DSN/TdT recycling digestion based cyclic amplification strategy for microRNA assay.

Sensitive and specific detection of microRNAs (miRNAs) is of great significance for early cancer diagnosis. Here we report a simple and sensitive fluorescence signal amplification strategy that based on DSN/TdT recycling digestion for miRNA detection. DSN initiates DNA digestion on 3'-phosphate-primer/miRNA heteroduplex which causes miRNA recycle. The digested DNA strands with 3'-OH ends enable TdT to synthesize a polydeoxyguanylic tails on the 3'-end. The DNAs with polydeoxyguanylic tails are converted to double-stranded-DNA prior to initiation of DSN/TdT recycling digestion. With the cooperation of TdT and DSN, a new round of digestion and extension is triggered, leading to massive fluorophores separating and signal amplification. The amplification strategy produces large amounts of 3'-OH probes that can be used directly for dsDNA enrichment and DSN digestion. Moreover, both DSN digestion and TdT extension are sequence-independent reaction without the need of complex sequences design. In addition, this strategy is utilized to analyze miRNA samples from MCF-7 cell lysates and Cu (II) ion samples, indicating its potential application in actual sample analysis. The method shows a promising analytical platform for DNA nicking-related studies and tumor biomarkers measuring in clinical diagnostics.

Telomerase-triggered DNAzyme spiders for exponential amplified assay of cancer cells.

A highly flexible electrochemical assay based on target-triggered DNAzyme spiders was proposed for the detection of telomerase. The DNAzyme-telomerase substrate primers (D-TSP) containing Cu2+-dependent DNAzymes serve as recognition elements, and primers of telomerase. Telomerase extracted from Hela cells recognize the D-TSP and elongated with DNA sequence repeats. A synthetic telomerase product hybridized with scaffold sequences of two DNAzyme-tethered probes on the basis of the mechanism of the proximity-ligation assay. The three-leg DNAzyme spiders has been assembled and initiated the autonomous hybridization/nicking/displacement cycles on substrate modified surface. The cleaved ferrocene-labeled fragements are adsorbed on gold surface leading to an increase in the electrochemical signal. As a result, the one input target, telomerase, release large amount of ferrocene-labeled DNA strands, achieving an exponential signal amplification and an excellent improvement in sensitivity over single molecule or two-component 'sandwich' binding complexes. Our proposed biosensor showed a nonlinear dependence with Hela cell numbers, ranging from 25 to 2000 with a detection limit of 10 cells. Telomerase activities from different cell lines were also successfully evaluated. Our electrochemical strategy based on target-triggered DNAzyme spiders was enzyme-free, PCR-free, simple in operation which indicated that it expected to expand the scope of DNA nanotechnology in the areas of clinical diagnosis.

Cleaved DNAzyme substrate induced enzymatic cascade for the exponential amplified analysis of L-histidine.

A novel strategy of cleaved DNAzyme substrate induced enzymatic cascade has been devised for the exponential amplified detection of L-histidine. The enzyme strand carries out hydrolytic cleavage of the substrate strand in the presence of L-histidine. The cleaved DNAzyme substrates introduce the polymerase/endonuclease reaction cycles as primers. The L-histidine acts as the activator for enzymatic cascade amplification generating a distinguishable fluorescence enhancement. A good nonlinear correlation (R=0.9994) between fluorescence intensity and the logarithm of the L-histidine concentration is obtained over the range from 50 nM to 1.0 mM. The detection limit was estimated as 30 nM. This efficient amplification of the fluorescence signal is attributed to the L-histidine induced cooperation of Klenow Fragment polymerase (exo(-)) and Nb.BbvCI endonuclease reaction. The activation of such enzymatic cascades through analyte-DNAzyme interactions has a substantial impact on the development of exponential amplified DNAzyme sensors.

Enzymatic cascade based fluorescent DNAzyme machines for the ultrasensitive detection of Cu(II) ions.

A novel enzymatic cascade based fluorescent DNAzyme machine has been developed for the amplified detection of copper (Cu(2+)) ions. This is the first attempt to carry out the combination of the self-cleaving DNAzyme and the polymerase/endonuclease reaction cycles involving cleaved substrate extension. In the presence of Cu(2+) ions, the enzyme strand carries out catalytic reactions to hydrolytic cleavage of the substrate strand. The cleaved DNAzyme substrates act as primers and trigger the Klenow Fragment polymerization. Nb.BbvCI endonuclease cuts the double-stranded niking site and thus opens a new site for a new replication. The replication regenerates the complete dsDNA to initiate another cycle of nicking, polymerization and displacement. Finally the fluorescence dye, SG, inserts into the DNA double helix to generate a distinguishable fluorescence enhancement. The Cu(2+) ions act as the activator for enzymatic cascade amplification generating multiple duplex structures in the nascent product. An increasing fluorescence is observed with increasing Cu(2+) ions concentration. A good nonlinear correlation (R=0.9997) was obtained between fluorescence intensity and the cubic logarithm of the Cu(2+) ions concentration over the range 0.50-200 nM. This nonlinear response phenomenon results in an efficient improvement of the sensitivity of our current proposed assay. The activation of such enzymatic cascades through analyte-DNAzyme interactions is not only valuable to activate the cooperation of enzyme networks, but also has a substantial impact on the development of amplified DNAzyme sensors.

Electrochemical aptameric sensor based on the Klenow fragment polymerase reaction for cocaine detection.

An electrochemical aptasensor based on Klenow fragment (KF) polymerase reaction that combines the aggregation of ferrocene-functionalized oligonucleotide has been developed successfully for cocaine detection. In the presence of cocaine, the recognition probe changed its hairpin conformation into the tripartite complex. The aptamer-cocaine complex gave a 3'-single-stranded tail sequence complementary to the surface-tethered capture probe. In KF polymerase reaction, the recognition probe served as a template for the extension of a capture probe. It requires a sample volume of 2 µL and is complete within 1 h. The ferrocene-appended oligonucleotide incorporated into the newly synthesized complementary probe leads to an electrochemical response. This sensitive detection of cocaine is due to a very low background signal and large signal enhancement up to 9-fold upon addition of analyte. It permits detection of as low as 200 µM cocaine. The simple and isothermal procedure does not require thermal cycling or special laboratory conditions, which makes it adaptable to low-cost and robust biosensing.

Biocatalytic growth of gold agglomerates on an electrode for aptamer-based electrochemical detection.

Here we describe the biocatalytic growth of high-density gold agglomerates on a gold electrode surface to form a carrier for aptamer probe immobilization. The present approach provides a simple strategy to promote the seed-mediated deposition of Au from AuCl(4)(-) onto surface-attached 12 nm diameter Au nanoparticles (AuNPs) in the presence of reductive coenzyme and surfactant. The growth process was studied by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). This nanostructured platform is effective and prospective toward the aptamer probe immobilization. For the nice performance of enhanced substrate, the aptamer-sensing interface showed excellent applicability under the investigations such as alternating current voltammetry (ACV) and surface-enhanced Resonance Raman scattering (SERRS) spectra.

Fluorescence aptameric sensor for strand displacement amplification detection of cocaine.

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement. Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double-stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical.

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection.

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34nM of HNE, and the response is linear in the tested concentration range of 0.34-68nM with the detection limit of 47pM.

[Preliminary discussion on the theory of body physiognomy in the Huangdineijing (Huangdi's Inner Classic)].

There are so many discussions on the body physiognomy in Huangdineijing (Huangdi's Inner Classic) that include not only the description of both difference of body physiognomy and its reasons, but also the discussions on physiological and pathological characteristics of different body physiognomy traits. Different physiological and pathological characteristics lead to different applicable therapeutic methods, so that Huangdineijing discusses the applicable therapeutic methods to different body physiognomy characteristics, especially elaborating on the difference between applicable acupuncture manipulation to those characteristics. Contents such as the above form the embryonic form of the theory of body physiognomy in Traditional Chinese Medicine (TCM).

Novel fluorescence enhancement IgE assay using a DNA aptamer.

In this paper, we demonstrate a fluorescence immunoglobulin E (IgE) assay probe based on a DNA aptamer. A Texas red-labeled short DNA strand (T-DNA) complementary with part of the IgE aptamer sequence was used to produce the fluorescence enhancement effected upon the binding of IgE to the aptamer. Another short DNA strand labeled with dabcyl quencher (Q-DNA) complementary with part of the aptamer sequence nearby the T-DNA location was used to lower the background fluorescence. The IgE can be detected in the concentration range from 9.2 x 10(-11) to 3.7 x 10(-8) mol L(-1) with a detection limit of 5.7 x 10(-11) mol L(-1).

United Nations system efforts to support the response to AIDS in China.

In the last two years, we have seen a remarkable intensification in the response to AIDS in China. A number of organizations have joined and contributed to the efforts of the Chinese government in responding the AIDS epidemic in China. This article specifically describes the role of the United Nations (UN) in supporting and strengthening those responses. Achievements of the United Nations highlighted in the article include: strengthened leadership and political commitment to respond to AIDS; improved HIV/AIDS surveillance and information; expanded prevention efforts; improved treatment, care and support to people living with HIV and increased resources for AIDS programs. Additional roles of the United Nations system in the near future include strengthening national leadership by supporting the 'three ones', i.e., one national plan on AIDS; one national coordinating authority for AIDS; and one monitoring and evaluation system for AIDS. In addition, the UN system is expected to strengthen alignment and harmonization of activities of all international organizations and improved accountability and oversight. Remaining challenges identified include increasing awareness of AIDS and reducing stigma and discrimination; reducing vulnerability and risk behaviour among specific groups; providing improved treatment, care and support for people living with HIV; promoting stronger engagement by civil society, and; addressing the gender dimensions of AIDS.