查找使用阿拉丁产品的科研文献

Engineering of exosome-triggered enzyme-powered DNA motors for highly sensitive fluorescence detection of tumor-derived exosomes

Yanyan Yu,Wei S. Zhang,Yuehua Guo,Huaping Peng,Min Zhu,Dandan Miao,Gaoxing Su

Tumor-derived exosomes containing multiple proteins originating from parent cancer cells have emerged as biomarkers for cancer diagnosis. Herein, we propose a three-dimensional DNA motor-based exosome assay platform for the selective and sensitive detection of exosomes. The DNA motor used gold nanoparticle (GNP) tracks, consisting of fluorescein-labeled substrate strands and aptamer-locked motor strands. Recognition of the target protein on exosomes by its aptamer unlocked the motor strand and triggered the DNA motor process. Powered by restriction endonuclease , the motor strands autonomously walked along the GNP track. Each movement step cleaved one substrate strand and restored one fluorescein molecule. For exosome detection, the proposed method displayed a broad dynamic range acrossing 5 orders of magnitude with the detection limit as low as 8.2 particles/μL in PBS. The method also exhibited good selectivity among different tumor-derived exosomes and performed well in complex biological samples. The capability to profile exosomal surface proteins efficiently endowed our DNA motor great potential for developing a simple and cost-effective device for clinical diagnosis.

Photoelectrochemical assay for DNA hydroxymethylation determination based on the inhibited photoactivity of black TiO2 nanosphere by ZnO

Chen Yan,Yin Huanshun,Sui Chengji,Fu Weizhang,Yang Yue,Ai Shiyun

A photoelectrochemical method was proposed for DNA hydroxymethylation determination using black TiO 2 (B-TiO 2 ) nanosphere as photoactive material and ZnO as photoactivity inhibitor. After hydroxymethylated DNA (5hmC-DNA) was captured on the probe modified B-TiO 2 /ITO electrode surface through hybridization, a glycosyl can be then transferred from uridine diphosphoglucose to 5hmC-DNA and formed a covalent structure with –CH 2 OH in the presence of T4 β-glucosyltransferase (β-GT). Afterwards, based on a series of covalent reaction, amino functionalized ZnO nanoparticles are further immobilized to the surface of the electrode. Due to the capacity to expend the irradiation light and the photogenerated electron of electron donor, the modified ZnO nanoparticles can result in a decreased photocurrent. The developed method shows wide linear ranges from 0.05-200 nM for hydroxymethylated DNA and 1-220 unit·mL −1 for T4-β-glucosyltransferase. The corresponding determination limits were 0.013 nM and 0.24 unit·mL −1 , respectively. The enzyme activity inhibited by 4-phenylimidazole was evaluated. This photoelectrochemical method shows high specificity for 5hmC-DNA (compared to 5fC, 5mC, m6A, control) and β-GT (compared to β-AGT, UGT2B7), and shows excellent stability for testing 5hmC (RSD = 2.75%). Schematic representation of photoelectrochemical method for DNA hydroxymethylation and β-glucosyltransferase detection based on the glycosylation reaction of –CH 2 OH in 5-hydroxymethylcytosine and the inhibition activity of ZnO to the photoactivity of black TiO 2 nanospheres.

Exquisitely designed magnetic DNA nanocompartment for enzyme immobilization with adjustable catalytic activity and improved enzymatic assay performance

Jiayi Song,Wenting He,Hao Shen,Zixin Zhou,Mengqi Li,Ping Su,Yi Yang

The enzymatic activity and practical applications of immobilized enzymes were severely influenced by their location orientation and microenvironment, which played the key roles in maintaining the active conformation and accelerating the reaction process of the desired enzymes. Herein, we presented a general approach to prepare magnetic DNA nanocompartments for encapsulation of metabolic model cascade multi-enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), through self-assembly. The enzymes were spatially confined in the well-designed DNA compartments, which were located at the gap areas of magnetic particles. Systematic studies indicated the proximity effect of the multi-enzymes and the high negative charge density microenvironment of the DNA compartments contributed to the high cascade activity together. In addition, the immobilization enzymes within magnetic DNA compartment remarkably increased the overall catalytic efficiency, operational stability, and reusability. The k cat of encapsulation multi-enzymes was 204 s −1 , which was 16.6-fold higher than that of free enzymes. The immobilized enzymes were further applied for on-line visual detection of glucose with excellent selectivity , and the immobilized enzymes also showed relative high cytotoxicity towards Hela cells, demonstrating their promising prospect for biocatalysis and cancer starvation therapy.

Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA walker and hybridization chain reaction amplification

Qu Xiaonan,Wang Jingfeng,Zhang Rufeng,Zhao Yihan,Li Shasha,Wang Yu,Liu Su,Huang Jiadong,Yu Jinghua

An ultrasensitive fluorescence sensing strategy for kanamycin (KANA) determination using endonuclease IV (Endo IV)-powered DNA walker, and hybridization chain reaction (HCR) amplification was reported. The sensing system consists of Endo IV-powered 3D DNA walker using for the specific recognition of KANA and the formation of the initiators, two metastable hairpin probes as the substrates of HCR and a tetrahydrofuran abasic site (AP site)-embeded fluorescence-quenched probe for fluorescence signal output. On account of this skilled design of sensing system, the specific binding between KANA and its aptamer activates DNA walker, in which the swing arm can move autonomously along the 3D track via Endo IV-mediated hydrolysis of the anchorages, inducing the formation of initiators that initiates HCR and the following Endo IV-assisted cyclic cleavage of fluorescence reporter probes. The use of Endo IV offers the advantages of simplified and accessible design without the need of specific sequence in DNA substrates. Under the optimal experimental conditions, the fluorescence biosensor shows excellent sensitivity toward KANA detection with a detection limit as low as 1.01 pM (the excitation wavelength is 486 nm). The practical applicability of this strategy is demonstrated by detecting KANA in spiked milk samples with recovery in the range of 98 to 102%. Therefore, this reported strategy might create an accurate and robust fluorescence sensing platform for trace amounts of antibiotic residues determination and related safety analysis. Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA Walker and hybridization chain, reaction amplification, Xiaonan Qu, Jingfeng Wang, Rufeng Zhang, Yihan Zhao, Shasha Li, Yu Wang, Su Liu*, Jiadong Huang, and Jinghua Yu, an ultrasensitive fluorescence sensing strategy for kanamycin determination using endonuclease IV-powered DNA walker, and hybridization chain reaction amplification is reported.

Preparation of Aptamer Responsive DNA Functionalized Hydrogels for the Sensitive Detection of α-Fetoprotein Using SERS Method

Qi Wang,Yongjun Hu,Ningjing Jiang,Junjie Wang,Meng Yu,Xiumei Zhuang

Hepatocellular carcinoma (HCC), which is one of the three major cancers, has attracted growing attention due to its high mortality, health care cost, and circumscribed therapeutic methods. Hence, the development of a fast, accurate, and flexible method to detect α-fetoprotein (AFP), the specific marker of HCC, is significant for diagnosis and treatment of cancer. Here, we constructed a novel SERS biosensing platform combining the target-responsive DNA hydrogel for the sensitive detection of AFP. The linker strand in DNA hydrogel is an aptamer that can specifically recognize AFP and accurately control the release of immunoglobulin G (IgG) encapsulated in hydrogel. In the presence of AFP, the hydrogels were disentangled and the IgG was released. Thereafter, the released IgG was captured by SERS probes and biofunctional magnetic beads through formation of sandwich-like structures, resulting in the signal of Raman tags decreasing in the supernatant after magnetic separation. Due to the ultrahigh sensitivity of the SERS biosensor, the proposed method has a wide detection linear range (50 pg/mL to 0.5 μg/mL) and a detection limit down to 50 pg/mL. Moreover, the sequence of the linker strand in the DNA hydrogel can be specifically encoded into a new aptamer that responds to other cancer markers. This convenient and inexpensive detection method provides a new strategy for the detection of tumor markers.

Antimicrobial Defensin and DNA Traps in Manila Clam Ruditapes philippinarum: Implications for Their Roles in Immune Responses

Han Yijing,Hu Gege,Chen Yuying,Chen Lizhu,Yu Daode,Zhang Qianqian,Yang Dinglong

Although defensins have been isolated from a variety of metazoan, their role in cellular immunity has not been answered. In the study, we found that the hemocytes of the Manila clams Ruditapes philippinarum release defensin (designated as Rpdef3) in response to Vibrio parahaemolyticus challenge. The antimicrobial Rpdef3 was proved to be involved in the extracellular traps (ETs) that hemocytes released in response to Vibrio challenge. Scanning electron microscopy observation proved the patterns how ETs eliminate invading bacteria. Furthermore, Rpdef3 involved in ETs had broad-spectrum antimicrobial effect on both Gram-negative bacteria and Gram-positive bacteria. ELISA assay revealed that Rpdef3 could bind lipopolysaccharides and peptidoglycan in a dose-dependent manner. As concerned to the antibacterial mechanisms, Rpdef3 can cause bacterial membrane permeabilization, leading to cell death. As a result, Rpdef3 might contribute to the trap and the elimination of invading Vibrio in clam ETs. Taken together, our study suggest that the formation of ETs is a defense mechanism triggered by bacterial stimulation, coupled with antibacterial defensin.

A versatile fluorescent nanobeacon lighted by DNA-templated copper nanoparticles and the application in isothermal amplification detection

Yuqi Wan,Qingli Chai,Yanyun Zou,Guobin Mao,Jinyang Chen

In this work, an environmentally-friendly and versatile nanobeacon was constructed by utilizing DNA-templated copper nanoparticles (CuNPs) as fluorescence signal source. As the key component of the nanobeacon, a hairpin DNA was designed to contain four segments: two segments for CuNPs template sequence, a target recognition segment and a blocking segment. At room temperature, the target recognition segment partly hybridizes with the blocking segment and thus prohibits the formation of double stranded DNA template, so that no CuNPs can be generated on the hairpin DNA. While a target is introduced, the specific binding of target with recognition sequence triggers off the conformational transformation of the hairpin DNA, which contributes to the formation of the CuNPs template. As a result, the in-situ generation of CuNPs gives birth to the fluorescence signal readout that can be used to identify the target. By reasonably varying the recognition sequence within hairpin DNA, a series of nanobeacons in response to corresponding targets, such as DNA, microRNA, thrombin, and ATP, were put forward with satisfactory sensitivity and selectivity. Moreover, this nanobeacon was also integrated with the strategy of enzyme-assisted target-recycling to realize signal amplification and ultrasensitive detection, which further demonstrated the versatility of the nanobeacon. This novel nanobeacon is expected to be a promising alternative to classical dye-labeled molecular beacon and provide new perspective on ultrasensitive fluorescence sensing.

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries

Mengqi Li,Hao Shen,Zixin Zhou,Wenting He,Ping Su,Jiayi Song,Yi Yang

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.

Construction of multiple enzyme metal–organic frameworks biocatalyst via DNA scaffold: A promising strategy for enzyme encapsulation

Jiayi Song,Wenting He,Hao Shen,Zixin Zhou,Mengqi Li,Ping Su,Yi Yang

The encapsulated enzyme system by metal–organic frameworks exhibited great potential in pharmaceuticals, enzyme fuel cells, and biocatalysis. However, the enzymatic activity and overall catalytic efficiency are severely hampered due to their enzyme leaching and causal enzyme location. Herein, for the first time, horseradish peroxidase (HRP) and glucose oxidase (GOx) used as model enzymes were cross-linked by rationally designed DNA scaffold network, and the multienzyme system has been successfully encapsulated into zeolite imidazolate framework-8 (ZIF-8). The prepared encapsulation multienzyme can efficiently suppress enzyme leaching from the MOFs skeleton, and the leaching amount was more than 10-times lower than the traditional MOFs encapsulation strategy. The well-tailored proximity and colocalization of cascade enzymes by the DNA scaffold network obviously improved the overall catalytic efficiency, enzymatic activity, and kinetic performance. The K m and k cat / K m for encapsulation multienzyme was 13.8 mM and 3.47 s −1  mM −1 , which was 0.27-fold lower and 9.9-fold higher than those of free enzymes, respectively, indicating a better substrate affinity. Furthermore, the multienzyme system exhibited excellent stability and reusability compared with free enzymes and control groups, which could preserve 95% initial activity at 50 °C after 75 min incubation and more than 87% of the original activity after 14 cycles. Finally, the immobilized multienzyme exhibited enhanced performance to detect low concentration of glucose (0.4 µM) with excellent selectivity. The outcome indicated that the encapsulated multienzyme could be extended for broad practical applications in biotechnology, biocatalysis, and biomedical engineering.

DNA-templated copper nanoclusters as a fluorescent probe for fluoride by using aluminum ions as a bridge

Pang Jiawei,Lu Yuexiang,Gao Xinyu,He Liuying,Sun Jingwei,Yang Fengyi,Hao Zixuan,Liu Yueying

A selective fluorescent on-off-on probe has have designed for the detection of fluoride (F − ) ions based on DNA-templated copper nanocluster (CuNCs) and by using aluminum(III) ions as a bridge. A 40-mer polythymine acts as a template for the reduction of Cu(II) to Cu(0) by ascorbic acid. This result is the formation of red fluorescent CuNCs, with excitation/emission peaks at 340/640 nm. After addition of Al 3+ ions, the fluorescence of CuNCs is quenched because the interaction of Al 3+ and DNA disturbs the formation of DNA-templated CuNCs. Fluorescence is restored on addition of fluoride to the system. This is due to the desorption of Al 3+ from the DNA and the formation of the Al(OH) 3 F − complex. This system displays a fast fluorometric response to fluoride, with high selectivity over other anions. Fluorescence increases linearly in the 2 to 150 μM F − concentration range, and the detection limit is 1.0 μM. This probe has been successfully used for the detection of F − ions in four brands of toothpaste. The method is rapid, cost-effective, selective, and does not require toxic solvents and reagents. Schematic presentation of a method for fluorometric determination of fluoride by using DNA-templated copper nanoclusters (CuNCs) and using aluminum(III) as a bridge. The red fluorescence of the CuNCs is quenched in the presence of Al(III) ions but restored after addition of fluoride.

Exploring the binding interaction between herring sperm DNA and sunitinib: insights from spectroscopic and molecular docking approaches

Jie-Hua Shi,Dong-Qi Pan,Kai-Li Zhou,Yan-Yue Lou

Backfilling rolling cycle amplification with enzyme-DNA conjugates on antibody for portable electrochemical immunoassay with glucometer readout

Lilin Ge,Bin Li,Houxi Xu,Wenyuan Pu,Hang Fai Kwok

A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA 2 conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA 2 -invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb 2 detection antibody conjugated with DNA 1 primer (DNA 1 -AuNP-pAb 2 ) in the presence of target ATP . Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA 2 conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1–100 ng mL −1 at a low detection limit of 0.087 ng mL −1 . Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.