A Magnetically Driven Tandem Chip Enables Rapid Isolation and Multiplexed Profiling of Extracellular Vesicles

The protein phenotypes of extracellular vesicles (EVs) have emerged as promising biomarkers for cancer diagnosis and treatment monitoring. However, the technical challenges in rapid isolation and multiplexed molecular detection of EVs have limited their clinical practice. Herein, we developed a magnetically driven tandem chip to achieve streamlined rapid isolation and multiplexed profiling of surface protein biomarkers of EVs. Driven by magnetic force, the magnetic nanomixers not only act as tiny stir bars to promote mass transfer and enhance reaction efficiency of EVs, but also transport on communicating vessels of the tandem chip continuously and expedite the assay workflow. We designed cyclic surface enhancement of Raman scattering (SERS) tags to bind with target EVs and then release them by exonuclease I, eliminating steric hindrance and amplifying the SERS signal of multiple protein biomarkers on EVs. Due to the excellent assay performance, six breast cancer biomarkers were detected simultaneously on EVs using only 10 μL plasma within 1.5 h. The unweighted SUM signature offers great accuracy in discriminating breast cancer patients from healthy donors. Overall, the dynamic magnetic driving tandem chip offers a new avenue to advance the clinical application of EV-based liquid biopsy.     

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with Ti^IV ions and a membrane-penetrating peptide, octa-arginine R₈⁺, which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to Ti^IV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.     

In Situ Amperometric Detection of Vesicles and Microgel Phases in an Aggregating System: Calcium Alginate

In situ amperometric characterization of an aggregating system in terms of molecular adsorption and single microparticle interactions at the electrode interface is demonstrated using a model system: alginate/Ca(II) in an aqueous electrolyte solution. Recording of chronoamperometric curves of oxygen reduction at the dropping mercury electrode is designed for detection of dip‐shaped signals of individual gel microparticles. By addition of Ca(II) decrease of alginate adsorption is accompanied by appearance of signals indicating vesicle type association of alginate molecules and microparticles of gel phase. AFM imaging provided evidence of initial stage in calcium alginate gel formation.     

Sensitive and Selective Detection of Dopamine Using a DNA Immobilized Ethylenedidamine/Polyglutamic Modified Electrode

DNA was attached on the surface of an ethylenedidamine/polyglutamic(En/PGA) modified glassy carbon electrode (GCE) to create a novel voltammetric sensor (DNA/En/PGA/GCE) for dopamine (DA). This modified electrode exhibited a linear voltammetric response for DA in the range from 1.0×10^?7 mol L^?1 to 1×10^?5 mol L^?1, with a detection limit of 2×10^?8 mol L^?1. The detection of DA was found to be unaffected by the presence of ascorbic acid, uric acid, serotonin and folic acid. The method proposed was applied to detect DA in pharmaceutical dosage and human blood serum with good satisfactory results.     

In Situ Analysis for Herbal Pieces of Aconitum Plants by Using Direct Analysis in Real Time Mass Spectrometry

In this study, an extend application was developed to in situ analyze the herbal pieces of Aconitum plants by Direct Analysis in Real Time Mass Spectrometry (DART‐MS). Nearly all aconitine‐type alkaloids can be desorbed and ionized in this method, including diester diterpenoid aconitines (DDAs), monoester diterpenoid aconitines (MDAs) and some other diterpenoid aconitines. The spectra of in situ analysis for the herbal pieces of aconitum plants are similar with that of their extracts. Radix Aconiti and Radix Aconiti Kusnezoffii can be distinguished from each other by the intensity differences of character fragment ions from MDAs, such as m/z 586, 572, and 556. The qualified and unqualified herbal pieces can be also identified by the relative abundances of DDAs. The RSD of the relative abundances of some character ions at m/z 556, 586, and 590 were 13.53%, 4.03%, and 12.03%, respectively. So this in situ analytical method can identify both the types of Aconitum preparata and their quality. It provides the following advantages in the analysis of Chinese herbs: fast detection without much pretreatment, high‐throughput analysis, and reduction of pollution without any organic solvent.     

Oxidation of Sanguinarine and Its Dihydro‐Derivative at a Pyrolytic Graphite Electrode Using Ex Situ Voltammetry. Study of the Interactions of the Alkaloids with DNA

This study describes the oxidation of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on the surface of a basal‐plane pyrolytic graphite electrode (PGE). Since both alkaloids strongly adsorb onto the surface of pyrolytic graphite, measurements were performed using ex situ voltammetric methods, adsorptive transfer (AdT) cyclic voltammetry (CV) and square‐wave voltammetry (SWV). Oxidation peaks of SG (peak A) and DHSG (peak A*) were observed around the potential of +0.7 V (vs. Ag/AgCl/3 M KCl), depending on the experimental conditions. The voltammetric peaks A and A* are probably related to the oxidation of N‐methylphenanthridine nitrogenous heterocycle of SG and oxidation of DHSG back to SG, respectively. The electrochemical results and optimized AdT SWV were subsequently applied to the study of the interactions of SG and DHSG with DNA in vitro. Analysis of the alkaloid/DNA interactions was based on observing heights of oxidation peaks A and A* after incubation of SG and/or DHSG with supercoiled (sc) DNA [pBSK_(?)]. Electrochemical study of the interactions was supported and complemented with measurements using gel electrophoresis (Topoisomerase I scDNA relaxation assay) and steady‐state and time‐resolved fluorescence spectroscopy. The results suggest that SG intercalates into the double‐stranded structure of scDNA (the SG/base pair ratio is max. 1/4) while increased binding affinity was observed for quaternary cation (SG⁺). DHSG which, unlike SG⁺, does not possess a strictly planar molecular structure, did not show intercalative DNA binding in any of the three methods applied.     

发夹型荧光分子探针用于DNA甲基化酶的活性分析及其在药物筛选中的应用

报道了一种基于发夹型荧光探针的甲基化酶活性的分析方法, 甲基化酶和相应的限制性内切酶的识别位点被设计在发夹型探针的茎部, 四甲基罗丹明(TAMRA)被连接在探针的5'端, 其荧光被连在3'端的熄灭基团4-(4'-二甲基对胺基偶氮苯)苯甲酸(DABCYL)所熄灭. 限制性内切酶可切割未发生甲基化修饰的探针, 导致探针的发夹结构遭到破坏, 引起TAMRA荧光信号的恢复. 根据荧光信号的恢复程度可实现对甲基化酶活性的分析. 在此基础上, 建立了一种简便、快速分析抗肿瘤药物对DNA甲基化酶活性的影响的方法, 为筛选针对基因甲基化异常引起的恶性肿瘤的治疗药物提供了一种新的思路和方法.     

Selective Imaging of HClO in the Liver Tissue In Vivo Using a Near-infrared Hepatocyte-specific Fluorescent Probe

Liver injury is typified by an inflammatory response. Hypochlorous acid (HClO), an important endogenous reactive oxygen species, is regarded as a biomarker associated with liver injury. Near-infrared (NIR) fluorescent probes with the advantage of deep tissue penetrating and low auto-fluorescence interference are more suitable for bioimaging in vivo. Thus, in this work, we designed and synthesized a novel NIR hepatocyte-specific fluorescent probe named NHF . The probe NHF showed fast response (<3 s), large spectral variation, and good selectivity to trace HClO in buffer solution. By employing N-acetylgalactosamine (GalNAc) as the targeting ligand, probe NHF can be actively delivered to the liver tissue of zebrafish and mice. It is important that probe NHF is the first NIR hepatocyte-specific fluorescent probe, which successfully visualized the up-regulation of endogenous HClO in the oxygen-glucose deprivation/reperfusion (OGD/R) model HepG2 cells and dynamically monitored APAP-induced endogenous HClO in the liver tissue of zebrafish and mice.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

基于高效液相色谱的喹啉类药物对刺参DNA甲基化水平的影响研究

建立了高效液相色谱/紫外法测定刺参组织全基因组DNA甲基化水平的方法,并运用此方法对喹噁啉药物处理过的刺参样品DNA甲基化水平进行分析。色谱条件为:色谱柱为ZORBAX SB-Aq(4.6 mm×250mm;5μm);柱温为30℃;检测波长为280 nm;进样量20μL;流动相为甲醇-7 mmol/L乙酸铵(7∶93),流速为1.0 mL/min。5-甲基脱氧胞苷和脱氧胞苷的质量浓度在0.05~1.0 mg/L范围内时,线性关系良好,相关系数(r)均为0.999 9。5-甲基脱氧胞苷和脱氧胞苷的检出限均为0.05 mg/L,在加标浓度为0.05,0.25,1.0 mg/L时,回收率为90.4%~100.6%,批内、批间相对标准偏差均小于4%。采用CTAB法提取刺参组织DNA,酶解后进行HPLC测定,结果表明:喹噁啉药物处理过的组织样品DNA甲基化水平低于对照组,该类药物通过改变DNA甲基化水平而影响基因的正常表达,可能是其产生遗传毒性的一种机制,建立的方法可以很好地用于全基因组DNA总甲基化水平的检测。     

In Situ Generation of Chiral N‐Dienyl Lactams in a Multicomponent Reaction: An Efficient and Highly Selective Way to Asymmetric Amidocyclohexenes

Chiral N‐dienyl lactams are crucial building blocks for the synthesis of complex organic compounds. However, their generation is rather challenging. This paper reports on a highly efficient and diastereoselective multicomponent methodology utilizing chiral a mides, a ldehydes, and d ienophiles (AAD reaction). The three components readily react under in situ generation of chiral N‐dienyl lactams which undergo a subsequent Diels–Alder reaction. Different chiral amides have been employed in the standard protocol delivering yields up to 94 %, and selectivities up to 90 % de. Moreover, DFT calculations were performed to explain the obtained selectivities.     

基于酶循环放大的DNA生物传感器用于检测食管鳞状细胞癌生物标志物蛋白PDGF-BB

食管鳞状细胞癌(ESCC)患者发病早期由于体内的肿瘤标志物种类少且含量低,难以在患病早期发现疾病从而快速反应并有效治疗.因此,找到一种合适的标志物对其进行快速高效检测是研究者亟待解决的问题.血小板衍生生长因子(PDGF-BB)在恶性肿瘤的早期诊断中具有极其重要的地位.该文基于酶循环放大荧光光谱设计了一种用于PDGF-B...     

In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as “clustered/complex DNA damage” and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.     

Fabrication of a Nitrate Selective Electrode for Determination of Nitrate in Fertilizers by Using Flow Injection Analysis System

A nitrate ion selective electrode (nitrate-ISE) based on pyrrole modification on a common pencil lead was proposed. The lab-made nitrate-ISE was easily constructed by pyrrole polymerization with nitrate ion as the dopant using cyclic voltammetry. The fabricated nitrate-ISE was then coupled with flow injection analysis (FIA) for an automatic system. The flow potentiometry provided working range of 1x10^-4 to 4x10^-3 mol L^-1 of nitrate and allowed sample throughput up to 50 samples h^-1. Linear regression analysis showed good agreement (r²=0.9961) with Nernstian response. This system was applied to determine nitrate-nitrogen (nitrate-N) in fertilizer samples.     

Electrochemical Detection of Nitrite in Meat and Water Samples Using a Mesoporous Carbon Ceramic SiO2/C Electrode Modified with In Situ Generated Manganese(II) Phthalocyanine

Mesoporous carbon ceramic SiO₂/50 wt % C (S_BET=170 m² g^?1), where C is graphite, were prepared by the sol‐gel method. The materials were characterized using N₂ sorption isotherms, scanning electron microscopy, and conductivity measurements. The matrix was used as support for the in situ immobilization of Mn(II) phthalocyanine (MnPc) on their surface. XPS was used to determine the Mn/Si atomic ratios of the MnPc‐modified materials. Pressed disk electrodes were prepared with the MnPc‐modified matrix, and tested as an electrochemical sensor for nitrite oxidation. The linear response range, sensitivity, detection limit and quantification limit were 0.79–15.74 µmol L^?1, 17.31 µA L µmol^?1, 0.02 µmol L^?1 and 0.79 µmol L^?1, respectively, obtained using cyclic voltammetry. The repeatability of the proposed sensor, evaluated in terms of relative standard deviation was 1.7 % for 10 measurements of a solution of 12.63 µmol L^?1 nitrite. The sensor employed to determine nitrite in sausage meat, river and lake water samples showed to be a promising tool for this purpose.     

Analysis of Free and Total Glycerol in Biodiesel Using an Electrochemical Assay Based on a Two‐Enzyme Oxygen‐Electrode System

In this work, an electroenzymatic methodology based on two coupled enzymatic activities (glycerokinase and glycerol‐3‐phosphate oxidase) was developed using an oxygen Clark‐type electrode for the determination of free and total glycerol in biodiesel samples. The enzymatic conversion of glycerol consumes oxygen, which is measured amperometrically in a Clark‐type electrode and correlated with the concentration of glycerol in the sample. The electroenzymatic method proposed showed a good linear correlation coefficient (R=0.9990) with a linear response in the concentration range of 6.25×10^?5 to 6.25×10^?4% (w/v) and limits of detection and quantification at 1.0×10^?5% and 3.0×10^?5% (w/v), respectively. Good correlations were found between the results obtained in this work and those by the gas chromatography technique (R=0.9994). The proposed method was shown to be promising for the analysis of glycerol in biodiesel samples, with a simple and inexpensive methodology compared with the gas chromatography technique.     

An Electrochemical DNA Biosensor for the Detection of the Apa I Polymorphism in the Vitamin D Receptor Gene Using Meldola's Blue as a Hybridization Indicator

Electrochemical detection of nucleic acid base mismatches related to Apa I single nucleotide polymorphism (SNP) in the vitamin D receptor gene was performed successfully using 7‐dimethyl‐amino‐1,2‐benzophenoxazinium salt (Meldola's blue, MDB) with 10.9 pmol/100 μL of detection limit. MDB reduction signals obtained from probe, mismatch(probe‐SNP containing target) and hybrid(probe‐target) modified pencil graphite electrode(PGE) increased respectively. The sensor was able to clearly distinguish perfect match from mismatch DNA in a 30 min. detection time. Several factors affecting on the hybridization and indicator response are studied to maximize sensitivity and selectivity. The advantages of the biosensor are discussed in comparison with previous electrochemical assays for DNA hybridization.     

Selective Determination of Verapamil in Pharmaceutics and Urine Using a Boron‐doped Diamond Electrode Coupled to Flow Injection Analysis with Multiple‐pulse Amperometric Detection

This work presents a simple and low‐cost method for fast and selective determination of Verapamil (VP) in tablets and human urine samples using a boron‐doped diamond working electrode (BDD) coupled to a flow injection analysis system with multiple pulse amperometric detection (FIA‐MPA). The electrochemical behaviour of VP in 0.1 mol L⁻¹ sulfuric acid showed three merged oxidation peaks at around +1.4 V and upon reverse scan, one reduction peak at 0.0 V (vs. Ag/AgCl). The MPA detection was performed applying a sequence of three potential pulses on BDD electrode: (1) at +1.6 V for VP oxidation, (2) at +0.2 V for reduction of the oxidized product and (3) at +0.1 V for cleaning of the working electrode surface. The FIA system was optimized with injection volume of 150 μL and flow rate of 3.5 mL min⁻¹. The method showed a linear range from 0.8 to 40.0 μmol L⁻¹ (R>0.99) with a low limit of detection of 0.16 μmol L⁻¹, good repeatability (RSD<2.2 %; n=10) and sample throughput (45 h⁻¹). Selective determination of VP in urine was performed at+0.2 V due to absence of interference from ascorbic and uric acids in this potential. The addition‐recovery tests in both samples were close to 100 % and the results were similar to an official method.