple and sensitive detection of pesticide residues in food. The principle of this biosensor exploited 4-mercaptophenylboronic acid (4-MPBA)-modified Ag/Au bimetallic nanoprobes as SERS signal probe to improve sensitivity and stability. The combination of AChE and choline oxidase (CHO) can hydrolyze acetylcholine (ATCh) to generate H2O2. The product of H2O2 selectively oxidizes the boronate ester of 4-MPBA, decreasing the Raman intensity of the B-O symmetric stretching. In the presence of OPs, it could inhibit the production of H2O2 by destroying the AChE activity, so the reduction of the SERS signal was also alleviated. Based on the principle, an Ag/Au bimetallic nanoparticle-based AChE SERS sensor was established without any complicated pretreatments. Benefiting from the synergistic effects of Ag/Au bimetallic hybrids, a linear detection range from 5×10-9 to 5×10-4 M was achieved with a limit of detection down to 1.7×10-9 M using parathion-methyl (PM) as the representative model of OPs. Moreover, the SERS biosensor uses readily available reagents and is simple to implement. Importantly, the proposed SERS biosensor was used to quantitatively analyze OP residues in apple peels. The levels of OPs detected in real samples by this method were consistent with those obtained using gas chromatography-mass spectrometry (GC-MS), suggesting the proposed assay has great potential applications for OPs in situ detection in food safety fields.

Influence of Ultra-High-Pressure Pretreatment Method on Chemical Constituents, Antioxidant and Cytoprotective Activities of Free, Esterified, and Bound Phenolics from Anneslea Fragrans Wall. Leaves.

Anneslea fragrans Wall., an edible and medicinal plant, is traditionally used to treat liver and gastrointestinal diseases. This paper aimed to investigate the influence of ultra-high pressure (UHP) pretreatment on the phenolics profiling, antioxidant, and cytoprotective activities of free (FP), esterified (EP), and bound (BP) phenolics from A. fragrans leaves. A total of 32 compounds were characterized and quantified. The davidigenin (44.46 and 113.37 mg/g extract) was the highest in A. fragrans leaves. The vitexin (9), afzelin (10), coreopsin (15), and davidigenin (28) were analyzed with MS2 fragment pathways. Results showed that UHP treated A. fragrans leaves had higher total phenolic (TPC) and total flavonoid (TFC) contents of FP, EP, and BP fractions than those in the raw leaves. Moreover, UHP pretreated A. fragrans leaves had higher scavenging activities on DPPH+• and ABTS+•, and inhibitory effects on the intracellular ROS generation in H2O2-induced HepG2 cells. UFP showed the highest inhibition of ROS production among the samples. Therefore, UHP pretreatment method might be used as an effective strategy for elevating the availabilities of A. fragrans leaves to develop functional foods.

Ratiometric Electrochemiluminescence Sensing of Carcinoembryonic Antigen Based on Luminol.

Ratiometric electrochemiluminescence (ECL) sensors can efficiently remove environmental interference to attain precise detection. Nonetheless, two eligible luminophores or coreactants were usually needed, increasing the complexity and restricting their practical application. In this study, a single luminophore of luminol with a single coreactant of H2O2 was employed to construct a dual-potential ratiometric ECL sensor for the detection of carcinoembryonic antigen (CEA). The produced palladium nanoclusters (Pd NCs) employing a DNA duplex as a template could not only stimulate luminol to produce cathodic ECL (Icathodic) but also quench its anodic ECL (Ianodic). During the detection process, CEA could damage the double-stranded structure and reduce the Pd NCs' amount, triggering a significant decrease in the ratio of Icathodic to Ianodic (Icathodic/Ianodic) and thereby achieving sensitive CEA's detection. Furthermore, the Icathodic/Ianodic was independent of the H2O2 concentration, which avoided a prejudicial effect from H2O2 decomposition and considerably enhanced the detection's reliability. The developed ratiometric ECL sensor demonstrated a sensitive detection toward CEA with a wide linear range from 100 ag/mL to 10 ng/mL and a detection limit of 87.1 ag/mL (S/N = 3). In conclusion, this study offers a new idea for constructing ratiometric ECL sensors based on a single luminophore and technical support for cancer's early diagnosis.

Thermally Hypsochromic or Bathochromic Emissions? The Silver Nuclei Does Matter.

Structural modulation of core-shell silver nanoclusters from the inside is a huge challenge but of great importance in their syntheses. Herein, two silver nanoclusters [Ag3 S9 @Ag42 ] (SD/Ag45b) and [Ag9 S9 @Ag42 ] (SD/Ag51a) are isolated in the presence of different kinds of sulfonic acids. Uniquely, SD/Ag45b and SD/Ag51a show typical core-shell structures with the similar Ag42 shell but different cores. The outer shell of 42 silver atoms comprises two Ag3 trigons at two poles encircled by three equatorial distorted square cupolas (J4 , Ag12 ). The core in SD/Ag45b is a silver trigon ligated by nine S2- ions (Ag3 S9 ), while a tricapped triangular prismatic Ag9 also ligated by the same amount of S2- ions (Ag9 S9 ) is observed in the inner core of SD/Ag51a. The electrospray ionization mass spectrometry (ESI-MS) indicates that the introduction of p-toluenesulfonic acid can realize the transformation from SD/Ag45b to Ag51 . SD/Ag45b and SD/Ag51a show inverse luminescence thermochromic behaviors in the near-infrared (NIR) region, mainly dictated by the inner silver cores. This work not only realizes the synthesis of new silver nanoclusters by core modulation but also provides a prototype to get molecular-level insight into the correlation between structure and luminescence thermochromism.

Spatially-extended 3D magnetic DNA nanodevice-based split-type photoelectrochemical strategy for sensitive and reliable miRNA detection in cancer cells.

Highly sensitive and reliable PEC detection of miRNAs still faces some challenges like the inaccuracy caused by coexisting interferences in the PEC system. Herein, we developed a split-type "turn-off" PEC biosensor based on spatially-extended 3D magnetic DNA nanodevices with high-order DNA amplifiers for sensitive detection of miRNAs in cancer cells.

Liquid-liquid interfacial self-assembled triangular Ag nanoplate-based high-density and ordered SERS-active arrays for the sensitive detection of dibutyl phthalate (DBP) in edible oils.

DBP, one of the phthalic acid esters (PAEs), is known as an endocrine disruptor and is toxic to humans in abnormal concentrations. Here, a high-density and ordered SERS substrate based on the self-assembly of triangular Ag nanoplate (TAgNP) arrays is developed for DBP detection. Benefiting from the ordered arrangement and sharp corners of TAgNPS, the arrays can provide sufficient and uniform hotspots for reproducible and highly active SERS effects. Using Rhodamine 6G (R6G) as a reporter molecule, the SERS enhancement factor (EF) of the TAgNP arrays was found to be as high as 1.2 × 107 and the relative standard deviation was 6.56%. As a trial for practical applications, the TAgNP array substrates were used for the detection of dibutyl phthalate (DBP) in edible oils. In this assay, edible oil samples were added to hexane as an organic phase for the formation of the TAgNP arrays, which caused DBP to be loaded at hotspots. DBP in edible oils could be identified at concentrations as low as 10-7 M. This SERS substrate based on the TAgNP arrays has great potential applications in the high sensitivity and reproducible detection of contaminants in food.

Lowly-aggregated perylene diimide as a near-infrared electrochemiluminescence luminophore for ultrasensitive immunosensors at low potentials.

In the electrochemiluminescence (ECL) field, most reported luminophores were focused on high-triggering potential and short wavelength, which was adverse for the ECL theory study and application at low potentials. Perylene diimide derivatives could emit near-infrared (NIR) ECL at low-triggering potential; however, they are always highly aggregated into a microrod structure and stacked together, which largely limited their application in biological fields such as bio-sensing and bio-imaging. To overcome these obstacles, we designed a novel perylene diimide molecule, namely N,N'-dicaproate sodium-3,4,9,10-perylenedicarboximide (PDI-COONa). This molecule self-assembled into a two-dimensional network nanostructure, which largely decreased the aggregation degree of PDI molecules and provided solid bases for designing lowly-aggregated PDI molecules. Also, the formed nanoluminophore produced strong emission at -0.26 V with an NIR wavelength 700 nm, which should be due to the excited J-type PDI-COO- dimers. Moreover, this network nanoluminophore well-dispersed on graphene oxide (GO) as an ECL nanomaterial to label secondary antibodies and fabricate a sandwiched immunosensor for alpha-fetoprotein (AFP) detection between 0 and -0.6 V. This immunosensor showed a wider linear response for AFP ranging from 0.1 fg mL-1 to 1 µg mL-1 with a low detection limit 0.0353 fg mL-1 compared with other immunosensors based on PDI microrod-modified GO ECL materials. The fabricated immunosensor also showed good feasibility in human serum samples.

A novel ratiometric electrochemical biosensing strategy based on T7 exonuclease-assisted homogenous target recycling coupling hairpin assembly-triggered double-signal output for the multiple amplified detection of miRNA.

A novel ratiometric electrochemical biosensing strategy based on T7 exonuclease (T7 Exo)-assisted homogenous target recycling coupling hairpin assembly triggered dual-signal output was proposed for the accurate and sensitive detection of microRNA-141 (miRNA-141). Concretely, in the presence of target miRNA, abundant signal transduction probes were released via the T7 Exo-assisted homogenous target recycling amplification, which could be captured by the specially designed ferrocene-labeled hairpin probe (Fc-H1) on -electrode interface and triggered the nonenzymatic catalytic hairpin assembly (Fc-H1 + MB-H2) to realize the cascade signal amplification and dual-signal output. Through such a conformational change process, the electrochemical signal of Fc (IFc) and MB (IMB) is proportionally and substantially decreased and increased. Therefore, the signal ratio of IMB/IFc can be employed to accurately reflect the true level of original miRNA. Benefiting from the efficient integration of the T7 Exo-assisted target recycle, nonenzymatic hairpin assembly and dual-signal output mode, the proposed sensor could realize the amplified detection of miRNA-141 effectively with a wide detection range from 1 fM to 100 pM, and a detection limit of 200 aM. Furthermore, it exhibits outstanding sequence specificity to discriminate mismatched RNA, acceptable reproducibility and feasibility for real sample. This strategy effectively integrated the advantages of multiple amplification and ratiometric output modes, which could provide an accurate and efficient method in biosensing and clinical diagnosis.

Simple and sensitive detection of deoxyribonucleic acid using a RecA-GFP fusion protein-DNA filament as probe.

A simple, rapid and highly sensitive method for detection of double-stranded DNA (dsDNA) was developed using a novel fluorescence probe composed of a RecA-GFP fusion protein that had specific recognition of ssDNA complexes (RecA-GFP-DNA filament). The RecA-GFP fusion protein not only had strong fluorescence, but could also occur by homologous recombination. In the presence of the target dsDNA, the complementary ssDNA of the RecA-GFP-DNA filaments invaded one end of the dsDNA chain. In addition, the other end of the ssDNA dissociated the RecA-GFP filaments. An assay of the probe showed a linear relationship with dsDNA concentration in the range 1-11 nM, with a correlation coefficient of 0.9923. The limit of detection for dsDNA was determined experimentally to be 0.3 nM (3δ). Compared with conventional methods, this method has the advantages of simple operation, high specificity, and high sensitivity.

Graphene-PtPd nanocomposite for low-potential-driven electrochemiluminescent determination of carcinoembryonic antigen using Ru(bpy)32.

As well known, the electrochemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) heavily relies on highly positive or negative triggered voltage, prejudicing the detection toward the bio-molecules. In this work, Ru(bpy)32+ could generate enhanced and stable ECL at a low potential of 0.05 V (vs. Ag/AgCl) on graphene-PtPd hybrid, attributing to its excellent electrocatalysis from the synergistic effect between Pt and Pd. The obtained low-potential-driven ECL could be quenched by MoS2 nanoflowers. Based on the quenching effect, a sandwich "signal-off" ECL immunosensor was fabricated to sensitively detect carcinoembryonic antigen (CEA). A linear calibration curve from 1 fg mL-1 to 1 ng mL-1 was obtained along with a low detection limit of 0.54 fg mL-1 (S/N = 3) under optimal conditions. The sensor showed satisfactory specificity, stability, and reproducibility and was successfully applied to determine CEA in actual samples. The recoveries ranged from 98.80 to 100.23%, and the relative standard deviation (RSD) was lower than 5%. Above all, this work explored new materials in low-potential-driven ECL system and provided a reliable sensing strategy for clinical applications.

Bioactivity-Guided Isolation of Phytochemicals from Vaccinium dunalianum Wight and Their Antioxidant and Enzyme Inhibitory Activities.

Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V.dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A-E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1-10). 6-O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds.

Correction: A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay.

Correction for 'A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay' by Fei Yin, et al., Analyst, 2019, 144, 5504-5510, DOI: 10.1039/C9AN01016E.

Multi-code magnetic beads based on DNAzyme-mediated double-cycling amplification for a point-of-care assay of telomerase activity.

Development of a reliable and facile telomerase activity assay with high specificity and sensitivity is a central challenge to make telomerase testing a routine part of medical care with respect to cancer. Herein, we propose a point-of-care (POC) assay of telomerase activity based on multi-code magnetic beads initiated by DNAzyme-mediated double-cycling amplification coupling with a glucometer. A designed DNAzyme-based telomerase substrate prime (DTSP) probe consists of three regions (AXB) for a DNAzyme catalytic sequence, poly-thymine (poly-T) linker and telomere primer sequence. In the presence of telomerase, telomerase elongates the primer site on the DTSP probe. Subsequent addition of the PbII cofactor activates the DNAzyme, which cleaves the elongated fragment at the RNA site, and releases the DTSP probe for repetitive cycling and the elongated fragment. The cleaved fragment as a trigger then continuously triggers the cyclic strand displacement reaction (CSDR) between MB-H1 and invertase-H2, constructing the second cycling and forming the MB-H1/invertase-H2 duplex. Owing to the double-cycling amplification, numerous invertases were coded onto the surface of MBs. After magnetic separation, the enriched multi-code MB catalytically hydrolyzes the sucrose substrate with multiple turnovers, generating a large amount of glucose for quantitative readout by personal glucose meters (PGM). Benefiting from the double-cycling amplification and magnetic separation, a high signal-to-background ratio was achieved, and the point-of-care assay realizes sensitive telomerase activity detection down to 5 HeLa cells with a significantly enhanced dynamic range without the need for enzymatic amplification. The designed DTSP probe highly ensured the specificity and reliability due to the high dependence of DNAzyme on the metal ion cofactor. Moreover, this method can be applied for the screening of telomerase inhibitors and the discrimination of cancer cells from normal cells. With the ability of simple operation, outstanding sensitivity, and excellent selectivity, this method offers a convenient and specific POCT strategy for telomerase activity detection, holding great potential in clinical diagnosis and drug discovery.

A dandelion-like liposomes-encoded magnetic bead probe-based toehold-mediated DNA circuit for the amplification detection of MiRNA.

The development of facile and sensitive miRNA quantitative detection methods is a central challenge for the early diagnosis of miRNA-related diseases. Herein, we propose a strategy for a liposome-encoded magnetic bead-based DNA toehold-mediated DNA circuit for the simple and sensitive detection of miRNA based on a toehold-mediated circular strand displacement reaction (TCSDR) coupled with a personal glucometer (PGM ). In this strategy, a glucoamylase-encapsulated liposomes (GELs)-encoded magnetic bead (GELs-MB) probe is designed to integrate target binding, magnetic separation, and signal response. Upon sensing the target miRNA-21, a GELs-MB probe-based toehold-mediated circular strand displacement reaction (TCSDR) was initiated with the help of fuel-DNA, constructing a DNA circuit system, and realizing target recycling amplification and the disassembly of the liposomes. The disassembled liposomes were finally removed via magnetic separation, and the encapsulated glucoamylase was liberated to catalyze amylose hydrolysis with multiple turnovers to glucose for a PGM readout. Benefiting from target recycling amplification initiated by the toehold-mediated DNA circuit and the liposome multiple-label amplification, a small quantity of target miRNA-21 can be transformed into a large glucose signal. The strategy realized the quantification of miRNA-21 down to a level of 0.7 fM without enzymatic amplification or precise instrumentation. Moreover, the high-density GELs-MB probe allows the sensitive detection of miRNA-21 to be accomplished within 1.5 h. Furthermore, this strategy exhibits the advantages of specificity and simplicity, since a toehold-mediated strand displacement reaction, magnetic separation and portable PGM were used. Importantly, this strategy has been demonstrated to allow the high-confidence quantification of miRNA. Therefore, with the advantages of low cost, ease of use, portability, and sensitivity, the reported method holds great potential for the early diagnosis of miRNA-related diseases.

A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay.

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment. Herein, a simultaneous detection platform of proteins and nucleic acids has been developed using a single substrate probe combining a label-free and background-eliminated fluorescence assay. Telomerase and telomerase RNA (TR) were chosen as the models. The molecular beacon (dU-BIO-HP) that contains deoxyuridine/biotin in its side arm, a TR recognition sequence in the loop and a telomerase substrate primer at the stem end was ingeniously designed. In the presence of telomerase, the stem of dU-BIO-HP is elongated by the addition of telomere repeats complementary to the assistant DNA. Furthermore, the formed dsDNA performed as engaging primers to initiate a SDA reaction, generating abundant G-quadruplex monomers. Similarly, on TR, the hybridization between TR and dU-BIO-HP can open its stem, triggering another SDA reaction, producing abundant short ssDNAs. With the G-quadruplex binding with ZnPPIX and ssDNA binding with SG for specific fluorescence responses, the label-free multiple detection can be achieved. In our strategy, the deoxyuridine of dU-BIO-HP acts as a barrier to block the DNA extension due to its strong inhibitory effects on DNA polymerase activity and to make sure that the two SDA reactions occurred independently. The biotin of dU-BIO-HP enables the reduction of the background from the binding between SG, ZnPPIX and dU-BIO-HP through streptavidin-biotin interaction. This method showed an excellent sensitivity with telomerase and TR detection limit of 2.18 HeLa cells per mL and 0.16 × 10-12 M, respectively. Furthermore, the telomerase and TR in different cell lines have been evaluated as powerful tools for biomedical research and clinical diagnosis.

Label-Free Fluorescent DNA Dendrimers for microRNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G-Quadruplex with Low Background Signal.

Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative approach for simple, sensitive, and selective miRNA quantification.