DNA methylation refers to the chemical modification process of obtaining a methyl group by the covalent bonding of a specific base in DNA sequence with S-adenosyl methionine (SAM) as a methyl donor under the catalysis of methyltransferase (MTase), which is related to the occurrence of multiple diseases. Therefore, the detection of MTase activity is of great significance for disease diagnosis and drug screening. Because reduced graphene oxide (rGO) has a unique planar structure and remarkable catalytic performance, it is not clear whether rGO can rapidly catalyze silver deposition as an effective way of signal amplification. However, in this study, we were pleasantly surprised to find that using H2O2 as a reducing agent, rGO can rapidly catalyze silver deposition, and its catalytic efficiency of silver deposition is significantly better than that of GO. Therefore, based on further verifying the mechanism of catalytic properties of rGO, we constructed a novel electrochemical biosensor (rGO/silver biosensor) for the detection of dam MTase activity, which has high selectivity and sensitivity to MTase in the range of 0.1 U/mL to 10.0 U/mL, and the detection limit is as low as 0.07 U/mL. Besides, this study also used Gentamicin and 5-Fluorouracil as inhibitor models, confirming that the biosensor has a good application prospect in the high-throughput screening of dam MTase inhibitors.

In this study, it is shown for the first time that a reduced graphene oxide (rGO) carrier has a 20-fold higher catalysis rate than graphene oxide in Ag + reduction. Based on this, a tumor microenvironment-enabled in situ silver-based electrochemical oncolytic bioreactor (SEOB) which switched Ag + prodrugs into in situ therapeutic silver nanoparticles with and above 95% transition rate is constructed to inhibit the growths of various tumors. In this SEOB-enabled intratumoral nanosynthetic medicine, intratumoral H 2 O 2 and rGO act as the reductant and the catalyst, respectively. Chelation of aptamers to the SEOB-unlocked prodrugs increases the production of silver nanoparticles in tumor cells, especially in the presence of Vitamin C, which is broken down in tumor cells to supply massive amounts of H 2 O 2 . Consequently, apoptosis and pyroptosis are induced to cooperatively contribute to the considerably-elevated anti-tumor effects on subcutaneous HepG2 and A549 tumors and orthotopic implanted HepG2 tumors in livers of nude mice. The specific aptamer targeting and intratumoral silver nanoparticle production guarantee excellent biosafety since it fails to elicit tissue damages in monkeys, which greatly increases the clinical translation potential of the SEOB system.

To explore dendritic cells/tumor-derived endothelial cells (DC/EC) fusion cells are potent stimulators of T cells to impact tumor progression. ECs were isolated from mice hepatoma cell line (H22) Xenograft, and dendritic cells were isolated from bone marrow of BALB/c mice, then the isolated ECs were cultured and detected the endothelial surface expression of CD105 by flow cytometry. The endothelial characteristics of ECs were detected by tube formation assay and Dil-Ac-LDL uptake assay. After the fusion with polyethylene glycol (PEG), we used DCs, ECs, DCs mixed ECs as the control groups, DC/EC fusion cells as the experimental group, Secretion of IFN-α and IFN-γ was evaluated, T lymphocyte proliferation and cytotoxic T lymphocytes (CTL) were detected in vitro. In vivo , T lymphocyte induced by five groups was injected to detect the effect of tumor progression. Purified ECs (CD105 + ) took the function of endothelial cells, then successfully fused with DCs. The DC/EC fusion cells were functional in stimulating the proliferation of T cells, which produced IFN-α and IFN-γ. In vivo , T cells stimulated by DC/EC fusion cells effectively repressed tumor growth. The fusion cells, which was capable of stimulating T cells, is indispensable for antitumor immunity.

Detection of human leukocyte antigens-A2-restricted p-hepatitis B virus (HBV) core antigen-specific cytotoxic T lymphocytes (CTLs) is important in the study of HBV immunopathogenesis and vaccine design. Currently, major histocompatibility complex (MHC) class I/peptide-(p) MHCI tetramers are considered the optimal tools to detect antigen-specific CTLs. However, the MHC-tetramer technique also has certain drawbacks and is under continuous development. The quantum dot (QD) bioconjugates nanotechnology with its unique inorganic-biological properties has been developing fast. However, QD/pMHC multimers have seldom been used for the identification of the C18–27 epitope, which is important in HBV infection. QD/pMHC multimers were synthesized by metal-affinity coordination and an avidin-biotin system. In the present study they were characterized by transmission electron microscopy, dynamic light scattering and fluorescence spectrophotometry. C18–27-specific CTLs were obtained by ex vivo expansion of CD8+ T cells. Cultured CTLs were tested for the secretion level of interferon (IFN)-γ by ELISA and for cytotoxicity by lactate dehydrogenase release assay. Then, the performance of phycoerythrin (PE)/pMHC tetramers and QD/pMHC multimers were compared by flow cytometry. The synthesized QD/pMHC multimers dispersed well and their emission spectrum exhibited only slight differences compared with original QDs. C18–27-specific CTLs not only secreted IFN-γ but also effectively targeted T2 cells pulsed with peptide C18–27. The frequencies of C18–27-specific CTLs determined by QD/pMHC multimers were higher compared with PE/pMHC tetramers. The present results suggested that QD/pMHC multimers may be able to characterize greater numbers of C18–27-specific CTLs with increased sensitivity compared to conventional strategies.

A highly sensitive non-enzymatic electrochemical sensor based on platinum nanoparticles/reduced graphene oxide-chitosan-ferrocene carboxylic acid nano-hybrids (Pt NPs/RGO-CS-Fc biosensor) was developed for the measurement of hydrogen peroxide (H2O2). The RGO-CS-Fc nano-hybrids was prepared and characterized by UV-vis spectrum, Fourier transform infrared spectroscopy, transmission electron microscopy, Raman spectrometer and electrochemical impedance spectroscopy. Under optimal experimental conditions, the Pt NPs/RGO-CS-Fc biosensor showed outstanding catalytic activity toward H2O2 reduction. The current response of the biosensor presented a linear relationship with H2O2 concentration from 2.0×10(-8)M to 3.0×10(-6)M with a correlation coefficient of R(2)=0.9968 and with logarithm of H2O2 concentration from 6.0×10(-6)M to 1.0×10(-2)M with a correlation coefficient of R(2)=0.9887, the low detection limit of 20nM was obtained at the signal/noise (S/N) ratio of 3. Moreover, the Pt NPs/RGO-CS-Fc biosensor exhibited excellent anti-interference capability and reproducibility for the detection of H2O2. The biosensor was also successfully applied for the detection of H2O2 from living cells containing normal and cancer cells. All these results prove that the Pt NPs/RGO-CS-Fc biosensor has the potential application in clinical diagnostics to evaluate oxidative stress of different living cells.

The invention discloses a biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification. The invention provides a kit for detecting leukemia cells; the kit comprises (a) a magnetic bead capture probe and (b) an aptamer functionalization Hemin-graphene composite nanomaterial, wherein the magnetic bead capture probe comprises a magnetic bead and a capture probe fixed on the magnetic bead; the capture probe is a single-stranded deoxyribonucleic acid (DNA) molecule shown by sequence 1; the aptamer functionalization Hemin-graphene composite nanomaterial is composed of graphene modified by Hemin, and an aptamer probe fixed on the graphene modified by the Hemin; the aptamer probe is a single-stranded DNA molecule shown by sequence 2. After the biosensing method is used, the target leukemia cells with lower concentration can be captured and enriched within a short time, and a detection signal is amplified, so that the leukemia cells can be rapidly, accurately and sensitively detected.

The invention provides an electrochemical detection method for catalytic silver deposit of reducing type oxidized graphene, as well as applications thereof. The method comprises the following steps: modifying a DNA probe, which can be identified specifically by a to-be-detected object, on a gold electrode, to obtain a working electrode; reacting the working electrode, the to-be-detected object with known specific activity, and standard sample solution of substances capable of cutting the DNA probe, which can be identified specifically by a to-be-detected object, into oligonucleotides single chains, loading the reacted electrode with reducing type oxidized graphene, performing silver deposit reaction together with silver ion-containing solution, dissolving out silver monomer deposited on the working electrode, determining the dissolution volt-ampere current value by utilizing a linear sweep voltammetry, and drawing a standard cure; according to the standard cure, calculating to obtain the specific activity of the to-be-detected sample. The established electrochemical detection method has the advantages of being labeling-free, safe for detection process, simple to operate, easy to miniature, wide to apply and the like.

OBJECTIVE:To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses. METHODS:Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. RESULTS:Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. CONCLUSIONS:The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

We report here a highly sensitive electrochemical sensing platform for Ag+ detection based on Ag+-induced conformational change of cytosine-rich single stranded DNA C-rich ssDNA probe and the controlled assembly of MWCNTs. In the protocol, the gold electrode was first modified with a dense 16-mercaptohexadecanoic acid self-assembled monolayer (MHA/SAM). The hydrophobic MHA/SAM isolated the electrode from the electroactive indicator in the aqueous solution, which resulted in the electronic transmission blocking. It was eT OFF state. In the presence of Ag+, C–Ag+–C coordination induced the conformational change of C-rich ssDNA probe from random-coil structure to fold into a hairpin structure, which cannot wrap on the surface of the MWCNTs. Then the “naked” MWCNTs can be assembled on the MHA/SAM gold electrode, mediating the electron transfer between the electrode and the electroactive indicator. It generated measurable electrochemical signals (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target Ag+ concentrations as low as 1.3nM. The linear response range for Ag+ detection was from 10 to 500nM. This method dose not need of electroactive molecules labeling on the C-rich ssDNA probe. Moreover, it has good selectivity to other environmentally relevant metal ions. Therefore, the developed electrochemical assay is an ideal method for Ag+ detection with some advantages including sensitivity, selectivity, simplicity, low-cost, and no requirement for probe label preparation. We expect that this strategy could be a generalized platform for DNA-based sensing.

A sensitive label-free "signal-on" electrochemical approach for detection of methyltransferases (MTase) activity is developed based on the signal transduction and amplification of single wall carbon nanotubes (SWCNTs). In this method, the oligonucleotide I is first self-assembled on the electrode via Au-S bonding. After hybridization with its complement ssDNA (oligonucleotide II), duplex strand DNA (dsDNA) probes containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I is then formed on the electrode. In the presence of Dam MTase and Dpn I, the dsDNA probes are methylated and subsequently cleaved into two dsDNA fragments. After heating, the remained dsDNA fragments on the electrode melted into ssDNA fragments. Then the SWCNTs can be controllably assembled on the ssDNA fragments remained on the electrode, mediating efficient electron transfer between the electrode and electroactive species. It generates measurable current signal (eT ON), which is related to the concentration of the Dam MTase. The resulting change in electron transfer efficiency is readily measured by differential pulse voltammetry at Dam MTase concentrations as low as 0.04 U/mL. This method does not need electroactive molecules labeling on the methylation-responsive DNA probes. The linear response of the developed facile signal-on electrochemical sensing system for Dam MTase is in the range of 0.1-1.0 U/mL. In addition, such a SWCNTs based electrochemical assay also has the ability to screen inhibitors for Dam MTase.

A novel electrogenerated chemiluminescence (ECL) sensor based on Ru(bpy)(3)(2+)-doped MCM-41 mesoporous silica nanoparticles (MSN) was developed. The Ru(bpy)(3)(2+) was first loaded into the sulfhydryl modified MSN through the electrostatic absorption. Then the Ru(bpy)(3)(2+)-doped MSN was immobilized onto the surface of Au electrode by Au-S interaction. The properties of electrochemistry and electrogenerated chemiluminescence (ECL) were also investigated. The amino structure of melamine is similar to tripropylamine, so we attempted to branch out the application of MCM-41 into the detection of melamine, the ECL sensor provided a new assay for the detection of melamine. Furthermore, the research provided a new approach for the immobilization of Ru(bpy)(3)(2+) onto the electrode surface.

This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modified "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modified "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7 h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase. (C) 2012 Elsevier B.V. All rights reserved.

A label-free, simple and fast electrochemical method for Klenow fragment exo-(KF-) detection was developed based on the properties of gold nanoparticles(AuNPs) such as the large specific surface, small size and the ability of binding much DNA. In this work, thiolated primer DNA was first modified on the surface of electrode. In the presence of template DNA and reporter DNA tethered with AuNPs(AuNPs-DNA), primer DNA and report DNA were hybridized with template DNA to form a sandwich-like hybridization mode, which brings AuNPs-DNA to the electrode surface. When put the modified electrode into the electrolyte containing [Ru(NH3)(6)](3+)(RuHex), the RuHex quantitatively binds to the DNA via electrostatic interaction. Since AuNPs was loaded with much reporter DNA that can bind a large number of RuHex, this offers the electrochemical signals amplification for the detection. In the presence of dNTPs and KF-, the extension reaction was triggered, which results in the dissociation of the AuNPs-DNA and release of abundant RuHex molecules. As a result, the electrochemical signal was reduced and the KF- was detected. Using this method, a detection limit as low as 5 U/mL for KF- was achieved.

In this paper, a novel label-free electrochemical strategy has been developed for assay of casein kinase II (CK2) activity and inhibition using TiO(2)/MWNTs nanocomposites. This detection system takes advantage of specific binding of the phosphate groups with TiO(2) nanoparticles and fast electron transfer rate of MWNTs. In this strategy, the synthesized TiO(2)/MWNTs nanocomposite was firstly deposited on the surface of a glassy carbon electrode (GCE). The presence of MWNTs not only increased the surface area of the electrode but also promoted electron-transfer reaction. In the presence of CK2, the kinase reaction resulted in the phosphorylation of peptide substrates. The phosphorylated peptides were subsequently captured to the surface of GCE modified with TiO(2)/MWNTs nanocomposite through specific binding of the phosphate groups with TiO(2) nanoparticles. Then the access of redox probe [Fe(CN)(6)](3-/4-) to electrode surface was blocked. As a result, the decrease peak currents were related to the concentrations of the CK2, providing a sensing mechanism for monitoring peptides phosphorylation. The electrochemical strategy can be employed to assay CK2 activity with a low detection limit of 0.07 U/mL. The linear range of the assay for CK2 was 0-0.5 U/mL. Furthermore, the interferences experiments of PKA and inhibition of CK2 have been also studied by using this strategy.

A sensitive and specific electrochemical assay for detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica nanocapsules (FcSH/SiNCs) amplification is described. In the protocol, a double aptamer sandwich structure was formed in the presence of thrombin, in which an aptamer-labeled FcSH/SiNCs for electrochemical detection, and a streptavidin-coated magnetic bead immobilized aptamer for rapid and specific separation of target protein. After separated from the sample mixture under a magnetic field, the sandwich complex was treated with NaOH to release the loaded ferrocenylhexanethiol (FcSH) from the silica nanocapsules (SiNCs). Differential pulse voltammetry (DPV) was employed to detect the released FcSH, which was related to the concentration of the thrombin. The method took advantage of sandwich binding of two affinity aptamers for increased specificity, high payload of FcSH in SiNCs for signal amplification, magnetic beads for fast magnetic separation. The peak current of released FcSH had a good linear relationship with the thrombin concentration in the range of 0.1–5nmol/L, and the detection limit of thrombin in the method was 0.06nmol/L. The detection was also specific for thrombin without being affected by other proteins, such as immunoglobulin G, bovine serum albumin, lysozyme and human serum albumin. The method has been used to detect thrombin in human serum albumin with minimum background interference.

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.

Site-specific delivery of drugs can significantly reduce drug toxicity and increase the therapeutic effect. Here, we report a one-pot synthesis of doxorubicin-doped silica nanoparticles (Dox/SiNPs) by using sodium fluoride (NaF) catalyzed hydrolysis of tetraethyl orthosilicate in a water-in-oil microemulsion. Through further surface chemical modification, carboxyl-terminated Dox/SiNPs (COOH-Dox/SiNPs) exhibiting high drug entrapment efficiency, strong fluorescence and long sustained release are obtained. Cell toxicity tests demonstrate that the COOH-Dox/SiNPs kill tumor cells effectively, while pure COOH-SiNPs are nontoxic. An aptamer is further conjugated to the nanoparticles for delivering loaded Dox to target cells. It is demonstrated that Dox/SiNPs modified with the aptamer sgc8c (sgc8c-Dox/SiNPs) could deliver loaded doxorubicin to CCRF-CEM cells with high specificity and excellent efficiency. Furthermore, ex vivo imaging studies show that the COOH-Dox/SiNPs are able to accumulate highly in the tumor areas, thanks to the enhanced permeability and retention (EPR) effects. Our data suggest that the sgc8c-Dox/SiNPs may be a useful new tumor therapy system.

A sensitive and simple signal-on electrochemical assay for detection of Dam methyltransferase (MTase) activity based on DNA-functionalized gold nanoparticles (AuNPs) amplification coupled with enzyme-linkage reactions is presented. This new assay takes advantage of the steric hindrance of AuNPs and the electrostatic repulsion between the negative-charge phosphate backbones of DNA modified on the AuNPs and redox probe [Fe(CN)(6)](3-/4-). In this method, the self-assembled ssDNA on the electrode is hybridized with its complement ssDNA modified on AuNPs to form dsDNA AuNPs bioconjugates containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I. Then, the AuNPs approach to the electrode and result in blockage of electronic transmission. It is eT OFF state. In the presence of Dam MTase and Dpn I, the specific sequence is methylated and cleavaged, which in turn release the DNA modified AuNPs from the electrode surface allowing free exchange of electrons. It generates a measurable electrochemical signal (eT ON). Differential pulse voltammetry (DPV) is employed to detect the recover current, which is related to the concentration of the Dam MTase. This method is simple, sensitive, nonradioactive and without use of gel-electrophoresis, PCR or chromatographic separation. Under optimized conditions, a linear response to concentration of Dam MTase range from 0.2U/mL to 10 U/mL and a detection limit of 0.12 U/mL are obtained. Furthermore, our new assay is a promising method to detect Dam MTase in the Luria-Bertani (LB) medium, as well as to screen inhibitors or drugs for Dam MTase.