A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.     

Functional Xeno Nucleic Acids for Biomedical Application

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.     

Detection of miRNA in Live Cells by Using Templated RuII‐Catalyzed Unmasking of a Fluorophore

Reactions templated by cellular nucleic acids are attractive for nucleic acid sensing or responsive systems. Herein we report the use of a photocatalyzed reductive cleavage of an immolative linker to unmask a rhodamine fluorophore, and its application to miRNA imaging. The reaction was found to proceed with a very high turnover (>4000) and provided reliable detection down to 5 pM of template by using γ‐serine‐modified peptide nucleic acid (PNA) probes. The reaction was used for the selective detection of miR‐21 in BT474 cells and miR‐31 in HeLa cells following irradiation for 30 min. The probes were introduced by using reversible permeation with streptolysin‐O (SLO) or a transfection technique.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Hybrid molecular probe for nucleic acid analysis in biological samples

The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.     

Oligonucleotide optical switches for intracellular sensing

Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or l-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.     

Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4',6-diamidino-2-phenylindol

PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4',6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4 pmol (0.3 ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250 pmol per band.     

Fluorescent hybridization probes for nucleic acid detection

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.     

Three copper(II) complexes constructed from a new 1,3,5-triazine derivative ligand

Three new complexes {[Cu(dpdapt)(Hhbd)] · 6H₂O}_n (1) (dpdapt = N,N′-di(2-pyridyl)-2,4-diamino-6-phenyl-1,3,5-triazine, Hhbd = 2-hydroxybutanedioicate dianion), [Cu(dpdapt)(SO₄)] · 2H₂O (2) and [Cu(dpdapt)(oxa)] · H₂O (3) (oxa = oxalate dianion) have been synthesized and structurally characterized. The non-covalent interactions of π–π stacking and hydrogen bonding extend complexes 1–3 into supramolecular architectures, where 1 self-assembles into a 1D polymeric chain by dicarboxylate bridges and exhibits a 3D framework with 1D open channels, while complexes 2 and 3 display 2D wavelike networks. Interestingly, in 1, the host framework encapsulates hexameric water clusters that are connected into 1D arrays by supramolecular association along the 1D open channels. The UV/vis, IR spectra, fluorescence and TG analysis for complexes 1, 2 and 3 are also discussed.     

BODIPY-based Fluorescent Probe for Fast Detection of Hydrogen Sulfide and Lysosome-targeting Applications in Living Cells

Hydrogen sulfide (H₂S) is recognized as an endogenous gaseous signaling agent in many biological activities. Lysosomes are the main metabolic site and play a pivotal role in cells. Herein, we designed and synthesized two new fluorescent probes BDP-DNBS and BDP-DNP with a BODIPY core to distinguish H₂S. The sensing mechanism is based on the inhibition-recovery of the photo-induced electron transfer (PET) process. Through comparing the responsive behaviors of the two probes toward H₂S, BDP-DNBS showed a fast response time (60 s), low limit of detection (LOD, 51 nM), high sensitivity and selectivity. Moreover, the reaction mechanism was demonstrated by mass spectrometry and fluorescence off-on mechanism was proved by density functional theory (DFT). Significantly, confocal fluorescence imaging indicated that BDP-DNBS was successfully used to visualize H₂S in lysosomes in living HeLa cells.     

Four Coordination Polymers Assembled with a New Biscarboxyl-functionalized Schiff Base Ligand

Based on a new biscarboxyl-functionalized Schiff base ligand 1,2-cyclohexanediamino-N,N'-bis[3-methoxyl-5-(p-carboxyl-phenylazo)] salicylidene(H₄L), four polymers,[(CH₃)₂NH₂]₂·[Mn₃(L)₂(H₂O)₄]·2DMF(CP1),[Fe₂(L)(H₂O)(DMF)](CP2),[(CH₃)₂NH₂]·[Cu(HL)]·H₂O(CP3) and[Ni₂(L)(teta)]·2DMF·H₂O(CP4), have been synthesized(teta=5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane). In CP1, both of the internal[N₂O₂] pocket and the external carboxylate groups of L^4- anion are ligated by Mn²⁺ ions, and the structure displays a layer. In CP2, the Fe²⁺ cations are linked by L^4- anions to form a binuclear double chain. CP3 displays a[Cu₂(HL)₂] dimer. In CP4, the Ni²⁺ ions are connected by L^4- anions to form a chain. The structures are further linked by hydrogen bonds to form 2D or 3D supramolecular architectures, respectively. CP1 exhibits adsorption ability to three organic dyes.     

Copper(I) 1,2,4-triazolates and related complexes: studies of the solvothermal ligand reactions, network topologies, and photoluminescence properties

One-pot solvothermal treatments of organonitriles, ammonia, and Cu(II) salts yielded Cu(I) and 3,5-disubstituted 1,2,4-triazolates. The organic triazolate components were derived from copper-mediated oxidative cycloaddition of nitriles and ammonia, in which a key intermediate 1,3,5-triazapentadienate was isolated as [Cu(II)(4-pytap)(2)] (4-Hpytap = 2,4-di(4-pyridyl)-1,3,5-triazapentadiene) via controlled solvothermal conditions. This intermediate could also be synthesized by Ni(II)-mediated reactions; however, the final triazoles were obtained only when Cu(II) was employed. Therefore, the reaction mechanism of these reactions was elucidated as follows: nitrile was first attacked by ammonia to form the amidine, which further reacted with another nitrile or self-condensed to yield 1,3,5-triazapentadiene, which was coordinated to two Cu(II) ions in its deprotonated form. A two-electron oxidation of the 1,3,5-triazapentadienate mediated by two Cu(II) ions gave one triazolate and Cu(I) cations. Other in situ ligand reactions, such as C-C bond cleavage and hydrolysis, were also found for the nitriles under these solvothermal conditions. Another remarkable feature of these crystalline Cu(I) triazolates is their simple, typical 3- or 4-connected network topologies. The self-assembly of these nets is presumably controlled by steric hindrance, which is subsequently applied to the rational design of the close-packed 2D networks [Cu(I)(tz)](infinity) and [Ag(I)(tz)](infinity) (Htz = 1,2,4-triazole), as well as the porous 3D network [Cu(I)(etz)](infinity) (Hetz = 3,5-diethyl-1,2,4-triazole). The interesting photoluminescence properties of these coinage d(10) metal complexes were also investigated.     

Two-Dimensional Supramolecular Polymerization of DNA Amphiphiles is Driven by Sequence-Dependent DNA-Chromophore Interactions

Two-dimensional (2D) assemblies of water-soluble block copolymers have been limited by a dearth of systematic studies that relate polymer structure to pathway mechanism and supramolecular morphology. Here, we employ sequence-defined triblock DNA amphiphiles for the supramolecular polymerization of free-standing DNA nanosheets in water. Our systematic modulation of amphiphile sequence shows the alkyl chain core forming a cell membrane-like structure and the distal π-stacking chromophore block folding back to interact with the hydrophilic DNA block on the nanosheet surface. This interaction is crucial to sheet formation, marked by a chiral “signature”, and sensitive to DNA sequence, where nanosheets form with a mixed sequence, but not with a homogeneous poly(thymine) sequence. This work opens the possibility of forming well-ordered, bilayer-like assemblies using a single DNA amphiphile for applications in cell sensing, nucleic acid therapeutic delivery and enzyme arrays.     

Luminescent Cyclometalated Platinum(II) Complex Forms Emissive Intercalating Adducts with Double‐Stranded DNA and RNA: Differential Emissions and Anticancer Activities

Luminescent metallo‐intercalators are potent biosensors of nucleic acid structure and anticancer agents targeting DNAs. There are few examples of luminescent metallo‐intercalators which can simultaneously act as emission probes of nucleic acid structure and display promising anticancer activities. Herein, we describe a luminescent platinum(II) complex, [Pt(C^N^N)(C≡NtBu)]ClO₄ ( 1 a , HC^N^N= 6‐phenyl‐2,2′‐bipyridyl), that intercalates between the nucleobases of nucleic acids, accompanied by an increase in emission intensity and/or a significant change in the maximum emission wavelength. The changes in emission properties measured with double‐stranded RNA (dsRNA) are different from those with dsDNA used in the binding reactions. Complex 1 a exhibited potent anticancer activity towards cancer cells in vitro and inhibited tumor growth in a mouse model. The stabilization of the topoisomerase I–DNA complex with resulting DNA damage by 1 a is suggested to contribute to its anticancer activity.     

单细胞核酸编码扩增成像分析

单细胞成像可在单细胞水平观测目标物位置、 确定目标物含量, 在生命科学与临床医学研究领域应用广泛. 核酸编码扩增技术利用特定分子反应将待测目标识别转化为核酸条码的扩增, 具有探针种类多、 易编程、 反应条件温和及信号放大效率高等特点, 在单细胞低丰度、 高灵敏、 多目标物成像中优势显著, 为理解细胞状态、 探索生命过程提供了新思路. 本文综合评述了核酸编码扩增在单细胞荧光成像领域的研究进展, 以目标物的编码方式为分类依据, 系统阐述了固定细胞原位成像和活细胞成像中不同目标物编码与扩增成像方式的区别, 并对活细胞成像中多重检测面临的问题以及未来发展前景进行了展望.     

Synthesis,structure determination,and magnetic properties of pentakis[ bis (hexafluoroacetylacetonato)1,2,4-triazinecopper(II)]

Reaction of bis(hexafluoroacetylacetonato)copper(II) hydrate with 1,2,4-triazine (tz) in dichloromethane yields pentakis[bis(hexafluoroacetylacetonato)triazinecopper(II)] [Cu(hfac)₂(tz)]₅ (hfac = hexafluoroacetylacetonate) (1). The complex crystallizes in the triclinic space group P-1, with cell parameters a = 11.4124(5), b = 13.3405(5), c = 16.1794(7) Å, α = 93.360(2)°, β = 108.700(2)°, γ = 100.293(2)° at 120(1) K. In the complex, the copper(II) ions show three types of coordination polyhedra: square planar, square pyramidal, and octahedral (4 + 2). The tz ligand also shows different coordination modes (bridging and monodentate). In addition, disorder is observed in the triazine molecule, either through non-crystallographic two-fold rotation about the longitudinal N,N-axis, or with respect to a crystallographic center of symmetry. The crystal structure of 1 consists of alternating trimers and dimers. The weak coordination of the tz molecules results in negligible magnetic exchange through the ring.     

Synthesis of CF3-containing tetrapeptide surrogates via Ugi reaction/dipolar cycloaddition sequence

A convenient synthetic pathway to novel functionalized tetrapeptides containing the 1,2,3-triazole moiety is described. The target molecules were obtained by the reaction of fluorinated α-amino acids or α-aminophosphonates with azidopeptides via Cu(I)-catalyzed Huisgen cycloaddition reaction (click chemistry). The synthesized tetrapeptides may find important applications in biomedical chemistry as potential drugs.     

2D Cu(I) and 3D mixed-valence Cu(I)/Cu(II) coordination polymers: Synthesis and structural characterization of [CuCl(pyz-H)2]·2H2O and [Cu2Cl2(pyz)(H2O)]·H2O (pyz-H = pyrazinic acid)

Two new coordination polymers of copper(I) chloride and pyrazinic acid (pyz-H), namely [CuCl(pyz-H)₂]·2H₂O (1) and [Cu₂Cl₂(pyz)(H₂O)]·H₂O (2) have been prepared and characterized by spectroscopic, magnetic and crystallographic methods. The overall physical measurements suggest that 1 is diamagnetic and contains monodentate N-pyrazinic acid, whereas 2 is paramagnetic and contains tridentate N,N′,O- chelating bridging pyrazinato anion. In the structure of 1 as elucidated by X-ray single crystal analysis, the asymmetric units [CuCl(pyz)₂] are linked together forming a zigzag chain with tetrahedral copper(I) environment. The two lattice water molecules form hydrogen bonds with the uncoordinated N atom and carboxylate group O atom of pyz-H molecules. The Cu–N bond lengths are 2.009(6) Å and Cu–Cl distances are 2.337(2) Å. Complex 2 has a three-dimensional structure with the chains [Cu(I)Cu(II)(C₅H₃N₂O₂)Cl₂(H₂O)] interconnected by [Cu(I)Cl₂N] tetrahedral unit and [Cu(II)NO₂Cl₂] polyhedra. The Cu(I)–Cl and Cu(I)–N distances are 2.327(2)–2.581(2) Å and 1.988(6) Å, respectively, whereas the Cu(II)–Cl and Cu(II)–N bond lengths are 2.258(2), 2.581(2) Å, and 2.017(6) Å, respectively. Hydrogen bonds of the type O–HO are formed between lattice and coordinated water, and carboxylate oxygens of pyrazinato ligand giving rise to a three-dimensional network. The Cl⁻ anions act as bridging ligands in both complexes. The magnetic data of complex 2 have been measured from 2 to 300 K and discussed.     

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a “sandwich” sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-μL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.