Modulation of Near-Infrared Mitochondria-Targetable fluorescent probe for H2S bioimaging through the modification of heavy atom iodine.

H2S is correlated with mitochondrial dysfunction, which results in the death of cells. Two near-infrared fluorescent probes, Mito-HS-1 and Mito-HS-2, were designed for mitochondrial H2S imaging. Initially, the synthesis protocol of expensive IR-780-based hemicyanine (HXPI) was optimized with an appreciate yield of 80 % as compared with 14-56 % previously reported. Iodine atom was introduced to HXPI to obtain iodine-HXPI whose Stokes shift was increased to be 90 nm. On account of the rapid and fast nucleophilic attack of H2S, HXPI-based Mito-HS-1 could be applied for the real time imaging of mitochondrial H2S. Besides some similar optical properties with Mito-HS-1, iodine-HXPI-based Mito-HS-2 exhibited wider linear range (3-150 µM), more stable fluorescent imaging and more favorable specificity in vitro. Both Mito-HS-1 and Mito-HS-2 could be used to image exogenous H2S in cells, with Mito-HS-2 showing fairly better signal-to-noise. Additionally, the Pearson correlation coefficient of two probes demonstrated that they could successfully monitor mitochondrial H2S in A549 cells and Hela cells.

Sensitive detection of transcription factor by coupled fluorescence-encoded microsphere with exonuclease protection.

Aberrant transcription factors (TFs) activities are closely related to the occurrence and development of various diseases. Herein, we presented a fluorescence-encoded microsphere-based approach for TFs detection coupling with common DNA footprinting assay. Target TFs specifically bound the binding sites of double-stranded DNA (dsDNA) probes which were conjugated to microspheres. Thus, the probes were protected from being hydrolyzed by exonuclease III (Exo III). Afterwards, biotins labeled on the probes reacted with streptavidin-phycoerythrin (SA-PE) to produce fluorescent signal; however, in the absence of target TFs, the dsDNA probes would be hydrolyzed by Exo III resulting in biotins falling off and thus fluorescence signal was not generated. This strategy can be used to detect nuclear factor-kappa B p50 (NF-κB p50) with a detection limit of 0.2 nM. The steric hindrance of microspheres overcome the disadvantage of Exo III that can nibble into the protein-bound DNA region. Meanwhile, the fluorescent label of microsphere was specific to each TF, enabling multiplex detection could be achieved by changing specific protein binding site of corresponding dsDNA probe. This method has been successfully applied for simultaneous detection of NF-κB p50, AP-1 and CREB in nuclear extract isolated from HeLa cells stimulated or unstimulated by TNF-α, showing great potential for biomedical researches and precise disease diagnosis.

Target-fueled catalytic hairpin assembly for sensitive and multiplex microRNA detection.

As a typical strand displacement-based DNA circuit, the catalytic hairpin assembly (CHA) has the potential to transduce and amplify signals for analytical applications, but little practice has been fulfilled in Luminex-based multiple microRNAs (miRNAs) detection. Here, we proposed a target-fueled CHA-based platform for sensitive and multiple miRNAs detection, by virtue of the multiplex characteristic of the Luminex xMAP platform. The cyclic use of target miRNA, which forms a substantial amount of H1-H2 duplexes, has amplified the fluorescent response to achieve sensitive sensing. Key experimental conditions including hairpin probe concentrations, reaction temperature, and concentration of SA-PE were optimized. Liver tumor-related miRNA-21, miRNA-122, and miRNA-222 could be simultaneously detected with LOD of 2 pM. Overall, the proposed method first combined CHA with the Luminex xMAP system to construct a sensitive sensing platform suitable for multiple miRNAs detection in real sample analysis, which could potentially be applied in biomedical research and clinical diagnosis. Graphical abstract.

Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.

Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper, we report an antibody free ELISA-like assay which could be used to analyze transcription factor NF-κB p50 with comparatively low cost and high throughput. This assay is based on the stabilization of a duplex DNA probe by binding with a transcription factor. The double-stranded DNA (dsDNA) probe immobilized on a 96-well plate was too short in length to stabilize its duplex structure at a relatively high temperature and would unwind into a single strand. In the presence of a target TF, the protein bound to a specific TF-binding site and prevented the specific dsDNA probe from being unzipped at the washing temperature, thus holding the chemiluminescence signal. This method could sensitively detect NF-κB p50 with a detection limit of 0.5 nM. The proposed strategy provides a convenient, cheap and high throughput detection method of TFs, which can potentially help the development of drug discovery and disease diagnosis.

A conformational switch-based fluorescent biosensor for homogeneous detection of telomerase activity.

As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Herein, we demonstrate the conformational switch-based fluorescence detection of telomerase activity using a redesigned RNA aptamer Spinach. Briefly, the original Spinach aptamer was extended at its 5' end and folded into an inactive conformation, where association with the small molecule fluorophore, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) was prevented. Only in the presence of telomerase, (TTAGGG)n repeats were added to the 3' end of the telomerase substrate primer, and the elongation products hybridized with inactive Spinach molecules, triggering its conformational switch and refolding it into the active, DFHBI-binding conformation. Moreover, the fluorescence signal was further amplified through a target recycling circuit, where Ribonuclease H (RNase H) specifically hydrolyzed the phosphodiester bonds of RNA in the DNA-RNA hybrid. The released telomere products could then hybridize to new inactive Spinach molecules and initiate multiple amplification cycles. The proposed fluorescent biosensor presented great performance for telomerase activity detection from 100 to 5 × 104 Hela cells with a detection limit of 100 cells. Besides, this new assay offers a good biosensing platform for differentiation of cancer cell lines from normal cell line and evaluation the inhibition efficiency of telomere-binding ligand, which is of great importance for telomerase-related cancer diagnosis and therapy.

Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs.

Multiple miRNA detection is often limited by sample, time-consuming, and complicated procedures. To address such challenges, we present a relatively simple and amplification-free fluorescent strategy based on hybridization-initiated exonuclease resistance for simultaneous detection of multiple miRNAs in a single tube. Single-stranded linear DNA probes were designed with dual roles of capture and reporter DNA, i.e. each DNA probe was labeled with biotin at the 3'-end for signal readout and amino at the 5'-end for probe immobilization. After target miRNAs specifically hybridized with the corresponding probes, the formation of double-stranded probe-target duplex protected the biotin on the probe from Exonuclease I digestion, thus resulting in high fluorescent intensities through the reaction between biotin and streptavidin-phycoerythrin. Coupling with the 3-plex fluorescent microsphere-based assay system, herein we successfully demonstrated the simultaneous and quantitative measurement of three sequence-specific miRNAs at concentration range of 2.5 pM to 1.25 nM and detection limits of 2 pM. To meet high throughput and rapid turnaround time requirements, we have also exemplified that, even shortening the total reaction time within 1 h, wider linear response range and lower detection limit were guaranteed. We further applied this assay to detect endogenous target miRNA levels from five kinds of cancer cell line and one normal cell line HEK 293T. This simple and rapid strategy will hold great potential for monitoring of multiple miRNAs biomarkers in biomedical research and early clinical diagnosis.

Duplex microRNAs assay based on target-triggered universal reporter hybridization.

In this paper, we designed and evaluated a duplex detection strategy for microRNAs (miRNAs) using universal probe-based target-triggered double hybridization and fluorescent microsphere-based assay system (xMAP array). In the absence of target miRNA, reporter DNA cannot hybridize stably with the immobilized capture DNA due to its low melting temperature. Only after adding target miRNA, can reporter probe hybridize with capture probe to form a stable three-component complex. This target-triggered stable hybridization makes this method possible for highly selective and sensitive detection of multiple miRNAs. We exemplified a quantitative detection of duplex miRNAs with a limit of detection of 40 pM. The xMAP array platform holds the potential of extending this approach to simultaneous detection of up to 100 miRNA targets. Considering the simplicity, rapidity and multiplexing, this work promised a potential detection of multiple miRNA biomarkers for early disease diagnosis and prognosis.

Bioluminescence Imaging of Carbon Monoxide in Living Cells and Nude Mice Based on Pd0-Mediated Tsuji-Trost Reaction.

Carbon monoxide (CO) is highly toxic and lethal to humans and animals because of its strong affinity for hemoglobin, while this "silent killer" is constantly generated in the body as a cell-signaling molecule of the gasotransmitter family in various pathological and physiological conditions. Up to now, designing fluorescent probes for real-time imaging of CO in living species is a continuous challenge due to background interference, light scattering, and photoactivation/photobleaching. Herein, a novel type of bioluminescence probe (allyl-luciferin) was synthesized and exploited to realize CO imaging with high signal-to-noise ratios. Based on Pd0-mediated Tsuji-Trost reaction, allyl-luciferin specifically reacted with CO to yield D-luciferin and thus generate a turn-on bioluminescence response, exhibiting high selectivity against bioactive small molecules such as reactive nitrogen, oxygen, and sulfur species. Furthermore, the new probe can be easily employed to detect exogenous CO in Huh7 cells and MDA-MB-231 cells, and CO production was enhanced greatly in these living cells after pretreatment with [Ru(CO)3Cl-(glycinate)] (CORM-3). Through the use of PdCl2-containing liposomes to improve poor membrane permeability of PdCl2, endogenous CO stimulated by heme was also seen clearly. In addition, the probe was successfully used to monitor exogenous and endogenous CO in nude mice. Overall, our data proved that the allyl-luciferin is a promising tool for exogenous and endogenous CO detection and imaging within living species. This is the first demonstration of bioluminescence imaging obtained by a probe for CO. We anticipate that the good imaging properties of allyl-luciferin presented in this study will provide a potentially powerful approach for illuminating CO functions in the future.

Target-initiated labeling for the dual-amplified detection of multiple microRNAs.

Herein we exploited a novel target-initiated labeling strategy for the multiplex detection of microRNAs (miRNAs) by coupling duplex-specific nuclease (DSN) with terminal deoxynucleotidyl transferase (TdT). In the presence of target miRNA, the immobilized and 3'-blocked capture probes hybridized with target and thus the formed DNA-RNA hybrid was recognized by DSN. DSN mediated the digestion of 3'-phosphated capture probes (CPs) in the hybrids and synchronously target was released and recycled for another round of hybridization and cleavage. The cleaved CP fragments with a free 3'-OH were then elongated and labeled with multiple biotin-dUTP nucleotides by TdT. Fluorescence reporter streptavidin-phycoerythin was finally added to react with the immobilized biotins and render fluorescence signals. This dual-amplification labeling strategy was successfully demonstrated to sensitively detect multiple miRNAs, taking advantage of DSN-mediated target recycling and TdT-catalyzed multiple signal modification with analysis by a commercial Luminex xMAP array platform. Our experimental results showed the simultaneous quantitative measurement of three sequence-specific miRNAs at concentrations from 1 pM to 2.5 nM. Attempts were also made to directly detect miRNAs in total RNA extracted from cancer cells. The dual-amplification labeling strategy reported here shows a great potential for the development of a method for the multiplexed, sensitive, selective, and simple analysis of multiple miRNAs in tissues or cells for biomedical research and clinical early diagnosis.

Probing cigarette smoke-induced DNA single-strand breaks and screening natural protective compounds by use of magnetic bead-based chemiluminescence.

Magnetic bead (MB)-based chemiluminescence (CL) ELISA can be a sample-thrifty, time-saving tool for evaluation of cigarette smoke-induced DNA single-strand breaks (SSBs) with high specificity. This article describes a novel approach using immobilized oligonucleotide on MBs to determine cigarette smoke-induced DNA SSBs and screen some protective natural compounds. Typically, fluorescein-labeled DNA (FAM-DNA) was immobilized on the MBs and then oxidized by the smoke in the absence or presence of natural compounds, and a part of FAM-DNA was fragmented due to cigarette smoke-induced DNA SSB and then detached from MBs whereas other non-broken FAM-DNA still remained on MBs. Then, any broken FAM-DNA fragments, complex tobacco smoke matrix, and other stuff related with natural compounds were conveniently washed away by a magnetic force, and thus possible interfering substances were completely removed. Finally, those remaining non-broken FAM-DNA on MBs were reacted with HRP-labeled anti-fluorescein antibody and then detected by CL ELISA. CL signal was converted to molar concentrations of the FAM-DNA by interpolation from a pre-determined standard linear calibration curve. The level of DNA SSBs induced by cigarette smoke was thus calculated using the method. A library of 30 natural products was subsequently screened, and two among them were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of DNA SSBs. Graphical abstract ᅟ.

Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection.

T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg(2+) and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg(2+) concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets.

Additive and enhanced fluorescence effects of hairpin DNA template-based copper nanoparticles and their application for the detection of NAD(.).

DNA is an excellent molecular scaffold for the preparation of copper nanoparticles (CuNPs). However, screening and designing an efficient template for the formation of fluorescent CuNPs remains a challenge. Herein, we synthesized CuNPs using a novel hairpin DNA template with a poly T loop and a random double strand stem which performs efficient in the formation of fluorescent CuNPs. Besides, the additive and enhanced effects between the loop and stem sequences on the fluorescence of induced CuNPs was first proposed and investigated. On comparing different sequences, we found that both the circular poly T sequence on the loop region and the stem sequence play important roles in this fluorescence enhancement effect. A label-free analytical system for the quantitative detection of NAD(+) was designed, demonstrating excellent sensitivity. Based on our design concept proposed herein, any random double-stranded DNA can be used for the design of efficient templates for the formation of fluorescent CuNPs in biochemical sensing.

Sesquiterpenoids and further diterpenoids from the rare Chloranthaceae plant Chloranthus sessilifolius.

Two new dimeric lindenane-type sesquiterpenoids (1 and 2, named chlorasessilifols A and B, resp.), one new ent-podocarpane-type C17 norditerpenoid (3), and one new ent-torarane-type diterpenoid (4), along with seven known terpenoids, were isolated from the whole plant of Chloranthus sessilifolius. The new structures were established by means of spectroscopic methods and/or observed cotton effects in the circular dichroism spectra. Among the isolates, 3α,7ß-dihydroxy-ent-abieta-8,11,13-triene (11) exhibited significant anti-neuroinflammatory activity by inhibiting the nitric oxide production in lipopolysaccharide-stimulated murine BV-2 microglial cells, with an IC50 value of 4.3 µM.

Visualization of in Vivo Hydrogen Sulfide Production by a Bioluminescence Probe in Cancer Cells and Nude Mice.

Hydrogen sulfide (H2S) has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon monoxide. However, its precise measurement in living cells and animals remains a challenge. In this study, a novel bioluminescence H2S probe was designed and synthesized by modifying the 6'-amino group of d-aminoluciferin into a 6'-azido group, which was highly selective against other reactive sulfur, nitrogen, and oxygen species. Our H2S probe azidoluciferin sensitively reacted with H2S to release d-aminoluciferin with a strong bioluminescence signal. On the basis of its high selectivity and sensitivity, the H2S probe was used to detect H2S production in live cancer cells and nude mice. The bioluminescence signal decreased in mice treated with propargylglycine, an inhibitor of H2S, suggesting that our H2S probe can detect endogenous H2S in real time, in vivo. Overall, the excellent sensing properties of the probe combined with its bioimaging capability make it a useful tool to study H2S biological roles.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction.

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

xMAP array microspheres based stem-loop structured probes as conformational switches for multiplexing detection of miRNAs.

We have designed and evaluated novel stem-loop-structured probes for fluorescence detection of multiple microRNA (miRNA) targets. In the initial stage, the probes are in a closed stem conformation, shielding sterically a biotin label from being accessible to a fluorescence reporter. After hybridizing with target miRNAs, the probes undergo a conformational switch, restoring accessibility of the biotin to streptavidin-phycoerythin (SA-PE) for signal readout. Apparently, the bulky nature of the reporter SA-PE facilitates shielding of the biotin label in the absence of the target, thereby the stem-loop-structured probes allow sensitive detection of unlabeled miRNA targets, and xMAP array microspheres further realize simultaneous detection of multiple analytes using one fluorescence dye, SA-PE, for final readout. Here we demonstrated a successful multiplex assay for quantitative measurement of miRNA21, miRNA222, miRNA20a, and miRNA223, which are associated with nonsmall cell lung cancer. The approach can be extended to detecting an increasing number of targets for various indications. We believe such advancements represent a significant improvement for early disease diagnosis and prognosis.

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide.

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD(+)), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5' end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3' end. Only in the presence of NAD(+), the probe was linked at 5' and 3' ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM-500 nM with a detection limit of as low as 1 pM NAD(+), and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD(+) level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD(+), taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences.

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification.

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3'-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.

Quantum dot-enhanced detection of dual short RNA sequences via one-step template-dependent surface hybridization.

A novel multiplexed method for short RNA detection is reported that employs a design strategy in which capture and reporter probes anneal to each other in the presence of a short RNA target via the formation of a stable three-component complex. Quantum dots (QDs) functionalized with reporter DNA are thus specifically bound onto a capture probe-modified 96-well plate by one-step hybridization for simple RNA detection. In comparison with conventional organic dye-modified reporter probes, the use of reporter DNA-modified QD conjugates increase the melting temperature and lead to the detection of short RNA without the need for a ligation reaction. Moreover, QD properties allow multiple short RNA sequences to be simultaneously determined via rapid and simple one-step hybridization, as exemplified herein. The present results clearly demonstrate that this new strategy can be used to detect dual-short RNA sequence at concentrations of 10 pM in 100 µL.