Duplex Probe Research Articles

A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)5NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay

Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S–CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S–CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.

Target-promoted autocatalytic hairpin assembly of bivalent DNAzymes for sensitive and label-free electrochemical metallothionein assay

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L−1 to μmol L−1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Magnetic graphene oxide nanocomposite as dual-mode genosensor for ultrasensitive detection of oncogenic microRNA

MicroRNA (miRNA) is a noncoding RNA that controls cellular functions and gene expression. Several oncogenic miRNAs that aberrantly expressed in prostate cancer have the potential to be used as biomarkers. We designed multifunctional nanosheets that can capture, detect, and quantify miRNA 183-5p from prostate cancer cells with the aid of a disposable printed electrode and a portable potentiostat. Magnetic reduced graphene oxide (MrGO) has been used as the starting nanocomposite to analyze miRNA. Three cationic dyes—toluidine blue (TBO), thionine, and neutral red—were used to modify MrGO and evaluate its impact on the electron transfer rate. MrGO modified with TBO had the fastest conductivity and a large electrochemically active surface area. Two strategies were used to detect miRNA. One used peroxidase-labeled amplification and the other used TBO as the redox probe intercalating in the miRNA-capture probe duplex. The intercalator method reduced the complications of using peroxidase-labeled probes and exhibited superior performance. The limits of miRNA detection in human serum and urine were 3.73 and 0.86 aM, respectively, with a linear range from 0.1 nM to over 1 aM. The assay time of the intercalator method, including wash, was less than 16 min, and only one sample droplet (5 μL) was needed for analysis. We provided dual-mode genosensors for miRNA detection, which might be used for point-of-care testing. The incorporation of MrGO, screen-printed carbon electrodes, and portable potentiostat can accelerate biomarker detection, simplify analysis, and reduce the time and cost of analysis.

Aptamer/proximity hybridization-based label-free and highly sensitive colorimetric detection of methotrexate via polymerization/nicking recycling amplifications

Methotrexate (MTX) is one of the commonly used therapeutic drugs for treating various tumors and autoimmune diseases. However, high dose usage of MTX may cause severe side effects and the monitoring of MTX is therefore critical. By coupling a new MTX aptamer-based proximity hybridization with polymerization/nicking reaction (PNR) recycling amplifications, we develop here a sensitive and label-free colorimetric approach for MTX detection in diluted human serums. The MTX molecules can bind and switch the conformation of aptamers in the DNA duplex probes to initiate subsequent proximity hybridization-induced PNR recycling processes for the yield of a great deal of G-quadruplexes with the assistance of two single-stranded assistant DNA sequences. Hemin subsequently combines with these G-quadruplexes to produce lots of G-quadruplex/hemin horseradish peroxidase (HRP) mimicking DNAzymes, which then catalyze intensified color transition of the substrate solution to exhibit highly magnified UV–Vis absorption for label-free and ultrasensitive detection of MTX at concentration as low as 5.66 nM in the range of 10 nM to 1 μM. High selectivity of the developed method also enables it to monitor low levels of MTX in diluted serum samples, which offers such a method enormous potentials for convenient and highly sensitive detection of other small molecule drugs for various clinical applications.

Construction of Bioluminescent Sensors for Label-Free, Template-Free, Separation-Free, and Sequence-Independent Detection of both Clustered and Isolated Damage in Genomic DNA.

DNA damage induced by endogenous/exogenous factors may cause various diseases, and the genomic DNA damage has become an important biomarker for clinical diagnosis and risk assessment, but it remains a great challenge to accurately quantify both clustered and isolated damage because of their random locations, large diversity, and low abundance. Herein, we demonstrate the development of bioluminescent sensors for label-free, template-free, separation-free, and sequence-independent detection of both clustered and isolated damage in genomic DNA based on the base-excision repair (BER) pathway and terminal transferase (TdT)-initiated template-free isothermal cyclic amplification. The damaged bases are cleaved by DNA glycosylase to generate a new 3'-OH terminus, and subsequently, TdT catalyzes the repeated incorporation of dTTPs into the 3'-OH terminus to produce poly-T structures which can hybridize with the signal probe to form a poly-T sequence/signal probe duplex. Under the lambda exonuclease hydrolysis, a large number of adenosine monophosphate (AMP) molecules are produced to generate a high bioluminescence signal through the cyclic interconversion of AMP-adenosine triphosphate (ATP)-AMP in the presence of luciferin and firefly luciferase. Moreover, the introduction of APE1-induced cyclic cleavage signal amplification can greatly improve the detection sensitivity. The proposed strategy can detect both clustered and isolated damage in genomic DNA with extremely high sensitivity and excellent specificity, and it can even distinguish 0.001% DNA damage in the mixture. Importantly, it can detect the cellular DNA damage with a detection limit of 0.011 ng and further extend to measure various DNA damage with the integration of appropriate DNA repair enzymes.

Cascade signal enhancement by integrating DNA walking and RCA reaction-assisted “silver-link” crossing electrode for ultrasensitive electrochemical detection of Staphylococcus aureus

The key factor to control the incidence rate of diseases caused by bacteria is rapid detection and early diagnosis. Herein, we proposed a new electrochemical bacterial sensor by coupling DNA walking and rolling circle amplification (RCA) reaction-assisted “silver-link” crossing electrode. Staphylococcus aureus (S. aureus) was detected using this proof-of concept strategy. Aptamer/DNA walker and auxiliary sequence (AS)/RCA reaction probe (RP) duplexes were modified on the electrode surface. The binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker and released DNA walker. With the help of Exo III, DNA walker moved along the electrode surface and AS in AS/RP duplex was continuously digested to release RP. By introducing phi29 DNA polymerase, RCA reaction was performed using RP as the reaction primer to form long single-strand RCA extension products between the electrodes. The “silver-link” crossing electrode was formed by metallization of “gene-link”, significant conductivity was thus acquired for bacteria detection. The limit of detection (LOD) was 10 CFU/mL and detection time was 2 h. The proposed sensor has high efficiency, good stability and low background signal, human serum and milk samples were successfully detected, which emerged a promising potential in the food monitoring and clinical diagnosis.

Aptamer-based and sensitive label-free colorimetric sensing of phenylalanine via cascaded signal amplifications

The concentration variation of phenylalanine (Phe), an essential amino acid in humans, can cause metabolism disorders and even mental disability. Sensitive and convenient monitoring of Phe is therefore important for disease diagnosis. We describe here the establishment of a new aptamer-based, sensitive and label-free colorimetric Phe detection strategy by integrating catalytic hairpin assembly (CHA) and Mg2+-dependent DNAzyme amplification cascades. The target Phe coordinates with pentamethylcyclopentadienyl rhodium(III) chloride dimer [(Cp*RhCl2)2] to form a complex that has a high affinity to the corresponding aptamer sequence. Upon its binding to the aptamers in DNA duplex probes, ssDNA strands are released to trigger subsequent CHA reactions for the formation of many DNAzymes, which cleave the substrate signal probes to liberate lots of CHA initiation strands and free G-quadruplexes to realize the cascaded amplifications. Hemin further associates with the many G-quadruplexes to yield hemin/G-quadruplex mimicking peroxidases, which catalyze solution of substrate to exhibit highly enhanced UV–vis adsorption for detecting Phe at 0.19 μM level. At the meantime, the monitoring of Phe in diluted serums with high selectivity has also been demonstrated by the developed method, indicating its potential for simple diagnosis of Phe-related diseases.

CRISPR/Cas12a-derived sensitive electrochemical biosensing of NF-κB p50 based on hybridization chain reaction and DNA hydrogel

Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.

Hypersensitive detection of transcription factors by multiple amplification strategy based on molecular beacon

Detection and quantification of specific targets at ultralow concentrations are crucial in biotechnological applications and biomedical diagnostics. However, traditional detection methods are time-consuming and complex. In this study, a simple, sensitive, and specific fluorescence-based strategy to detect transcription factors (TFs) was developed by combining molecular beacon (MB) with exonuclease III (Exo III). The strategy was validated using nuclear factor-kappa B (NF-κB) p65 as a model case. In this assay, the TF concentration is determined by estimating the concentration of the nucleic acid released through the reactions of TFs with DNA-1/DNA-2 duplex probe and Exo III; thus, this method can overcome the limitations of the traditional methods. The nucleic acid is then cyclically amplified in the presence of MB and Exo III, and the sequence fragments containing the fluorescent group are released. This method is sensitive, expandable, and suitable for the direct detection of TFs in crude nuclear extracts of cancer cells. Furthermore, it can provide key test data for studies of coronavirus disease 2019-induced acute respiratory distress syndrome and related research.

An "off-on" electrochemiluminescence biosensor coupled with strand displacement-powered 3D micromolecule walking nanomachine for ultrasensitive detection of adenosine triphosphate.

Athree-dimensional (3D) micromolecule walking nanomachine propelled by strand displacement was developed to establish a novel switching electrochemiluminescence (ECL) biosensor for ultrasensitive detection of adenosine triphosphate (ATP). Generally, walking nanomachines reported previously were limited to DNA walkers, while the proposed 3D walking nanomachine focused on the micromolecule walker. Firstly, TiO2 and silver nanoparticles (Ag NPs) functionalized N-(4-aminobutyl)-N-(ethylisoluminol) (Ag-ABEI) were deposited onto the electrode surface to offer an enhanced ECL signal, resulting from the double catalytic effect of TiO2 and Ag NPs for H2O2. Following, dopamine (DA)-labeled DNA duplex probes (S1/S2-DA) immobilized onto the modified electrode cut down the original ECL signal due to the quenching of DA toward ABEI (signal-off). Target ATP walker moved along the 3D DNA track, simultaneously releasing numerous DNA3, which was applied to displace S2-DA, resulting in the quenched ECL intensity recovery (signal-on). As a result, the biosensor showed a low limit of detection down to 0.5 nM (S/N = 3) and was successfully employed to determine ATP in human serum samples. Thus, the established biosensing strategy holds great potential for biochemical studies and clinical diagnosis.

Outside Front Cover: Biotechnology Journal 3/2020

A label-free and enzyme-free uracil DNA glycosylase (UDG) activity assay is described. In the upper left bubble, detection duplex probe serving as substrate for UDG releases a catalyst strand (blue strand) due to the UDG-catalyzed degradation of uracil, which subsequently promotes the toehold-mediated strand displacement (TMSD) reaction in the upper right bubble. As a result, active split G-quadruplexes are produced leading to the colorimetric response in the bottom bubble. This is reported by Youna Kim, Yeonkyung Park, Chang Yeol Lee, Hyun Gyu Park in article 1900420.

Multi-channel collection of G-quadruplex transducers for amplified signaling of Pax-5 based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe

Nucleic acids-sensing methods provide the potential to enable and hasten early disease detection and to rationalize treatments, especially for cancers. In this work, a colorimetric label-free sensing system based on target-triggered split-to-intact remodeling of dual-G-rich duplex probe (Dual-GDP) has been developed for amplified signaling of Pax-5 gene. In this sensing system, the Dual-GDP is splited into one hairpin-structured G-rich probe (HGP) and one duplex-shaped G-rich probe (DGP), which can be re-built to an intact form through target gene triggered accurate base-pairing. Significantly, this unique split-to-intact remodeling behavior allows the occurrence of a well-designed active isothermal amplification that can amplify target response with seven stages to multi-channel accumulate numbers of G-quadruplex transducers. When specifically combined with hemin, the formed G-quadruplex-hemin complexes, also known as DNAzymes, can strongly catalyze the oxidization of TMB to a colored TMB+ and robustly reflect the target gene even in complex biological environments. We demonstrate that this method offers simplified operations and improved assay abilities for Pax5 gene analysis. We envision these findings might provide new insights to exploit versatile oligonucleotide probes and nucleic acid-based amplification strategies for diverse bioanalytical applications.

Nanopore Resistive Pulse Sensing with Multiple Alpha-Hemolysin Pores Improves the Detection Limit of Microrna

The concentration profile of microRNAs in body fluids has been related to a variety of diseases, including specific cancers. By conjugation with a complementary DNA probe, specific miRNA species can be enumerated by nanopore resistive pulsing. This is possible because a miRNA-DNA probe duplex temporarily blocks a nanopore of ∼1.5 nm diameter, causing a decrease in the electrical current by displacing electrolyte ions. Under the influence of the electrophoretic driving force, the duplex dissociates after ∼10-1000 ms, and both the miRNA and the DNA molecules transit through the pore. The pore is now open again and can capture another duplex, with the frequency of the duplex pore translocations corresponding to the miRNA concentration in the solution. Biological porins are attractive because of their reproducible self-assembly, but the lipid bilayer matrix prevents the application of voltages >120 mV. It has previously been shown that the electrophoretic driving force can also be enhanced by an electrolyte concentration gradient of 0.5 / 4 M KCl, but few pulses were observed below 1 nM duplex concentration. Here, we demonstrate that the frequency can be further increased by incorporating multiple pores in the same bilayer. With an electrolyte gradient of 0.2 / 1 M KCl, it was possible to perform resistive pulse sensing with up to 100 alpha-hemolysin pores in an aperture-suspended DPhPC bilayer. We characterize the effect of electrolyte salt gradients on the stability of bilayer-incorporated alpha-hemolysin and quantify the resistive pulses for duplex concentrations in the range 0.01-100 nM. The achieved ∼10-fold increase in frequency, with respect to single-pore sensing with a 0.5 / 4 M KCl gradient, demonstrates the potential of multi-pore sensing to achieve miRNA detection at clinically relevant concentrations.

Determination of the concentration of transcription factor by using exonuclease III-aided amplification and gold nanoparticle mediated fluorescence intensity: A new method for gene transcription related enzyme detection

Herein, we report a new probe for the determination of the concentration of NF-κB p50, one kind of DNA-binding transcription factors (TFs), by using Exonuclease III (Exo III)-aided amplification and gold nanoparticle mediated fluorescence intensity. Since TFs play critical roles in various biological processes, the detection of TFs can provide a lot of useful biological information for studding gene expression regulation related disease. In our system, in the presence of transcription factor, Exo III based amplification reaction was trigged. This enzymatic digestion results in the release of intermediate DNA and ultimately liberating the fluorophore (which, separated from the quencher of AuNP and BHQ2, now fluoresces). The released intermediate DNA then hybridizes with another strand3, whence the cycle starts anew. So, the fluorescence intensity reflects the NF-κB p50 concentration with a detection limit of 1.32 pM. Importantly, this method might be further extended to selectively detect various dsDNA-binding proteins by simply changing the binding-site sequences of strand1/strand2 duplex probes.

Intravascular Ultrasound for Endovascular Aneurysm Repair Compared to Peroperative Angiography. A Pilot Study

Introduction - The aim of this study was to compare intravascular ultrasound (IVUS)-assisted procedures for endovascular aneurysm repair (EVAR) and thoracic endovascular aneurysm repair (TEVAR) with standard angiography-assisted procedures. Methods - From June 2015 to June 2017, 90 consecutive patients underwent endovascular aneurysm repair. Forty procedures were IVUS-assisted (35 EVAR and 5 TEVAR) with X-ray exposure limited to completion angiography for security because IVUS probe does not incorporate a duplex probe. Fifty procedures were angiography-assisted (45 EVAR and 5 TEVAR). The IVUS-assisted procedures were performed by vascular surgeons following a basic Duplex Ultrasound training. The primary study endpoints were the median duration of X-ray exposure, the median duration of the procedure and the amount of contrast medium administered. Secondary endpoints were the operative mortality and morbidity, arterial access complications and incidence of endoleaks. The median length of follow-up was 9 months [6-30 months]. Results - IVUS-assisted procedures required fewer contrast media compared to standard angiography assisted procedures (60 ± 20 ml vs. 120 ± 40 ml, p < .01). Median duration of X-ray exposure was significantly lower in IVUS-assisted procedures (24 ±15 minutes vs 40 ±30 minutes, p < .01). The median duration of the procedure was comparable in both groups (120 ± 30 minutes vs. 140 ± 30 minutes, p = 0.7). No change in renal clearance was observed for IVUS-assisted procedures (97.85ml/min preoperative vs 97.8 ml/min postoperative, p = 0.8) . No postoperative mortality, morbidity and arterial access complications occurred. Early type II endoleaks were observed in 10 patients (11%), 6 in the angiography-assisted group (12%) and 4 in the IVUS-assisted group (10%). These endoleaks were not associated with sac enlargement ≥ 5 mm diameter and did not require any additional treatment. Conclusion - Compared to standard angiography-assisted EVAR, IVUS-assisted EVAR significantly reduces renal load with contrast media as well as X-ray exposure time, while preserving the high efficiency of endovascular aneurysm repair. IVUS allows for precise device sizing and identification of critical vessel origins. Confirmation from large prospective studies with improved IVUS-Duplex probes is needed before IVUS-assisted EVAR could become a solo standard practice.

Imaging NF-κB activity in a murine model of early stage diabetes.

Early pro-inflammatory signaling in the endocrine pancreas involves activation of NF-κB, which is believed to be important for determining the ultimate fate of β-cells and hence progression of type 1 diabetes (T1D). Thus, early non-invasive detection of NF-κB in pancreatic islets may serve as a potential strategy for monitoring early changes in pancreatic endocrine cells eventually leading to T1D. We investigated the feasibility of optical imaging of NF-κB transcription factor activation induced by low-dose streptozocin (LD-STZ) treatment in the immunocompetent SKH1 mouse model of early stage diabetes. In this model, we showed that the levels of NF-κB may be visualized and measured by fluorescence intensity of specific near-infrared (NIR) fluorophore-labeled oligodeoxyribonucleotide duplex (ODND) probes. In addition, NF-κB activation following LD-STZ treatment was validated using immunofluorescence and transgenic animals expressing NF-κB inducible imaging reporter. We showed that LD-STZ-treated SKH1 mice had significantly higher (2-3 times, P<.01) specific NIR FI in the nuclei and cytoplasm of islets cells than in non-treated control mice and this finding was corroborated by immunoblotting and electrophoretic mobility shift assays. Finally, using semi-quantitative confocal analysis of non-fixed pancreatic islet microscopy we demonstrated that ODND probes may be used to distinguish between the islets with high levels of NF-κB transcription factor and control islet cells.

Peroperative Intravascular Ultrasound for Endovascular Aneurysm Repair versus Peroperative Angiography: A Pilot Study in Fit Patients with Favorable Anatomy

The aim of this study was to compare intravascular ultrasound (IVUS) assistance for endovascular aortic aneurysm repair (EVAR) to standard assistance by angiography. From June 2015 to June 2017, 173 consecutive patients underwent EVAR. In this group, 69 procedures were IVUS-assisted with X-ray exposure limited to completion angiography for safety purposes because an IVUS probe does not yet incorporate a duplex probe (group A), and 104 were angiography-assisted procedures (group B). All IVUS-assisted procedures were performed by vascular surgeons with basic duplex ultrasound (DUS) training. The primary study endpoints were mean radiation dose, duration of fluoroscopy, amount of contrast media administered, procedure-related outcomes, and renal clearance expressed as the glomerular filtration rate (GFR) before and after the procedure. Secondary endpoints were operative mortality, morbidity, and arterial access complications. Mean duration of fluoroscopy time was significantly lower for IVUS-assisted procedures (24±15min vs. 40±30min for angiography-assisted procedures, P<0.01). Moreover, mean radiation dose (Air KERMA) was significantly lower in IVUS-assisted procedures (76m Gy [44-102] vs. 131mGy [58-494]), P<0.01. IVUS-assisted procedures required fewer contrast media than standard angiography-assisted procedures (60±20mL vs. 120±40mL, P<0.01). The mean duration of the procedure was comparable in the two groups (120±30min vs. 140±30min, P=0.07). No difference in renal clearance before and after the procedure was observed in either of the two groups (99.0±4/97.8±2mL/min in group A and 98.0±3/97.6±5mL/min in group B) (P=0.28). The mean length of follow-up was ninemonths (6-30months). No postoperative mortality, morbidity, or arterial access complications occurred. No type 1 endoleak was observed. Early type II endoleaks were observed in 21 patients (11%), 12 in the angiography-assisted group (11%) and nine in the IVUS-assisted group (12%). They were not associated with sac enlargement ≥5mm diameter and therefore did not require any additional treatment. Compared with standard angiography-assisted EVAR, IVUS significantly reduces renal load with contrast media, fluoroscopy time, and radiation dose while preserving endograft deployment efficiency. Confirmation from a large prospective study with improved IVUS probes will be required before IVUS-assisted EVAR alone can become standard practice.

Colorimetric Assay for Uracil DNA Glycosylase Activity Based on Toehold-Mediated Strand Displacement Circuit.

Herein, a novel enzyme-free and label-free strategy for colorimetric assay of uracil DNA glycosylase (UDG) activity, which relies on a target-activated toehold-mediated strand displacement (TMSD) circuit is described. The strategy employs a detection duplex probe composed of a uracil-containing strand (US) and a catalyst strand (CS). UDG present in a sample will cleave uracil bases within US and destabilize the detection duplex probe, which then leads to the dissociation of the detection duplex, releasing CS. The free CS promotes the TMSD reaction, consequently liberating a G-quadruplex DNAzyme strand (GS) which is initially caged by a blocker strand (BS). Notably, a fuel strand (FS) is supplemented to recycle the CS to promote another cycle of TMSD reaction. As a consequence, a large number of GSs are activated by UDG activity and a distinct colorimetric signal is produced through the oxidation of ABTS promoted by the peroxidase mimicking activity of the liberated GSs. Based on this design principle, UDG activity down to 0.006 U mL-1 with excellent selectivity is successfully determined. The practical applicability of this assay is also demonstrated by reliably determining UDG activities in human serum.