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Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020)., Competing Interests: G.W.Y. is co-founder, member of the Board of Directors, equity holder, and paid consultant for Locana and Eclipse Bioinnovations. G.W.Y. is a Distinguished Visiting Professor at the National University of Singapore. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies. M.C. and G.W.Y. are inventors on a patent disclosure to UCSD on technologies described in this manuscript. H.Y.C. is an inventor on a patent owned by Stanford University on in vivo SHAPE. H.Y.C. is a co-founder of Accent Therapeutics and Boundless Bio and an advisor for 10× Genomics, Arsenal Biosciences, and Spring Discovery. H.Y.C. is an Investigator of the Howard Hughes Medical Institute., (© 2021.)

RNA is a pivotal element of the cell which is most of the time found in complex with protein(s) in a cellular environment. RNA can adopt three-dimensional structures that may form specific binding sites not only for proteins but for all sorts of molecules. Since the early days of molecular biology, strategies to probe RNA structure have been developed. Such probes are small molecules or RNases that most of the time specifically react with single strand nucleotides. The precise reaction or cleavage site can be mapped by reverse transcription. It appears that nucleotides in close contact or in proximity of a ligand are no longer reactive to these probes. Carrying the RNA probing experiment in parallel in presence and absence of a ligand yield differences that are known as the ligand "footprint." Such footprints allow for the identification of the precise site of the ligand interaction, but also reveals RNA structural rearrangement upon ligand binding. Here we provide an experimental and analytical workflow to carry RNA footprinting experiments.

Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Micro RNA (miR) are regulatory non-coding RNA molecules, which contain a small number of nucleotides ~18-28 nt. There are many various miR sequences found in plants and animals that perform important functions in developmental, metabolic, and disease processes. miRs can bind to complementary sequences within mRNA molecules thus silencing mRNA. Other functions include cardiovascular and neural development, stem cell differentiation, apoptosis, and tumors. In tumors, some miRs can function as oncogenes, others as tumor suppressors. Levels of certain miR molecules reflect cellular events, both normal and pathological. Therefore, miR molecules can be used as biomarkers for disease diagnosis and prognosis. One of these promising molecules is miR-21, which can serve as a biomarker with high potential for early diagnosis of various types of cancer. Here, we present a novel design of miR detection and demonstrate its efficacy on miR-21. The design employs emissive properties of DNA-silver nanoclusters (DNA/AgNC). The detection probe is designed as a hairpin DNA structure with one side of the stem complimentary to miR molecule. The binding of target miR-21 opens the hairpin structure, dramatically modulating emissive properties of AgNC hosted by the C 12 loop of the hairpin. "Red" fluorescence of the DNA/AgNC probe is diminished in the presence of the target miR. At the same time, "green" fluorescence is activated and its intensity increases several-fold. The increase in intensity of "green" fluorescence is strong enough to detect the presence of miR-21. The intensity change follows the concentration dependence of the target miR present in a sample, which provides the basis of developing a new, simple probe for miR detection. The detection strategy is specific, as demonstrated using the response of the DNA/AgNC probe towards the scrambled miR-21 sequence and miR-25 molecule. Additionally, the design reported here is very sensitive with an estimated detection limit at ~1 picomole of miR-21.

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO 3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm -1 . With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Near-infrared (NIR) fluorescence imaging has been proved as an effective modality in identifying the tumor border and distinguishing the tumor cells from healthy tissue during the oncological surgery. Developing NIR fluorescent probes with high tumor to background (T/B) signal is essential for the complete debulking of the tumor, which will prolong the survival rate of tumor patients. However, the nonspecific binding and "always-on" properties of the conventional fluorescent probes leads to high background signals and poor specificity. Method : To address this problem, glutathione (GSH)-responsive, two disulfide-bonded dicyanine dyes ( ss-diCy5 and ss-diNH800CW ) were synthesized. As synthesized dyes are quenched under normal physiological conditions, however, once reached to the tumor site, these dyes are capable of emitting strong fluorescence signals primarily because of the cleavage of the disulfide bond in the tumor microenvironment with high GSH concentration. Besides, the GSH-responsive behavior of these dyes was monitored using the UV-vis and fluorescence spectroscopy. The diagnostic accuracy of the aforementioned dyes was also tested both in tumor cells and 4T1-bearing mice. Results : The fluorescence signal intensity of disulfide dicyanine dyes was quenched up to 89% compared to the mono cyanine dyes, thus providing a very low fluorescence background. However, when the disulfide dicyanine dye reaches the tumor site, the dicyanine is cleaved by GSH into two mono-dyes with high fluorescence strength, thus producing strong fluorescent signals upon excitation. The fluorescent signal of the dicyanine was enhanced by up to 27-fold after interacting with the GSH solution. In vivo xenografts tumor studies further revealed that the fluorescence signals of aforementioned dyes can be quickly recovered in the solid tumor. Conclusion : In summary, the disulfide dicyanines dyes can provide a promising platform for specific tumor-activatable fluorescence imaging with improved T/B value., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan ® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.

DNA methylation is an epigenetic regulation of gene expression, which has drawn great attention in biomedical research due to its association with various diseases. A robust, inexpensive platform to detect and quantify the methylation status in a specific genomic region is necessary. In this study, an on-chip analytical technique of cytosine methylation with droplets in a microchannel is proposed. Genomic DNA samples are encapsulated into a series of droplets and transported through a detection region, where a stable temperature gradient is created. As the temperature is elevated from 60 °C to 85 °C, the DNA samples denature and the associated fluorescence signals decay, with the relationship being acquired as the melting curve. The droplets serve as discrete reactors for conducting DNA melting curve analysis in the liquid phase, thereby eliminating the need for immobilization of reagents. Due to a high heating rate and greater enhanced thermal stability, this microchip allows larger melting temperature differences for the samples at different percentages of methylated DNA. It has an enhanced discrimination ability and lower volume consumption, compared to the commercial qPCR machine. This chip enables quantification of the methylation levels of the pluripotent stem cell factor Oct-4 in its distal enhancer (DE) region, with a designed probe after bisulfite treatment and asymmetric PCR.

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal's position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design., (Copyright © 2016 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are linked to a large number of human diseases and disorders. In this work, we developed a colorimetric method for rapid, ultrasensitive miRNA detection via isothermal exponential amplification reaction (EXPAR)-assisted gold nanoparticle (AuNP) amplification. The sensing probe designed with a tandem phosphorothioate modification in the backbone of the polyadenines at the 5' terminus was employed to directly assemble onto the surface of AuNP with high adsorption affinity. The recognition domain at the 3' terminus of the sensing probe hybridizes with target miRNAs to trigger EXPAR with exponential signal amplification. With the amplification reaction with the action of DNA polymerase, the sensing probe gradually detaches from the AuNP, resulting in the aggregation of bare AuNPs in the high-salt reaction environment due to lack of DNA protection. The presence of AuNP aggregation is conveniently measured by UV-vis spectroscopy. Our proposed method could provide a linear detection range from 50fM to 10nM with a detection limit of ∼46fM within 60min, and also discriminate a single-nucleotide difference between homologous miRNAs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ultrasensitive, rapid and selective diagnostic probes are urgently needed to overcome the limitations of traditional probes for norovirus (NV). Here, we report the detection of NV genogroup II via nucleic acid hybridization technology using a quantum dot (QD)-conjugated molecular beacon (MB) probe. To boost the sensitivity of the MB assay system, an ultrasensitive QD fluorophore with unique optical properties was synthesized, characterized and exploited as a fluorescence signal generator. Alloyed thioglycolic (TGA)-capped CdZnSeS QDs with a high photoluminescence (PL) quantum yield (QY) value of 92% were synthesized, and a modified silanization method was employed to encapsulate the thiol-capped QDs in a silica layer. The resulting highly luminescent alloyed SiO2-coated CdZnSeS QDs had a remarkable PL QY value of 98%. Transmission electron microscopy and dynamic light scattering confirmed the monodispersity of the alloyed nanocrystals, and zeta potential analysis confirmed their colloidal stability. Powder X-ray diffraction and PL lifetime measurements confirmed the surface modification of the QDs. The alloyed TGA-capped and SiO2-coated CdZnSeS QD-conjugated MB bioprobes detected extremely low concentrations of NV RNA. Ultrasensitive detection of low concentrations of NV RNA with a limit of detection (LOD) of 8.2copies/mL in human serum and a LOD of 9.3 copies/mL in buffer was achieved using the SiO2-coated CdZnSeS QD-MB probes, an increase in sensitivity of 3-fold compared with the detection limit for NV RNA using TGA-capped CdZnSeS QD-MBs. The additional merits of our detection system are rapidity, specificity and improved sensitivity over conventional molecular test probes., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel surface plasmon resonance (SPR) biosensor, coupled with the magnetic bioseparation technique, was constructed and used to the determination of human IgG. Carboxyl-functionalized graphene oxide (cGO) sheet was employed as the sensing film for the efficient immobilization of capture antibody (Ab1). Nanoconjugates (FHAb2), obtained by binding detection antibody (Ab2) to the nanohybrids containing Fe3O4 nanoparticles (Fe3O4 NPs) and hollow gold sphere nanoparticles (HGNPs), were used to specifically collect the target analytes from sample solutions and serve as labels. Owing to the notable plasmonic fields spreading over inner and outer surfaces, HGNPs played key roles in amplifying the SPR response signals originating from the dielectric changes on the sensing films during the binding of Ab1 and human IgG-Ab2FH complexes. In addition, FHAb2 were also used as "vehicles" for the rapid delivery of the separated and enriched target analytes from sample solutions to the sensor surface via an external magnet. In the present method, taking advantages of the magnetic field-driven mass transfer and the significant signal amplification effect of FHAb2, the separation and analysis of human IgG in serum samples are quite effective and sensitive. The limit of detection was 1.88ngmL(-1), which is about 260-fold lower than that obtained by routine SPR biosensors with sandwich assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nanog is among the most important indicators of cell pluripotency and self-renew, so detection of Nanog is critical for tumor assessment and monitoring of clinical prognosis. In this work, a novel method for Nanog detection is proposed by using electrochemical technique based on target-induced conformational change of an electrode-bound DNA pseudoknot. In the absence of Nanog, the rigid structure of the pseudoknot will minimize the connection between the redox tag and the electrode, thus reducing the obtained faradaic current. Nevertheless, the Nanog binding may liberate the flexible single-stranded element that transforms the DNA pesudokont into DNA hairpin structure due to steric hindrance effect, thus making the electrochemical tag close to the electrode surface. Consequently, electron transfer can be enhanced and very well electrochemical response can be observed. By using the proposed method, Nanog can be determined in a linear range from 2nM to 25nM with a detection limit of 163 pM. Furthermore, the proposed method can be directly used to assay Nanog not only in purified samples but also in complex media (cell extracts), which shows potential applications in Nanog functional studies as well as clinical diagnosis in the future., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent years have witnessed great progress in developing functional nucleic acids (FNAs)-based sensors for the detection of heavy metal ion. In this review, four types of the FNAs that most widely-used in heavy metal ions detection were briefly introduced and a dozen of recently published review articles which summarized those FNAs-based sensors were introduced. Particularly, according to the degree of automation and system integration, those FNAs-based sensors which belong to the lab-on-a-chip (LOC) category were reviewed in more detail by classifying them into six types such as microfluidic LOC system, microchip, lateral flow dipstick, personal glucose meter, microfluidic paper-based analytical devices (μPADs) and disc-based analytical platform. After gave a brief description of the sensing strategies, properties, advantages or disadvantages of these FNAs-based sensors, existing problems and future perspectives were also discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Foodborne pathogens pose a significant threat to human health worldwide. The identification of foodborne pathogens needs to be rapid, accurate and convenient. Here, we constructed a nanoparticle cluster (NPC) catalyzed signal amplification biosensor for foodborne pathogens visual detection. In this work, vancomycin (Van), a glycopeptide antibiotic for Gram-positive bacteria, was used as the first molecular recognition agent to capture Listeria monocytogenes (L. monocytogenes). Fe3O4 NPC modified aptamer, was used as the signal amplification nanoprobe, specifically recognize to the cell wall of L. monocytogenes. As vancomycin and aptamer recognize L. monocytogenes at different sites, the sandwich recognition showed satisfied specificity. Compared to individual Fe3O4 nanoparticle (NP), NPC exhibit collective effect-enhanced catalytic activity for the color reaction of chromogenic substrate. The change in absorbance or color could represent the concentration of target. Using the Fe3O4 NPC-based signal amplification method, L. monocytogenes whole cells could be directly assayed within a linear range of 5.4×10(3)-10(8) cfu/mL and a visual limit of detection of 5.4×10(3) cfu/mL. Fe3O4 NPC-based method was more sensitive than the Fe3O4 NP-based method. All these attractive characteristics of highly sensitivity, visual and labor-saving, make the biosensor possess a potential application for foodborne pathogenic bacteria detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Thymine DNA glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation, which also plays an essential role in DNA demethylation. In this work, the novel iridium(III) complex 1 with an anchor tail was synthesized and employed to construct a G-quadruplex-based assay for detecting TDG activity in aqueous solution by using the mismatched base excising property of TDG with T4 DNA ligase and phi29 DNA polymerase, in concert with the rolling circle amplification (RCA) strategy. The assay achieved a detection limit of 0.048UmL(-)(1) (0.012ngmL(-1)), and showed high selectivity towards TDG even in the presence of other proteins and enzymes. Additionally, the assay could function in diluted cellular debris., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Simultaneous and multiplexed detection of microRNAs (miRNAs) in a whole exosome is developed, which can be utilized as a PCR-free efficient diagnosis method for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs from parental cells. Because they circulate throughout bodily fluids, exosomal biomarkers offer great advantages for diagnosis in many aspects. In general, PCR-based methods can be used for exosomal miRNA detection but they are laborious, expensive, and time-consuming, which make them unsuitable for high-throughput diagnosis of diseases. Previously, we reported that single miRNA in the exosomes can be detected specifically using an oligonucleotide probe or molecular beacon. Herein, we demonstrate for the first time that multiple miRNAs can be detected simultaneously in exosomes using miRNA-targeting molecular beacons. Exosomes from a breast cancer cell line, MCF-7, were used for the production of exosomes because MCF-7 has a high level of miR-21, miR-375, and miR-27a as target miRNAs. Molecular beacons successfully hybridized with multiple miRNAs in the cancer cell-derived exosomes even in the presence of high human serum concentration. In addition, it is noteworthy that the choice of fluorophores for multiplexing biomarkers in an exosome is crucial because of its small size. The proposed method described in this article is beneficial to high-throughput analysis for disease diagnosis, prognosis, and response to treatment because it is a time-, labor-, and cost-saving technique., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, a novel Ru complex and hollow gold nanoparticles branched-poly(N-(3-aminopropyl)methacrylamide) hydrogel composites (pNAMA-Ru-HGNPs) were prepared and used as electrogenerated chemiluminescence (ECL) signal probe to construct aptasensor for ultrasensitive detection of thrombin (TB). Herein, [Ru(phen)2(cpaphen)](2+) linked N-(3-aminopropyl)methacrylamide and hollow gold nanoparticles functionalized N-(3-aminopropyl)methacrylamide were used as two polymer monomers to prepare pNAMA-Ru-HGNPs composites via free-radical polymerization. The obtained hydrogel composite, containing amount of Ru complex and HGNPs, were used as effective tag-carriers for the immobilization of thrombin binding aptamer II (TBA II) to form the pNAMA-Ru-HGNPs labeled TBA II (pNAMA-Ru-HGNPs-TBA II). For building the interface of the aptasensor, dendritic gold nanoparticles reduced by poly(ethyleneimine) (PEI@DGNPs) were modified on the carbon nanotube-nafion (CNTs-Nf) coated electrode through electrostatic adsorption, which was used not only as matrix for immobilization of thrombin binding aptamer I (TBA I) but also as enhancer to amplify the ECL signal because PEI is an efficient co-reactant of Ru complex. Target TB was sandwiched between pNAMA-Ru-HGNPs-TBA II and TBA I, resulting in the ECL signals relevant to the TB concentrations. Combining the novel pNAMA-Ru-HGNPs containing amount of Ru complex as the ECL signal probe and PEI@DGNPs as the enhancer for signal amplification, the sandwich ECL aptasensor was constructed for the detection of TB with a wide range of 1.0 fM to 10 pM and a low detection of 0.54 fM., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We devised and synthesized a fluorogenic substrate of β-lactamases as a probe to detect the activity of the enzymes. Fluorescence of the probe emitted upon treatment of a β-lactamase and increased proportionally to the concentration of the enzyme, demonstrating its sensing property for the activity of the enzyme. We also showed that the probe could be utilized to assay the enzyme and to determine kinetic parameters of the enzyme. Moreover, the probe was able to detect resistance to the third-generation oxyimino-cephalosporin-derived antibiotics such as cefotaxime and ceftazidime. In particular, the probe could identify the ceftazidime-resistance in bacteria that was not detectable using conventional pH-sensing materials, indicating the practical utility of the probe., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, a novel strategy for modulating the fluorescence of gold nanoclusters (Au NCs) is developed. The fluorescence of bovine serum albumin (BSA) protected Au NCs is firstly quenched by KMnO4 and then restored by ascorbic acid (AA) due to the deterioration/restoration of the surface structure. Based on which, a novel "switch-on" fluorescent assay for probing the activity of alkaline phosphatase (ALP) is developed with a detection limit as low as 0.002 U/L. In addition, this testing protocol is also expanded to the detection of the inhibitor of ALP and mouse IgG (as a model), the detection limits are 15 ng/mL for the inhibitor of 2,4-Dichlorophenoxyacetic acid (2,4-DA) and 1.5 pg/mL for mouse IgG. The present method paves a new way to develop convenient, sensitive, and selective metal NCs-based fluorescent "turn-on" probes with promising applications in versatile biosensing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide (NAD(+)) based on a dumbbell-like DNA probe designed for both ligation reaction and digestion reaction with low background noise. SYBR Green I (SG I), a double-helix dye, was chosen as the readout fluorescence signal. In the absence of NAD(+), the ligation reaction did not occur, but the probe was digested to mononucleotides after the addition of exonuclease I (Exo I) and exonuclease I (Exo III), resulting in a weak fluorescence intensity due to the weak interaction between SG I and mononucleotides. In the presence of NAD(+), the DNA probe was ligated by Escherichia coli DNA ligase, blocking the digestion by Exo I and Exo III. As a result, SG I was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved. This method was simple in design, fast to operate, with good sensitivity and selectivity which could discriminate NAD(+) from its analogs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Presented here is a novel implementation of polypropylene capillary-channeled polymer (C-CP) films, functionalized for bioaffinity separations and implemented as a platform for lateral flow (immuno) assays. The parallel ∼80 μm × 80 μm channels pass test solutions down the 30 mm film length via spontaneous wicking action, setting up the possibility for immobilizing different capture agents in the respective channels. The base-film modification process is divided into two steps: ultraviolet light treatment to improve hydrophillicity of the polypropylene substrate and the physical adsorption of a functionalized lipid tethered ligand (LTL) as a selective capture agent. The entire modification procedure is performed under ambient conditions in an aqueous solution without extreme pH conditions. In this demonstration, physical adsorption of a biotinylated-LTL onto the UV-treated PP surface selectively captures Texas Red-labeled streptavidin (SAv-TR) in the presence of enhanced green fluorescence protein (EGFP), which passes without retention in less than 5 s. In addition to the fluorescence imaging of the protein solutes, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to confirm the formation of the LTL-SAv conjugates on the channel surface as well as to demonstrate an alternative means of probing the capture step. The present effort sets the groundwork for further development of C-CP films as a parallel, multi-analyte LFA platform; a format that to-date has not been described.

Clinical microbiology automation is currently limited by the lack of an in-plate culture identification system. Using an inexpensive, printed, disposable colorimetric sensor array (CSA) responsive to the volatiles emitted into plate headspace by microorganisms during growth, we report here that not only the presence but the species of bacteria growing in plate was identified before colonies are visible. In 1894 trials, 15 pathogenic bacterial species cultured on blood agar were identified with 91.0% sensitivity and 99.4% specificity within 3 hours of detection. The results indicate CSAs integrated into Petri dish lids present a novel paradigm to speciate microorganisms, well-suited to integration into automated plate handling systems.

Molecular self-assembly with nucleic acids can be used to fabricate discrete objects with defined sizes and arbitrary shapes. It relies on building blocks that are commensurate to those of biological macromolecular machines and should therefore be capable of delivering the atomic-scale placement accuracy known today only from natural and designed proteins. However, research in the field has predominantly focused on producing increasingly large and complex, but more coarsely defined, objects and placing them in an orderly manner on solid substrates. So far, few objects afford a design accuracy better than 5 nm, and the subnanometre scale has been reached only within the unit cells of designed DNA crystals. Here, we report a molecular positioning device made from a hinged DNA origami object in which the angle between the two structural units can be controlled with adjuster helices. To test the positioning capabilities of the device, we used photophysical and crosslinking assays that report the coordinate of interest directly with atomic resolution. Using this combination of placement and analysis, we rationally adjusted the average distance between fluorescent molecules and reactive groups from 1.5 to 9 nm in 123 discrete displacement steps. The smallest displacement step possible was 0.04 nm, which is slightly less than the Bohr radius. The fluctuation amplitudes in the distance coordinate were also small (±0.5 nm), and within a factor of two to three of the amplitudes found in protein structures.

We present a new implementation of ion imaging for the study of surface scattering processes. The technique uses a combination of spatial ion imaging with laser slicing and delayed pulsed extraction. The scattering velocities of interest are parallel to the imaging plane, allowing speed and angular distributions to be extracted from a single image. The first results of direct scattering of N2 from a clean, single-crystal Au(111) surface are reported, and the speed resolution is shown to be competitive with current state-of-the-art time-of-flight methods for velocity measurements while providing simultaneous measurements of in-plane angular distributions.

A new complex between coumarin-based ligand (CL) and copper ion has been prepared and applied to be an ON-OFF-ON reversible fluorescence chemosensor for the detection of Cys with high sensitivity and specificity. In HEPES buffer, CL displayed high affinity towards Cu(2+) ion over other physiological and environmental metal ions, accompanied with a 98.4% of fluorescence quenching. In the presence of Cys, the detachment of Cu(2+) ion of CL-Cu(2+) ensemble led to the liberation of the fluorophore, CL, and thus fluorescence was recovered. The results of absorption and emission spectroscopy analyses confirmed that the fluorescence turn ON response of CL-Cu(2+) ensemble was selective towards Cys only with high sensitivity (detection of limit, 7.2×10(-7) M). Confocal microscopy studies indicated that the lipophilic CL targets the endoplasmic reticulum (ER) of live cells, where it could be functioned as a fluorescent chemosensor for visualization of Cys in this organelle. Quantitative fluorescence detection of Cys in ER was successfully realized by flow cytometry analysis. The developed chemosensor was further applied to detect Cys in real urine samples with great recoveries ranges from 95.41% to 107.40%., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this paper, we developed a simple, low-cost and sensitive DNA sequences detection biosensor based on a label-free molecular beacon (MB) whose DNA hairpin structure terminal has a guanine-rich sequence that can enhance fluorescence of silver nanoclusters (Ag NCs). Without hybridization between hairpin probe and target DNA, the Ag NCs presented bright fluorescence for the proximity of guanine-rich sequences (GRSs). After binding with target DNA, the hairpin shape was destroyed which results in a decrease of the Ag NCs fluorescence intensity. With this biosensor, we detected three disease-related genes that were the human immunodeficiency virus (HIV) gene, hepatitis B virus (HBV) gene and human T-lymphotropic virus type I (HTLV-I) gene. The detection limits based on S/N of 3 were 4.4 nM, 6.8 nM and 8.5 nM for HIV gene, HBV gene and HTLV-I gene, respectively. Our sensor was also of high selectivity and could distinguish even one nucleotide mismatched target., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the unique features of herpesvirus infection is latent infection following an initial exposure, which is characterized by viral genome persistence in a small fraction of cells within the latently infected tissue. Investigation of the mechanisms of herpesvirus latency has been very challenging in tissues with only a small fraction of cells that are latently infected. Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important and deadly pathogen of koi and common carp, Cyprinus carpio. Acute infection can cause up to 100% mortality in exposed fish, and fish that survive the infection become latently infected. KHV becomes latent in a small percentage of B lymphocytes and can reactivate under stressful conditions. During latency, KHV ORF6 transcript is expressed in the latently infected B lymphocytes. In order to study KHV latent infection in cells that are only latently infected, a nanoflare probe specific to ORF6 RNA was used to separate KHV latently infected cells from total peripheral white blood cells (WBC). Using the ORF6 nanoflare probe, less than 1% of peripheral WBC was isolated from KHV latently infected koi. When this enriched population of WBC was examined by real-time PCR specific for KHV, it was estimated that about 1-2 copies of viral genome persists in the sorted cells. In addition, KHV ORF6 transcript was shown to be the major transcript expressed during latency by RNA-seq analysis. This study demonstrated that an RNA nanoflare probe could be used to enrich latently infected cells, which can subsequently be used to investigate the molecular mechanisms of KHV latency., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vitrectomy is a standard ophthalmic procedure to remove the vitreous body from the eye. The biomechanics of the vitreous affects its duration (by changing the removal rate) and the mechanical forces transmitted via the vitreous on the surrounding tissues during the procedure. Biomechanical characterization of the vitreous is essential for optimizing the design and control of instruments that operate within the vitreous for improved precision, safety, and efficacy. The measurements are carried out using a magnetic microprobe inserted into the vitreous, a method known as magnetic microrheology. The location of the probe is tracked by a microscope/camera while magnetic forces are exerted wirelessly by applied magnetic fields. In this work, in vitro artificial vitreous, ex vivo human vitreous and ex vivo porcine vitreous were characterized. In addition, in vivo rabbit measurements were performed using a suturelessly injected probe. Measurements indicate that viscoelasticity parameters of the ex vivo human vitreous are an order of magnitude different from those of the ex vivo porcine vitreous. The in vivo intra-operative measurements show typical viscoelastic behavior of the vitreous with a lower compliance than the ex vivo measurements. The results of the magnetic microrheology measurements were validated with those obtained by a standard atomic force microscopy (AFM) method and in vitro artificial vitreous. This method allows minimally-invasive characterization of localized mechanical properties of the vitreous in vitro, ex vivo, and in vivo. A better understanding of the characteristics of the vitreous can lead to improvements in treatments concerning vitreal manipulation such as vitrectomy.

A novel ultrasensitive immunoassay method was developed by combination of the enzymatically catalytic gold deposition with the prepared gold nanoprobe and the gold stripping analysis at an electrochemical chip based immunosensor. The immunosensor was constructed through covalently immobilizing capture antibody at a carbon nanotube (CNT) modified screen-printed carbon electrode. The gold nanoprobe was prepared by loading signal antibody and high-content glucose oxidase (GOD) on the nanocarrier of gold nanorod (Au NR). After sandwich immunoreaction, the GOD-Au NR nanoprobe could be quantitatively captured onto the immunosensor surface and then induce the deposition of gold nanoparticles (Au NPs) via the enzymatically catalytic reaction. Based on the electrochemical stripping analysis of the Au NR nanocarriers and the enzymatically produced Au NPs, sensitive electrochemical signal was obtained for the immunoassay. Both the GOD-induced deposition of Au NPs by the nanoprobe and the sensitive electrochemical stripping analysis on the CNTs based sensing surface greatly amplified the signal response, leading to the ultrahigh sensitivity of this method. Using carcinoembryonic antigen as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a detection limit down to 4.2 pg/mL was obtained. In addition, this immunosensor showed high specificity and satisfactory reproducibility, stability and reliability. The relatively positive detection potential excluded the conventional interference from dissolved oxygen. Thus this electrochemical chip based immunosensing method provided great potentials for practical applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A double-water-in-oil-emulsion procedure was designed to synthesize cadmium, lead and copper alginate nanobeads less than 200n m diameter under mild conditions. The cadmium, lead and copper alginate nanobeads can be activated to immobilize biomacromolecules and can directly produce distinctive electrochemical signals. Using the novel alginate nanobeads labeled with antibodies as electrochemical probes, a sandwich-type immunosensor was constructed using AFP, CEA and PSA as model analytes. This proposed immunosensor shows wide linear range with detection limits of 0.01, 0.0086 and 0.0075 ng mL(-1) for AFP, CEA and PSA, respectively. Analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). It suggested that the alginate nanobeads electrochemical probes could be generally extended to other multiple analytes detection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We have synthesized a series of luminescent iridium(III) complexes and investigated their ability to act as luminescent split G-quadruplex probes. After screening, the iridium(III) complex 1 [Ir(2-phenylquinoline)2(3,4,7,8-tetramethyl-1,10-phenanthroline)]PF6 was validated as a highly-selective G-quadruplex probe and was utilized to construct a label-free intermolecular G-quadruplex-based assay for the selective and sensitive detection of LMP1 gene deletion. This "mix-and-detect" assay is simple and selective, and could detect down to 10 nM of the target gene in aqueous solution with a linear range from 10 to 500 nM. We also investigated the performance of our split G-quadruplex-based sensing platform for LMP1 gene deletion in the presence of cellular debris, demonstrating the robustness of this sensing system in biological samples. Comparative assays were also performed using either organic dyes or labeled oligonucleotides as signal-transducing agents., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A novel biosensor platform was developed for detection of microRNAs (miRNAs) based on graphene quantum dots (GQDs) and pyrene-functionalized molecular beacon probes (py-MBs). Pyrene was introduced to trigger specifically fluorescence resonance energy transfer (FRET) between GQDs and fluorescent dyes labeled on py-MBs, and the unique fluorescent intensity change produced a novel signal for detection of the target. The platform realized detection of miRNAs in a wide range from 0.1 nM to 200 nM with great discrimination abilities, as well as multidetection of different kinds of miRNAs, which paved a brand new way for miRNA detection based on GQDs.

A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0U/µL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg(2+) and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04µM and 4.34nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

Simple and accurate detection of prion proteins in biological samples is of utmost importance in recent years. In this study, we developed a label-free electrical detection-based field effect transistor (FET) biosensor using thiamine as a probe molecule for a non-invasive and specific test of human prion protein detection. We found that thiamine-immobilized FETs can be used to observe the prion protein oligomer, and might be a significant test for the early diagnosis of prion-related diseases. The thiamine-immobilized FET was also demonstrated for the detection of prion proteins in blood serum without any complex pre-treatments. Furthermore, we designed a dual-ligand binding approach by the addition of metal ions as a second ligand to bind with the adsorbed prion protein on the thiamine-immobilized surface. When the prion attached to metal ions, the additional positive charge was induced on the gate surface of the FET. This approach was capable of amplifying the magnitude of the FET response and of enhancing the sensitivity of the FET biosensor. Detection of prion proteins has achieved the required concentration for clinical diagnosis in blood serum, which is less than 2 nM. In summary, this FET biosensor was successfully applied to prion detection and proved useful as a simple, fast, sensitive and low-cost method towards a mass-scale and routine blood screening-based test., (Copyright © 2014 Elsevier B.V. All rights reserved.)

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.

Submolecular imaging by atomic force microscopy (AFM) has recently been established as a stunning technique to reveal the chemical structure of unknown molecules, to characterize intramolecular charge distributions and bond ordering, as well as to study chemical transformations and intermolecular interactions. So far, most of these feats were achieved on planar molecular systems because high-resolution imaging of three-dimensional (3D) surface structures with AFM remains challenging. Here we present a method for high-resolution imaging of nonplanar molecules and 3D surface systems using AFM with silicon cantilevers as force sensors. We demonstrate this method by resolving the step-edges of the (101) anatase surface at the atomic scale by simultaneously visualizing the structure of a pentacene molecule together with the atomic positions of the substrate and by resolving the contour and probe-surface force field on a C60 molecule with intramolecular resolution. The method reported here holds substantial promise for the study of 3D surface systems such as nanotubes, clusters, nanoparticles, polymers, and biomolecules using AFM with high resolution.

Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 k(B)T) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology., (© 2014 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nervous necrosis virus (NNV) and iridovirus are highly infectious pathogens that can cause lethal diseases in various species of fish. These infectious diseases have no effective treatments and the mortality rate is over 80%, which could cause dramatic economic losses in the aquaculture industry. Conventional diagnostic methods of NNV or iridovirus infected fishes, such as virus culture, enzyme-linked immunosorbent assays and nucleic acid assays usually require time-consuming and complex procedures performed by specialized technicians with delicate laboratory facilities. Rapid, simple, accurate and on-site detection of NNV and iridovirus infections would enable timely preventive measures such as immediate sacrifice of infected fishes, and is therefore critically needed for the aquaculture industry. In this study, a microfluidic-based assay that employ magnetic beads conjugated with viral deoxyribonucleic acid (DNA) capturing probes and fluorescent DNA molecular beacons were developed to rapidly detect NNV and iridovirus. Importantly, this new assay was realized in an integrated microfluidic system with a custom-made control system. With this approach, direct and automated NNV and iridovirus detection from infected fishes can be achieved in less than 30 min. Therefore, this molecular-beacon based microfluidic system presents a potentially promising tool for rapid diagnosis of fish pathogens in the field in the future., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.

X-ray nanoprobes require coherent illumination to achieve optic-limited resolution, and so will benefit directly from diffraction-limited storage rings. Here, the example of high-resolution X-ray fluorescence tomography is focused on as one of the most voracious demanders of coherent photons, since the detected signal is only a small fraction of the incident flux. Alternative schemes are considered for beam delivery, sample scanning and detectors. One must consider as well the steps before and after the X-ray experiment: sample preparation and examination conditions, and analysis complexity due to minimum dose requirements and self-absorption. By understanding the requirements and opportunities for nanoscale fluorescence tomography, one gains insight into the R&D challenges in optics and instrumentation needed to fully exploit the source advances that diffraction-limited storage rings offer.