Photoactivated DNA Walker Based on DNA Nanoflares for Signal-Amplified MicroRNA Imaging in Single Living Cells.

Specific and sensitive detection and imaging of cancer-related miRNA in living cells are desirable for cancer diagnosis and treatment. Because of the spatiotemporal variability of miRNA expression level during different cell cycles, signal amplification strategies that can be activated by external stimuli are required to image miRNAs on demand at desired times and selected locations. Herein, we develop a signal amplification strategy termed as the photoactivated DNA walker based on DNA nanoflares, which enables photocontrollable signal amplification imaging of cancer-related miRNA in single living cells. The developed method is achieved via combining photoactivated nucleic acid displacement reaction with the traditional exonuclease III (EXO III)-assisted DNA walker based on DNA nanoflares. This method is capable of on-demand activation of the DNA walker for dictated signal amplification imaging of cancer-related miRNA in single living cells. The developed method was demonstrated as a proof of concept to achieve photoactivated signal amplification imaging of miRNA-21 in single living HeLa cells via selective two-photon irradiation (λ = 740 nm) of single living HeLa cells by using confocal microscopy equipped with a femtosecond laser.

Photoactivated Biosensing Process for Dictated ATP Detection in Single Living Cells.

The subcellular distribution of adenosine 5'-triphosphate (ATP) and the concentration of ATP in living cells dynamically fluctuate with time during different cell cycles. The dictated activation of the biosensing process in living cells enables the spatiotemporal target detection in single living cells. Herein, a kind of o-nitrobenzylphosphate ester hairpin nucleic acid was introduced as a photoresponsive DNA probe for light-activated ATP detection in single living cells. Two methods to spatiotemporally activate the probe in single living cells were discussed. One method was the usage of the micrometer-sized optical fiber (about 5 µm) to guide the UV light (λ = 365 nm) to selectively activate the photoresponsive DNA probe in single living cells. The second method involved a two-photon laser confocal scanning microscope to selectively irradiate the photoresponsive DNA probes confined in single living cells via two-photon irradiation (λ = 740 nm). ATP aptamer integrated in the activated DNA probes selectively interacted with the target ATP, resulting in dictated signal generation. Furthermore, the photoactivated biosensing process enables dictated dual-model ATP detection in single living cells with "Signal-ON" fluorescence signal and "Signal-OFF" electrochemical signal outputs. The developed photoactivated biosensor for dictated ATP detection with high spatiotemporal resolution in single living cells at a desired time and desired place suggests the possibility to monitor biomarkers during different cell cycles.

Spatiotemporal patterning of photoresponsive DNA-based hydrogels to tune local cell responses.

Understanding the spatiotemporal effects of surface topographies and modulated stiffness and anisotropic stresses of hydrogels on cell growth remains a biophysical challenge. Here we introduce the photolithographic patterning or two-photon laser scanning confocal microscopy patterning of a series of o-nitrobenzylphosphate ester nucleic acid-based polyacrylamide hydrogel films generating periodically-spaced circular patterned domains surrounded by continuous hydrogel matrices. The patterning processes lead to guided modulated stiffness differences between the patterned domains and the surrounding hydrogel matrices, and to the selective functionalization of sub-regions of the films with nucleic acid anchoring tethers. HeLa cells are deposited on the circularly-shaped domains functionalized with the MUC-1 aptamers. Initiation of the hybridization chain reaction by nucleic acid tethers associated with the continuous hydrogel matrix results in stress-induced ordered orthogonal shape-changes on the patterned domains, leading to ordered shapes of cell aggregates bound to the patterns.

Near-infrared light-activated membrane fusion for cancer cell therapeutic applications.

The spatiotemporal stimulation of liposome-liposome or liposome-membrane fusion processes attracts growing interest as a means to mimic cell-cell interactions in nature and for using these processes for biomedical applications. We report the use of o-nitrobenzyl phosphate functionalized-cholesterol tethered nucleic acid-modified liposomes as functional photoresponsive units for inducing, by NIR-irradiation, spatiotemporal liposome-liposome or liposome-membrane fusion processes. The liposomes are loaded with upconversion nanoparticles (UCNPs) and their NIR irradiation (λ = 980 nm) yields luminescence at λ = 365 nm, providing a localized light-source to deprotect the o-nitrobenzyl phosphate groups and resulting in the fragmentation of the nucleic acid structures. In one system, the NIR-triggered fusion of two liposomes, L1 and L2, is exemplified. Liposome L1 is loaded with UCNPs and Tb3+ ions, and the liposome boundary is functionalized with a cholesterol-tethered, o-nitrobenzyl phosphate caged hairpin nucleic acid structure. Liposome L2 is loaded with 2,6-pyridinedicarboxylic acid, DPA, and its boundary is modified with a cholesterol-tethered nucleic acid, complementary to a part of the caged hairpin, associated with L1. NIR-irradiation of the L1/L2 mixture resulted in the photocleavage of the hairpin structure, associated with L1, and the resulting fragmented nucleic acid associated with L1 hybridized with the nucleic acid linked to L2, leading to the fusion of the two liposomes. The fusion process was followed by dynamic light scattering, and by monitoring the fluorescence of the Tb3+-DPA complex generated upon the fusion of the liposomes and their exchange of contents (fusion efficiency 30%). In a second system, the fusion of the liposomes L1, loaded with UCNPs and doxorubicin (DOX), with HeLa cancer cells functionalized with nucleic acid tethers, complementary to the hairpin units associated with the boundary of L1, and linked to the MUC-1 receptor sites associated with the HeLa cells, through a MUC-1 aptamer unit is exemplified. The effect of DOX-loaded L1/HeLa cell fusion on the cytotoxicity towards HeLa cells is addressed. The NIR UCNP-stimulated cleavage of the o-nitrobenzyl phosphate caged hairpin units associated with L1 leads to the fragmentation of the hairpin units and the resulting nucleic acid tethers hybridize with the nucleic acid-modified HeLa cells, resulting in the liposome-HeLa cell fusion and the release of DOX into the HeLa cells. Selective spatiotemporal cytotoxicity towards HeLa cells is demonstrated (ca. 40% cell killing within two days). The study presents a comprehensive stepwise set of experiments directed towards the development of NIR-driven liposome-liposome or liposome-membrane fusion processes.

Near-Infrared Light Activated Nucleic Acid Cascade Recycling Amplification for Spatiotemporally Controllable Signal Amplified mRNA Imaging.

The expression level and subcellular distribution of mRNA dynamically changed during the different cell circles. Spatiotemporally controllable signal amplification methods capable of controlling the when and where of the amplification process could allow the sensitive mRNA imaging of selected living cells at dictated time-intervals of the cell life-cycle. However, the present methods for amplified mRNA imaging are hard to control the where and when of the signal amplification due to the lack of an effective strategy to precisely trigger and control the signal amplification process. Herein, we present a conceptual study termed as photocontrollable nucleic acid cascade recycling amplification which uses near-infrared (NIR) light to precisely control and trigger the whole process. This strategy is achieved by integrating photocontrollable nucleic acid displacement reaction with exonuclease III (EXO III) assisted nucleic acid cascade recycling amplification and combination with upconversion nanoparticles (UCNPs), thus resulting in a NIR light activatable signal amplification. As a proof of concept, we demonstrate this developed NIR light triggered signal amplification process in selected living cancer cells for spatiotemporally controllable signal amplified mRNA imaging.

Two-Photon Lithographic Patterning of DNA-Coated Single-Microparticle Surfaces.

The spatially defined functionalization of microparticles with asymmetric shape-controlled nucleic acid patterns is a major challenge in materials science. The asymmetric patterning of microparticles is important to allow the controlled fabrication of crystalline lattices or controlled aggregates of microparticles. We present the combination of two-photon lithography and photocleavable o-nitrobenzylphosphate ester nucleic acid coating-modified microparticles as a versatile means to asymmetrically pattern single microparticle surfaces. The two-photon patterning of microparticles with predesigned nucleic acid structures of different sizes (700 nm to 2.8 µm) and shapes (circles, rings, triangles, and squares) are demonstrated. In addition, complex patterned domains consisting of two different asymmetric nucleic acid domains are fabricated by the controlled Z-positioning of the microparticles in respect to the two-photon irradiation sources. In addition, the two-photon lithographic patterning of the photocleavable DNA coating allows the generation of functional nucleic acid domains for the photostimulated activation of the catalytic hybridization assembly (CHA) of branched nucleic acid structures on single microparticles.

Photoactivated Specific mRNA Detection in Single Living Cells by Coupling "Signal-on" Fluorescence and "Signal-off" Electrochemical Signals.

The spatiotemporal detection of a target mRNA in a single living cell is a major challenge in nanoscience and nanomedicine. We introduce a versatile method to detect mRNA at a single living cell level that uses photocleavable hairpin probes as functional units for the optical (fluorescent) and electrochemical (voltammetric) detection of MnSOD mRNA in single MCF-7 cancer cells. The fluorescent probe is composed of an ortho-nitrophenylphosphate ester functionalized hairpin that includes the FAM fluorophore in a caged configuration quenched by Dabcyl. The fluorescent probe is further modified with the AS1411 aptamer to facilitate the targeting and internalization of the probe into the MCF-7 cells. Under UV irradiation, the hairpin is cleaved, leading to the intracellular mRNA toehold-stimulated displacement of the FAM-functionalized strand resulting in a switched-on fluorescence signal upon the detection of the mRNA in a single cell. In addition, a nanoelectrode functionalized with a methylene blue (MB) redox-active photocleavable hairpin is inserted into the cytoplasm of a single MCF-7 cell. Photocleavage of the hairpin leads to the mRNA-mediated toehold displacement of the redox-active strand associated with the probe, leading to the depletion of the voltammetric response of the probe. The parallel optical and electrochemical detection of the mRNA at a single cell level is demonstrated.

Prewetting dichloromethane induced aqueous solution adhered on Cassie superhydrophobic substrates to fabricate efficient fog-harvesting materials inspired by Namib Desert beetles and mussels.

Namib Desert beetles harvest water from harsh environments by using their hydrophilic-hydrophobic dorsal surfaces. Generally, Cassie-state superhydrophobic materials are chosen as substrates to prepare bioinspired (super)hydrophilic/(super)hydrophobic patterned surfaces. However, due to the low adhesion and strong repellency, aqueous solution cannot be directly set on Cassie superhydrophobic materials until the dropping volume is larger than 6.5 µL. Therefore, arranging a (super)hydrophilic substance on Cassie superhydrophobic substrates to construct (super)hydrophilic/superhydrophobic patterned surfaces still remains a challenge. In this work, by prewetting with dichloromethane (DCM), the mussel-inspired hydrophilic and bio-adhesive dopamine solution (DA) could be dripped onto a Cassie superhydrophobic Cu surface with an ultralow volume of 0.1 µL, whereby low surface tension DCM would "cloak" the high surface tension DA. Along with DCM volatility, DA was adhered on the Cassie superhydrophobic surface and would then self-polymerize into hydrophilic polydopamine domains, thus hydrophilic/superhydrophobic patterned surfaces with efficient water collection could be successfully developed inspired by Namib Desert beetles and mussels. The bioinspired materials show the potential for real-world industrialization in a large scale, which is of great significance for providing living security for those living in areas with no access to fresh water.

Determination of Sunset Yellow Based on Its Quenching Effect on the Fluorescence of Acridine Orange.

Acridine orange (AO) is widely applied as an organic fluorescent probe. In this work, AO was reacted with sunset yellow (SY) to form an ion-association complex in pH 3.4 Britton-Robinson (BR) buffer solution medium. This resulted in the fluorescence quenching of the former and helped to detect the latter with the maximum excitation wavelengths (λex) and emission wavelengths (λem) near 490 and 530 nm, respectively. The assay exhibits high sensitivity and selectivity with a detection limit of 0.002 µmol L-1 and the remarkable quenching of fluorescence was proportional to the concentration of SY in the range of 0.008 - 9.0 µmol L-1. Herein, this finding was utilized to develop a new strategy for simple, rapid, sensitive and selective detection of SY by combining AO based on fluorescence quenching. In addition, the optimum reaction conditions and the effect of foreign substances were studied. The reasons for fluorescence quenching were also investigated, which showed the quenching of fluorescence of AO with SY was a static quenching process. Furthermore, the proposed method was applied in a real sample analysis with satisfactory results.

Determination of norfloxacin in food by an enhanced spectrofluorimetric method.

BACKGROUND: Using a norfloxacin (NFLX)-Nd3+ -cetyltrimethylammonium bromide (CTAB) system for the detection of NFLX, a simple and sensitive method based on fluorescence enhancement was developed. RESULTS: In pH 7.0 buffer solution, NFLX reacted with Nd3+ to form a complex, which resulted in fluorescence enhancement of NFLX, and the maximum emission peak shifted from 415 nm for NFLX to 450 nm for NFLX-Nd3+ . Moreover, the fluorescence intensity increased further when the surfactant CTAB was added to NFLX-Nd3+ . Under the optimum conditions, the fluorescence intensity of the NFLX-Nd3+ -CTAB system was linearly correlated with the NFLX concentration in the range 0.038-10 µmol L-1 , with a correlation coefficient (R2 ) of 0.9997. The detection limit (3σ/k) was 0.021 µmol L-1 , indicating that this method can be applied to detect trace NFLX levels. The mechanism of fluorescence enhancement is discussed. The method was used to detect NFLX in fish and chicken samples with satisfactory results. CONCLUSION: The present results indicate that this method has the potential for fast and real-time determination of NFLX in food samples © 2016 Society of Chemical Industry.

Resonance Rayleigh scattering technique for simple and sensitive analysis of tannic acid with carbon dots.

Carbon dots (CDs) are raising a substantial amount of attention owing to their many unique and novel physicochemical properties. Herein one-pot synthesized CDs, to the best of our knowledge, were first served as the robust nanoprobe for detection tannic acid (TA) based on resonance Rayleigh scattering technique. The as-prepared CDs can combine with TA via hydrogen bond, resulting in remarkable enhancement of scattering signal with no changes in the fluorescence of CDs. Therefore, a novel protocol for TA determination was established and this strategy allowed quantitative detection of TA in the linear range of 0.2-10.0µmolL-1 with an excellent detection limit of 9.0nmolL-1. Moreover, the CDs based nanoprobe can be applied to the determination of TA in water sample with satisfactory results. Our study can potentially influence our current views on CDs and particularly impressive and offers new insights into application of CDs beyond the traditional understanding of CDs.

A Simple and Sensitive Method for Auramine O Detection Based on the Binding Interaction with Bovin Serum Albumin.

A simple, rapid and effective method for auramine O (AO) detection was proposed by fluorescence and UV-Vis absorption spectroscopy. In the BR buffer system (pH 7.0), AO had a strong quenching ability to the fluorescence of bovin serum albumin (BSA) by dynamic quenching. In terms of the thermodynamic parameters calculated as ΔH > 0 and ΔS > 0, the resulting binding of BSA and AO was mainly attributed to the hydrophobic interaction forces. The linearity of this method was in the concentration range from 0.16 to 50 µmol L(-1) with a detection limit of 0.05 µmol L(-1). Based on fluorescence resonance energy transfer (FRET), the distance r (1.36 nm) between donor (BSA) and acceptor (AO) was obtained. Furthermore, the effects of foreign substances and ionic strength were evaluated under the optimum reaction conditions. BSA as a selective probe could be applied to the analysis of AO in medicines with satisfactory results.

Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0µmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046µmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

Study on erythrosine-phen-Cd(II) systems by resonance Rayleigh scattering, absorption spectra and their analytical applications.

In pH 7.0-8.0 KH2PO4-Na2HPO4 buffer solution, Cd(II) reacted with 1,10-phenanthroline to form chelate cation [Cd(phen)3]2+, which further reacted with anion of erythrosine to form ternary ion-association complex through electrostatic attraction and hydrophobic effect. This process could result in remarkable absorption spectra change and produce obvious fading reaction at 528 nm. Absorbance change (ΔA) of system was directly proportional to the concentration of Cd(II). Hereby, a highly sensitive spectrophotometric method for the determination of Cd(II) was established. The molar absorption coefficient was 2.29×10(5) L mol(-1) cm(-1) and the detection limit of Cd(II) was 26.5 ng mL(-1). Furthermore, the resonance Rayleigh scattering (RRS) of this system with two peaks located at 371 and 590 nm enhanced significantly, and second-order scattering (SOS) and frequence doubling scattering (FDS) of this system changed notably at 640 and 350 nm, respectively. Under the optimum conditions, the scattering intensities (ΔIRRS, ΔIDWO-RRS, ΔISOS and ΔIFDS) had good linear relationship with the concentration of Cd(II) in certain ranges. The detection limits of Cd(II) were 1.27 ng mL(-1), 1.39 ng mL(-1), 4.03 ng mL(-1), 5.92 ng mL(-1) and 14.7 ng mL(-1) for dual-wavelength overlapping resonance Rayleigh scattering (DWO-RRS), RRS (371 nm), RRS (590 nm), SOS and FDS, respectively. In addition, the suitable reaction conditions and effects of coexisting substances were investigated. The methods had been successfully applied to the determination of Cd(II) in environmental water samples. The recovery range was between 93.0% and 103.0% and the relative standard deviation (RSD) was between 2.5% and 4.3%. The results were in agreement with those obtained from atomic absorption spectroscopy.