We reported one-step engineering of intrinsically fluorescent silver nanoclusters–aptamer assemblies that would allow the development of facile and specific luminescent labels for target tumor cell recognition and analysis.

A strategy of combining physical embedding and covalent crosslinking was developed to encapsulate cysteamine-capped quantum dots (QDs) into agarose hydrogel microbeads (AHM). Cysteamine-capped QDs were encapsulated into the pores of agarose hydrogel microbeads by virtue of hydrogen bonding between the amino groups of cysteamine and hydroxyl groups of agarose, resulting in more than 6.0 × 107 QDs per microbead. Polyethylenimine (PEI) and oxalaldehyde were then introduced to form a covalently crosslinked network to further stabilize the encapsulation. The resulting hybrid hydrogel microbeads exhibited high doping capacity and negligible QDs leakage, and enabled optical multicolor barcoding.

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti4+) 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 Ti4+. 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 U mL−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.Highlights► We develop an electrochemical strategy for assay T4 PNK activity and inhibition. ► The electrochemical assay is based on ferrocene-functionalized SWCNT. ► Titanium ion (Ti4+) mediated signal transition. ► Signal amplification of single wall carbon nanotubes. ► Researching on the interactions between proteins and nucleic acids.

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.

Here we report a facile switchable fluorescent QD probe for F− ions, which is based on the hydrogen bonding-driven aggregation and the analyte-triggered disaggregation.

We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD+) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD+ related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD+, resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD+ with high selectivity against other NAD+ analogs. This novel assay can also provide a convenient and robust way to analyze NAD+ in biological samples such as cell lysate. As NAD+ plays an essential role in many biochemical processes, this method can be used to investigate NAD+ related life processes. For instance, the effect of calorie restriction on the intracellular NAD+ level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD+ assay holds wide applicability as an analytical tool in biochemical and biomedical research.

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.

A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection ofE. coli O157:H7 in PB buffer at levels as low as 7 cells mL−1. The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL−1 can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.

A one-step sensitive method using dynamic light scattering (DLS) was introduced for direct detection of adenosine with high selectivity. For the first time, DLS was used to detect small molecules directly and quantitatively.

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 0.2 U/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.

An electrochemical method for nicotinamide adenine dinucleotide (NAD+) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold–thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD+, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD+-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD+ range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD+ from its analogues.

It was difficult to detect small molecules directly using conventional surface plasmon resonance (SPR) biosensors since the changes of refractive index, which was resulted by binding small molecules, were usually small. In this paper, split aptamer fragments were used for the construction of SPR biosensor to determine small molecule such as adenosine with high sensitivity. An aptamer for adenosine was designed to be two flexible ssDNA pieces, one was tethered on Au film and the other was modified on Au nanoparticles (AuNPs). In the presence of adenosine, two ssDNA pieces reassembled into the intact aptamer structure and the AuNPs-labeled adenosine–aptamer complex was formed on the Au film. Then, the resonance wavelength shift was enhanced obviously, due to the electronic coupling between the localized plasmon of AuNPs and the surface plasmon wave associated with Au film. The results confirmed that this biosensor could detect adenosine with high sensitivity and selectivity. The limitation of detection (LOD) of this SPR biosensor was ca. 1.5 pM, which was an approximately ca. 2–3 order of magnitude lower than that of those SPR biosensors which utilized competitive methods.

Cocaine is one of the most abused drugs in the United States and is potentially dangerous when consumed in excess. Its detection is thus important in many areas in the fight against drug trafficking. We have developed an amplified detection method for cocaine based on a strand-displacement polymerization reaction using aptamer recognition. The system mainly consists of a hairpin probe with Cy5 labeled on its 3′ end, a primer with FAM labeled on its 5′ end, and polymerase. The aptamer sequence is integrated into the 5′-section of the hairpin probe. The primer is designed complementary to the 3′ end of the hairpin probe, which is also part of the hairpin stem region. The cocaine induced reaction cycle generates product for detection and thus for signal amplification. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent. We believe that this strategy will be useful for the development of analytical schemes for a variety of aptamers for small molecules, metal ions, and proteins. This simple scheme employing the strand-displacement polymerization reaction may find wide application in forensic analysis, environmental monitoring, and clinical diagnostics.Highlights► The detection limit of this mehod is 200 nM in about 16 min and the specificity of this approach is excellent. ► It could easily be used for real-time monitoring of the entire reaction. ► It is a universal strategy which may find wide application with other aptamers.

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 0.1–5 nmol/L, and the detection limit of thrombin in the method was 0.06 nmol/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.

In this paper, we explore the possibility of using ultrasmall near-infrared (NIR) gold nanoclusters (AuNCs) as novel contrast imaging agents for tumor fluorescence imaging in vivo. The fluorescence imaging signal of the tail vein administrated AuNCs in living organisms can spectrally be well distinguished from the background with maximum emission wavelength at about 710 nm, and the high photostability of AuNCs promises continuous imaging in vivo. The uptake of AuNCs by the reticuloendothelial system is relatively low in comparison with other nanoparticle-based contrast imaging agents due to their ultrasmall hydrodynamic size (∼2.7 nm). Through the body weight change analysis, the results show that the body weight of the mice administrated with AuNCs has not been changed obviously in comparison with that of the control mice injected with PBS. Furthermore, using MDA-MB-45 and Hela tumor xenograft models, in vivo and ex vivo imaging studies show that the ultrasmall NIR AuNCs are able to be highly accumulated in the tumor areas, thanks to the enhanced permeability and retention (EPR) effects. And the tumor-to-background ratio is about 15 for 6 h postinjection. The results indicate that the ultrasmall NIR AuNCs appear as very promising contrast imaging agents for in vivo fluorescence tumor imaging.

In this paper, a novel biocompatible and long-life lysosome labeling and tracking method based on dye entrapped silica nanoparticles (DSiNPs) has been put forward. Through colocalization studies using LysoTracker Green as the standard lysosome marker, it has been demonstrated that DSiNPs selectively accumulated in lysosomes of Hela cells and the photostability of DSiNPs associated with lysosomes was detectable, at least, 30 times as long as that of LysoTracker Green involved in lysosomes. By comparison with LysoTracker Green and Alexa 488-dextran, the fluorescence of DSiNPs could be detected over a 5-day postrecultivation period and the staining pattern in lysosomes could be well retained after cell fixation and permeabilization. In addition, results from MTT assays showed that DSiNPs did not affect the viability of Hela cells at the concentration for lysosome labeling. Primary applications of DSiNPs were then further performed in lysosome tracking in chloroquine-treated Hela cells, and lysosome labeling of differnet cell lines, including MCF-7 cells, MEAR cells, and MSC cells. These results indicated that DSiNPs, therefore, can be used as a biocompatible, long-life, and highly photostable lysosome marker for lysosome-related studies.

A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination.

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification and prior sample purification. To address these limitations, we report here the design, synthesis, and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by a steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), showing the generality of our approach. Therefore, the competition-mediated pyrene-switching aptasensor is promising to have potential for clinical and forensic applications.

There is increasing interest in developing bioconjugated carriers for the cellular delivery of bioactive molecules to stem cells, since they can allow modulation of stem cell differentiation. The present study reported biocompatible silica nanoparticle−insulin conjugates for rat mesenchymal stem cell (RMSC) adipogenic differentiation in vitro. A systematic study was first carried out on the biocompatibility of the SiNPs with RMSCs. The cell viability assay was performed to screen the SiNP concentration for creating little cytotoxicity on RMSCs. Furthermore, transmission electron microscopy (TEM) and adipogenesis and osteogenesis assays revealed that the pure SiNPs had no effect on cellular ultrastructures, adipogenic differentiation, and osteogenic differentiation. Under the optimized SiNP concentration with little cytotoxicity on RMSC and no effects on the RMSC phenotype, SiNP−insulin conjugates were prepared and used for RMSC adipogenic differentiation. Results showed that RMSCs had the ability to differentiate into adipocytes when cultured in the presence of insulin-conjugated SiNPs. This work demonstrated that the biological activity of insulin conjugated to the SiNPs was not affected and the SiNPs could be used as biocompatibile carriers of insulin for RMSC adipogenic differentiation, which would help to expand the new potential application of SiNPs in stem cell research.

This report has described a convenient genotyping method capable of detecting point mutations directly in human genomic DNA based on the combination of ligase chain reaction (LCR) and microbead-enrichment technique. LCR primers, including a biotin-labeled common primer and two fluorescence-labeled allele-specific primers, are designed for two alleles of a mutated site. When genomic DNA carries the mutated site, the common primer and allele-specific primer are ligated to form exponential amplified biotin-labeled fluorescence ligation products. These ligated products are enriched by streptavidin-coated microbeads, and genotypes are identified conveniently according to the fluorescence color of microbeads using fluorescent microscopy. Due to amplification of LCR process and enrichment of microbeads, the detection limit of the proposed method is as low as 10−15 mol/L templates. The method provides a convenient and simple strategy to detect point mutation directly in human genome. We have confirmed the efficiency of this approach with the identification of β-globin gene point mutation, which results in the reduced production of globin in an inherited hemoglobin disorder thalassemia disease.

A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5′-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 103 and 107 cfu/mL.

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H2O2, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.

We investigated the quenching properties of poly(phenylene ethynylene) with anionic alkoxyl sulfonate side groups, PPE-SO3, by tris(2,2-bipyridyl)dichlororuthenium(II) (Rubpy)-doped silica nanoparticles (SiNPs) in water, methanol, and the surfactant Triton X-100 aqueous solution. SiNPs demonstrated hyper-efficient quenching characterized by the Stern−Volmer constants in the range of 109−1010 M−1, which is about 100−10 000 times more efficient compared to the Rubpy dye. More importantly, quenching by SiNPs was found to be more efficient when the polymer existed as a nonaggregated state, such as in methanol solution and in the surfactant solution. Investigations on confocal fluorescence images and time-resolved fluorescence decays were carried out to study the quenching mechanism.

In this article, the dielectrophoretic (DEP) assembly of chemically-modified silica nanoparticles (SiNPs) was introduced. Five types of surface-modified SiNPs, including OH-SiNPs, COOH-SiNPs, CH3HPO2-SiNPs, PEG-SiNPs, and NH2-SiNPs, have been investigated. After applying an ac field with relatively high intensity and frequency, it was shown that only COOH-SiNPs and CH3HPO2-SiNPs could be self-assembled on the microelectrodes by the DEP forces. The results indicated that the anionic group modification could obviously enhance the DEP self-assembly of SiNPs on the microelectrodes. Then the DEP assembly of CH3HPO2-SiNPs was selected as a representative to be investigated further. By using Rubpy dye doped in the core of the CH3HPO2-SiNPs, the assembly process was visualized in real time by inverse fluorescence microscopy. Precise control over the frequency of the applied ac field showed that the DEP forces can assemble CH3HPO2-SiNPs from aqueous suspensions into submicrowires, and it was found that the number of assembled submicrowires between the microelectrode gaps could be well controlled with reversibility. Furthermore, the DEP assembly process of CH3HPO2-SiNPs was sensitive to the pH of the dispersed medium. These findings would provide a way to circumvent the difficulty in controlling the dielectrophoretic assembly process of nanoparticles and offer application opportunities for the DEP assembly of chemically modified SiNPs.

With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and biofunctionalized nanoparticles, a series of novel principles, methods and techniques have been exploited for bioanalytical and biomedical studies. This review mainly discusses the applications of molecular beacon probes and biofunctionalized nanoparticles-based technologies for real-time, in-situ, highly sensitive and highly selective protein analysis, including the nonspecific or specific protein detection and separation, protein/DNA interaction studies, cell surface protein recognition, and antigen-antibody binding process-based bacteria assays. The introduction of molecular beacon probes and biofunctionalized nanoparticles into the protein analysis area would necessarily advance the proteomics research.

This communication describes a facile and mild chemical etching method for resizing of CdTe nanocrystals, which provides a possible approach for tailoring the size-dependent properties of the nanocrystals.

A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. Hg2+ was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of Hg2+ between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing Hg2+ ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1−1.6 μM, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter.

Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe. The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time. Compared with traditional approach, this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavage reaction, but also offered abundant dynamic data for choosing potential gene therapeutic agents. It provides a new tool for DNAzyme research, as well as a new insight into research on human disease diagnosis. Based on this method, 8–17deoxyribozyme (8–17DNAzyme) against hepatitis C virus RNA (HCV-RNA) was designed and the cleavage process was studied in real time.

The design, synthesis, characterization and application of biologically synthesized nanomaterials have become an important branch of nanotechnology. In this paper, we report the extracellular synthesis of gold nanoparticles using Barbated Skullcup (BS) herb (a dried whole plant of Scutellaria barbata D. Don) as the reducing agent. After exposing the gold ions to BS herb extract, rapid reduction of gold ions is observed leading to the formation of gold nanoparticles in solution. UV–vis spectrum of the aqueous medium containing gold nanoparticles showed a peak at around 540 nm. Transmission electron microscopy (TEM) micrograph analysis of the gold nanoparticles indicated that they were well-dispersed and ranged in size 5–30 nm. When the gold nanoparticles were modified on the glassy carbon electrode (GCE), it could enhance electronic transmission rate between the electrode and the p-nitrophenol.

An assay for rapid and direct detection of DNA/mRNA using cationic fluorescent polymer based on one-dimensional microfluidic beads array (1-D chip) has been developed. The cationic water-soluble polythiophene derivatives can easily transduce hybridization events into measurable optical signal due to the conformational changes of the conjugated backbone, when mixed with single-stranded or double-stranded oligonucleotides. In this paper, the polymer was introduced into 1-D chip for fluorescence detection of nucleic acids, and demonstrated its capability on rapid detection of p53 complementary DNA (cDNA) with different concentration. Using this system, we have evaluated the mRNA expression changes of three tumor-associated genes (p53, c-myc and cyclin-d1) in human nasopharyngeal carcinoma CNE2 cell lines before and after 5-flouorouracil (5-FU) stimuli. These results were validated by the conventional reverse transcriptase-PCR. The most important advantage of this assay is not needed target or report labeling prior to hybridization, which makes the experiment process easy to handle and low-cost for multi-target measurement.

In this study, GFP mRNA in COS-7 cell and GFP-transfected COS-7 cell was detected in real time using phosphorothioate-modified molecular beacon based on living cell imaging method. Results showed that phosphorothioate-modified molecular beacon still kept the advantages of molecular beacon, such as, excellent selectivity, high sensitivity, and no separation detection. In addition, this modification could significantly increase the nuclease resistance of molecular beacon. Phosphorothioate-modified molecular beacon can efficiently reduce the false positive signal and improve the accuracy of living cell mRNA detection.

The biodistribution and urinary excretion of different surface-modified silica nanoparticles (SiNPs) in mice were investigated in situ using an in vivo optical imaging system. Three types of surface-modified SiNPs, including OH-SiNPs, COOH-SiNPs, and PEG-SiNPs with a size of ∼45 nm, have been prepared with RuBPY doped for imaging purposes. Intravenous (iv) injection of these SiNPs followed by fluorescence tracing in vivo using the Maestro in vivo imaging system indicated that OH-SiNPs, COOH-SiNPs, and PEG-SiNPs were all cleared from the systemic blood circulation, but that both the clearance time and subsequent biological organ deposition were dependent on the surface chemical modification of the SiNPs. Thus, for instance, the PEG-SiNPs exhibited relatively longer blood circulation times and lower uptake by the reticuloendothelial system organs than OH-SiNPs and COOH-SiNPs. More interestingly, in vivo real-time imaged dominant signal in bladder and urine excretion studies revealed that all three types of iv-injected SiNPs with a size of ∼45 nm were partly excreted through the renal excretion route. These conclusions were further confirmed through ex vivo organ optical imaging and TEM imaging and energy-dispersed X-ray spectrum analysis of urine samples. These findings would have direct implications for the use of SiNPs as delivery systems and imaging tools in live animals. Furthermore, our results demonstrate that the in vivo optical imaging method is helpful for in vivo sensing the biological effects of SiNPs by using luminescent dye doped in the silica matrix as a synchronous signal.

The effect of a pulsed electric field (PEF) on Staphylococcus epidermidis was investigated by using an atomic force microscopy (AFM) image and force measurement under liquid. The changes in the bacterial envelope were probed in situ before and after applying different dosages of PEF. Our results indicated that PEF induced the changes of bacterial peptidoglycan layer and the exposure of plasma membrane. This conclusion was further confirmed by two control experiments: the effect of the lysozyme or heat on the bacterial envelope. Furthermore, our results demonstrated that AFM analysis including image and force measurement is helpful to explain the relationship between the chemical composition change of the cellular envelope and the external stimulation.

A simple, convenient and effective method based on the surface plasmon resonance (SPR) technique was introduced for recognition of single-base mismatch DNA (smDNA) by Au nanoparticle (AuNPs)-assisted electroelution. In this method, target DNA, including perfectly matched DNA and smDNA, hybridized with the DNA probes immobilized on Au film and AuNPs, then the Au film was negatively charged. Owing to the difference in stability between single-base mismatch and perfect match DNA, effective distinction between complementary DNA (cDNA) and smDNA was achieved in the presence of an electric field: double-stranded DNA (dsDNA) formed between smDNA targets and DNA probes was denatured by the repulsion force acting on the negatively-charged DNA-linked AuNPs, while the perfectly matched duplex was not influenced. However, if the AuNPs were absent, the effects of cDNA and smDNA were not distinguishable. The effects of electric field intensity and mismatch sites were also investigated. All of the operations were performed under mild conditions. The results showed that AuNP-assisted electroelution may be exploited for the construction of biosensors with high selectivity.

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360–480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N = 3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site “sandwich” immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per μl sample can be determined specifically. The expression changes of p53, c-myc and β-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.

The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.

Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon (MB) specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.

In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.

The specific interaction between angiogenin and aptamer has been investigated by using AFM. The specificity of the interaction is revealed by comparing the binding probability of aptamer to other elements in a series of control experiments. The results have shown that there is specific interaction force between angiogenin and aptamer. Moreover, the single molecular pull-off force between angiogenin and aptamer has also been determined using the Poisson statistical method to be 133.7±11.7 pN. These findings obtained are helpful to the better revelation of recognition mechanism between angiogenin and aptamer, which provided basis for further understanding the inhibition of the aptamer to angiogenic activity.

A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly(N-isopropylacrylamide) (PNIPAm). The permeation ability of the membranes at different temperatures was investigated using sodium fluorescein and quantum dots as probes. The results showed that the pore diameter of nanotube was changed due to the reversible response of PNIPAm-modified membranes to temperature, and then the permeation ability of the membranes was changed. The permeation of fluorescence probes was slow and even almost blocked at 25°C (below the lower critical solution temperature, LCST), since PNIPAm formed expanded structures and decreased the pore size. While at 40°C (above the LCST), the permeation was increased, since PNIPAm became compact structures and the pore diameter was increased. Furthermore, the permeation ability of the temperature-sensitive nanotube array membranes could be adjusted reversibly and it is possible to use the membranes in nanofluidic devices, nanogates, etc.

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen–antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.

A sensitive method for rapid angiogenin (Ang) detection based on fluorescence resonance energy transfer (FRET) has been described. A dual-labeled probe based on high affinity aptamer for Ang was constructed. As donor and acceptor, 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TMR) were labeled at 5′- and 3′-termini of the aptamer probe, respectively. The dual-labeled probe showed obvious fluorescence changes due to the specific binding between aptamer and Ang. By monitoring the fluorescence intensity of donor and acceptor, quantitative Ang detection could be achieved. This assay is highly specific and sensitive, with a detection limit of 2.0 × 10−10 mol L−1 and a linear range of 5.0 × 10−10 to 4.0 × 10−8 mol L−1 Ang. Ang in serum samples of health and lung cancer were also detected.

A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of ∼0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.

Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.

As a highly conserved damage repair protein, uracil–DNA glycosylase (UDG) mainly catalyzes the excision of uracil from DNA to sustain the genome integrity. Here a novel method for monitoring the uracil removal in real time is introduced. Double-stranded DNA probes modified with uracil residues that can occur in fluorescent resonance energy transfer (FRET) were used as substrates and detecting probes in a homogeneous solution. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also was used to accurately determine the kinetic constant of UDG. The limit of detection of UDG was 0.033 U/ml. The KM and Kcat were 0.11 μM and 4 s−1, respectively. In addition, the method was applied to investigate the influence of chemical drugs on UDG activity. The results showed that 10 mM fluorouracil (5-FU) and gentamicin are inhibitors to UDG. The in vitro detection of UDG in A549 cells showed that the activity of UDG was four times greater after the cells were treated with cisplatin. These results showed that this method can monitor uracil removal in real time and conveniently assay UDG activity with ultrasensitivity and excellent specificity in the homogeneous solution. This method is also amenable to high-throughput drug screening in vitro.

Great efforts have been made on the early diagnosis and molecular mechanism research of tumour metastasis in recent years. In this paper, based on the one-dimensional microfluidic beads array, a novel platform for tumour metastasis-associated genes profiling has been developed by depositing nucleic acids functional beads in the microchannel. This platform is sensitive (limit of detection: 0.02 nmol/L) and can perform mRNAs analysis without PCR. Two human colon cancer cell lines (primary and metastatic) from the same patient were used as a model, and transcriptional expression profiling of multiple tumour metastasis-associated genes in these two cell lines was successfully achieved. Furthermore, the results obtained on the beads array were validated by RT-PCR. This novel beads array has advantages of high sensitivity, little sample consumption, short assay time, low cost and high throughput capability. It holds the potential in early diagnosis and mechanism research of tumour metastasis.

A simple and efficient approach for the rapid isolation of plasmid DNA from crude cell lysates has been described. The approach took advantage of the amino-modified silica coated magnetic nanoparticles (ASMNPs) with positive zeta potential at neutral pH and superparamagnetism under the external magnetic fields. As a demonstration, the pEGFP-N3 plasmid has been concentrated and isolated from the E. coli DH5α transformed with pEGFP-N3 plasmid through electrostatic binding between the positive charge of the amino group of ASMNPs and the negative charge of the phosphate groups of the plasmid DNA. Then the pEGFP-N3 plasmid has been released easily and quickly from the pEGFP-N3 plasmid–ASMNPs complexes with 3 M NaCl. The entire procedure could be carried out by the aid of external magnetic fields in 15 min and eliminate the need of phenol, cesium chloride gradients or other noxious reagents and complexes operation. Moreover, the pEGFP-N3 plasmid obtained by this approach retains biological activity that can be suitable for restriction enzyme digestion and cells transfection with expression of green fluorescence protein.

Melting curve analysis is a powerful tool for detecting single-base mutations that may be linked to genetic diseases. Current existing methods provide insignificant melting point difference for some point mutations with the risk of wrong genotyping results, causing great limitations to their applications in clinic diagnosis. Here, we have developed an enhanced melting point difference approach to genotype single-base mutations using DNA ligase. Ligase covalently joins an allele-specific discriminating probe and a signal probe flanked the mutation site to form a long duplex, resulting in an enhanced melting temperature. CD17 and Ivs-2-654 point mutations of β-globin gene in thalassemia disease were identified by using this approach, and the homozygotes and heterozygotes were scored accurately and conveniently. To the best of our knowledge, the use of ligase to improve the differences of melting temperature between various genotypes has not been reported. This method will provide a promising tool for clinical diagnosis of gene-mutant diseases.

Bio-functioned fluorescent silica nanoparticles have been synthesized for cell labeling and cell differentiation and have shown great promise as novel fluorescent probes. The galactose-conjugated fluorescent nanoparticles (GCFNPs) have been obtained by the conjugation of amino-modified fluorescent silica nanoparticles with lactobionic acid (LA) through EDAC linkage. The GCFNPs retain excellent biological activity and can be used in bioanalysis as an immunofluorescence assay. The specific identification of target cells from the background cells have been directly demonstrated in a simple model system by a laser confocal scanning microscope, because the specific and non-specific labeling can simultaneously visualized in a given microscopic field of view. The flow cytometric analysis has proved that GCFNPs can effectively recognize target cells in the mixed cell system. The demonstration of precise identification of few liver cancer cells in the blood confirmed the excellent capability of GCFNPs in identifying specific cells in a large host cell background. The nanoparticle's excellent photostability, good biocompatibility and significant signal amplification make them well-suited for the identification of individual cells sensitively for a variety of biomedical studies such as cancer metastasis and stem cell progeny in vivo.

In this paper, the relationship of intracellular acidification and apoptosis in Hela cells induced by vincristine sulfate has been studied by use of the ratiometric pH nanosensors that have been developed by our group, employing fluorescein isothiocyanate (FITC) doped as the pH-sensitive dye and Tris(2,2′-bipyidyl) dichlororuthenium(II) hexahydrate (RuBpy) doped as reference dye. The pH change of the Hela cells induced by vincristine sulfate has been monitored in vivo, in situ and real time by use of the ratiometric pH nanosensors. The experimental results show that the pH of the apoptotic Hela cells induced by vincristine sulfate has been acidified from 7.11 to 6.51, and the percentage of intracellular acidification is correlated with the induced concentration and incubation time of the vincristine sulfate. The further study of the percentage of intracellular acidification and the percentage of apoptosis of Hela cells at the same time reveals that apoptosis of Hela cells induced by vincristine sulfate is preceded by intracellular acidification. These results would provide theoretical foundation for the therapy of cancer through interfering the pH of cells by use of vincristine sulfate or other anti-cancer drugs.

Cy5 dye is widely used as a biomarker in the research fields of life science because of its excitation at wavelengths above 600 nm where autofluorescence of bio-matter is much reduced. However, Cy5 dye could not be encapsulate into silica directly to form stable nanoparticles by using of the traditional methods. In this paper, an improved method had been developed to prepare Cy5 dye doped core-shell silica fluorescent nanoparticles (SFNPs), employing biomolecules conjugated Cy5 as the core material and silica coating produced from the hydrolysis TEOS (tetraethyl orthosilicate) in the water-in-oil microemulsion. To obtain stable Cy5 dye doped SFNPs with core-shell structure, five kinds of biomolecules with different iso-electric point (pI) have been selected to conjugate Cy5 for preparation of core-shell SFNPs. Results demonstrated that very bright and photostable Cy5 doped core-shell SFNPs could be both prepared by use of positive polysine conjugated Cy5 or IgG conjugated Cy5 as the core material, respectively. IgG conjugated Cy5 doped core-shell SFNPs was selected as a demonstration to be characterized and applied as a near-infrared fluorescent marker in cell recognition. The results showed that Cy5 doped core-shell SFNPs prepared by conjugating with a positive biomolecules IgG as the core material were luminescent and stable. About 110 Cy5 dye molecules could be doped in one nanoparticle with size of 42 ± 5 nm. The breast cancer cells had been selectively recognized by use of the near-infrared fluorescent marker based on the Cy5–IgG doped core-shell SFNPs. And the results demonstrated that this Cy5 doped core-shell SFNPs fluorescence marker was superior to the pure Cy5 dye marker for cell recognition in photostability and detection sensitivity.

The catalytic growth of Au nanoparticles (AuNPs) has been employed in several analytical methods for improving the detection sensitivity, or integrated with the enzyme reactions for the quantitative detection of the respective substrates. However, the catalytic growth of Au nanoparticles do not work in some situations, such as surface plasmon resonance (SPR), electrochemistry, where metal matrices were used, because metal matrices used in these techniques, e.g. Au, are susceptible to metal deposition, which increased the background seriously. In this work, a SiO2 layer was vapor-deposited on the gold film. The inhibition of metal deposition by this SiO2 layer was investigated by SPR sensor. The results showed that the SiO2 layer could avoid the deposition of metal on Au film. With the low background achieved by SiO2-coated Au films, sensitive detection of DNA hybridization using the catalytic growth of Au nanoparticles enhanced SPR was demonstrated. The work described here maybe helpful for the development of sensitive bioanalytical methods.

NAD+-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD+-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 × 10−4 Weiss Unit mL−1 to 0.4 Weiss Unit mL−1 and the detection limit of 4.0 × 10−4 Weiss Unit mL−1.

In this paper, an improved recovery method for target ssDNA using amino-modified silica-coated magnetic nanoparticles (ASMNPs) is reported. This method takes advantages of the amino-modified silica-coated magnetic nanoparticles prepared using water-in-oil microemulsion technique, which employs amino-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. The nanoparticles have a silica surface with amino groups and can be conjugated with any desired bio-molecules through many existing amino group chemistry. In this research, a linear DNA probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. A target ssDNA(I) (5′-TMR-CGCATAGGGCCTCGTGATAC-3′) has been successfully recovered from a crude sample under a magnet field through their special recognition and hybridization. A designed ssDNA fragment of severe acute respiratory syndrome (SARS) virus at a much lower concentration than the target ssDNA(I) was also recovered with high efficiency and good selectivity.

A novel optical chemical sensor based on the dynamic liquid drops combined with flow injection-solid phase has been developed for continuous and sequential determinations of the mixture of Vitamin B1, Vitamin B2 and Vitamin B6. The design and characteristics of the sensor system are described. The dynamically growing and falling drops serve as windowless optical cells. The adsorption, desorption and quantitative determinations of VB1, VB2 and VB6 were carried out based on selective adsorption of Sephadex CMC-25 and different fluorescence characteristics of VB2, VB6 in the basic solutions. The optimum analytical conditions for compound Vitamin B assay have been established. Under these conditions, linear calibration curves were obtained over the range of 0.01–8.00, 0.01–10.00 and 0.01–3.00 μg/ml for VB1, VB2 and VB6 with the limits of determination of 0.008, 0.005 and 0.006 μg/ml, respectively. This method has been applied in the determination of synthetic mixture of VB1, VB2, VB6 and Vitamin B compound tablets with satisfactory results. The technique, which has been described, provides a simple, effective and sensitive method to assay the biological samples.

A strategy of combining physical embedding and covalent crosslinking was developed to encapsulate cysteamine-capped quantum dots (QDs) into agarose hydrogel microbeads (AHM). Cysteamine-capped QDs were encapsulated into the pores of agarose hydrogel microbeads by virtue of hydrogen bonding between the amino groups of cysteamine and hydroxyl groups of agarose, resulting in more than 6.0 × 107 QDs per microbead. Polyethylenimine (PEI) and oxalaldehyde were then introduced to form a covalently crosslinked network to further stabilize the encapsulation. The resulting hybrid hydrogel microbeads exhibited high doping capacity and negligible QDs leakage, and enabled optical multicolor barcoding.

Here we report a facile switchable fluorescent QD probe for F− ions, which is based on the hydrogen bonding-driven aggregation and the analyte-triggered disaggregation.

A one-step sensitive method using dynamic light scattering (DLS) was introduced for direct detection of adenosine with high selectivity. For the first time, DLS was used to detect small molecules directly and quantitatively.

This communication describes a facile and mild chemical etching method for resizing of CdTe nanocrystals, which provides a possible approach for tailoring the size-dependent properties of the nanocrystals.