Label-free fluorescence turn-on detection of microRNA based on duplex-specific nuclease and a perylene probe

A novel fluorescence turn-on microRNA (miRNA) detection method based on duplex-specific nuclease (DSN) and a perylene probe is presented in this study. A positively charged perylene derivative (compound 1) was used as the fluorescent probe. Compound 1 exhibits strong monomer fluorescence in an aqueous buffer solution. It is well known that single-stranded DNA is a polyanion in nature. Thus, it can induce the aggregation of compound 1 through strong electrostatic, hydrophobic and π−π stacking interactions. As a result, the fluorescence of compound 1 was efficiently quenched. When the target miRNA was added, the formation of DNA-RNA hybridized duplex initiated the cleavage of the DNA strand by DSN cycle reaction, which resulted in disaggregation of compound 1. A fluorescence turn-on signal was detected, and a novel miRNA sensing method was therefore established. The presented method is label-free, simple, cost effective, sensitive and selective.     

Cationic conjugated polymers as signal reporter for label-free assay based on targets-mediated aggregation of perylene diimide quencher

Herein, we reported a cationic conjugated polymers-based new biosensor with label-free and fluorescence turn-on strategy by virtue of targets regulated aggregation and quenching ability of perylene diimide derivatives.     

Terbium fluorescence as a sensitive,inexpensive probe for UV-induced damage in nucleic acids

Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb³⁺). Single-stranded oligonucleotides greatly enhance the Tb³⁺ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb³⁺/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb³⁺, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb³⁺/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb³⁺/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.     

A novel fluorescence probe for cilnidipine assay

A general route for preparation of dye-encapsulated polymer particles via an emulsion polymerization process has been described. 1-Naphthaleneboronic acid (NBA) was encapsulated, the resultant particles were used as a fluorescence probe for cilnidipine assay based on fluorescence quenching. The sensitivity of NBA-encapsulated probe to cilnidipine was largely improved in comparison with that of free NBA. The probe showed a linear response toward cilnidipine over the concentration range of 2.0 × 10⁻⁷ to 1.1 × 10⁻⁵ mol l⁻¹, with high sensitivity, fast response time, and good selectivity.     

新的吲哚类同型二聚体荧光探针测定蛋白质的分析研究

采用新的水溶性吲哚类同型二聚体探针Ⅰ,建立了一种新的蛋白质荧光分析体系.在酸性条件下,蛋白质分子能与吲哚探针发生结合作用,体系荧光明显增强并向长波方向发生红移,荧光强度与蛋白质质量浓度呈良好的线性关系,在最优条件下,牛血清白蛋白BSA的线性响应范围0.80～25.00 μg/mL,检出限(3σ/K)为0. μg/mL;用于模拟样品的测定,1.00,2.00和5.00 μg/mL的样品测定回收率为99.0%～102.0%,相对标准偏差1.8%～2.8%;与国家标准方法比较,结果测定偏差为1.0%～4.1%.     

A label-free fluorescence sensor for probing the interaction of oligonucleotides with target molecules

This paper describes a universal, label-free fluorescence sensor that gauges the interaction of oligonucleotides with their targets. The sensor is based on supramolecular assemblies formed by oligonucleotides (polyanions) and small molecule cation surfactant in aqueous solution. The environmentally dependent dye pyrene, encapsulated in the apolar interiors of the assemblies via hydrophobic interactions works as the fluorescence probe. Target binding causes the conformational change of the oligonucleotides, which results in disorganization of supramolecular assemblies, release of pyrene into the aqueous solution and subsequent quenching of its fluorescence. The kinetic processes (including the formation of supramolecular assemblies and the release of pyrenes after adding the targets) were investigated. The fluorescence decreases of pyrenes are proportional to the concentrations of targets within the linear ranges. This label-free fluorescence system is simple, convenient, low cost, and can serve as an alternative tool for interaction studies of oligonucleotides with their targets, especially with small molecular targets.     

Electrocatalytic assay of mercury(II) ions using a bifunctional oligonucleotide signal probe

Engineered nucleic acid probes containing recognition and signaling functions find growing interest in biosensor design. In this paper, we developed a novel electrochemical biosensor for sensitive and selective detecting of Hg²⁺ based on a bifunctional oligonucleotide signal probe combining a mercury-specific sequence and a G-quadruplex (G4) sequence. For constructing the electrochemical Hg²⁺ biosensor, a thiolated, mercury-specific oligonucleotide capture probe was first immobilized on gold electrode surface. In the presence of Hg²⁺, a bifunctional oligonucleotide signal probe was hybridized with the immobilized capture probe through thymine–mercury(II)–thymine interaction-mediated surface hybridization. The further interaction between G4 sequence of the signal probe and hemin generated a G4–hemin complex, which catalyzed the electrochemical reduction of hydrogen peroxide, producing amplified readout signals for Hg²⁺ interaction events. This electrochemical Hg²⁺ biosensor was highly sensitive and selective to Hg²⁺ in the concentration of 1.0 nM to 1 μM with a detection limit of 0.5 nM. The new design of bifunctional oligonucleotide signal probes also provides a potential alternative for developing simple and effective electrochemical biosensors capable of detecting other metal ions specific to natural or artificial bases.     

Preparation and application of functionalized nanoparticles of CdS as a fluorescence probe

CdS nanoparticles have been prepared and modified with mercaptoacetic acid. The functionalized nanoparticles are water-soluble and biocompatible. They could be used as a fluorescence probe in the determination of bovine serum albumin (BSA), which was proved to be a simple, rapid and specific method. In comparison with single organic fluorophores, these nanoparticle probes are brighter, more stable against photobleaching, and do not suffer from blinking. Under the optimum conditions, the response is linearly proportional to the concentration of BSA between 0.1 and 3.2 μg ml⁻¹, and the limit of detection is 0.08 μg ml⁻¹.     

A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid

A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3′-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully.     

Preparation and application of functionalized nanoparticles of CdSe capped with 11-mercaptoundecanoic acid as a fluorescence probe

A new fluorescence probe, CdSe nanocrystal has been prepared and modified with 11-mercaptoundecanoic acid [HS-(CH₂)₁₀-COOH]. The functionalized nanoparticles were characterized using transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectroscopy and photoluminescence (PL) spectroscopy. The results demonstrate that CdSe is dispersed homogeneously in aqueous solution and well protected from the environmental oxygen. They can be used as a new fluorescence probe for lysozyme, which was simple, rapid and specific. Under the optimum condition, the response is linearly proportional to the amount of lysozyme from 0.20 to 26.0 μg ml⁻¹, and the limit of detection is 0.115 μg ml⁻¹. The proposed method has been applied to the determination of lysozyme in egg white, with the recovery of 96-105%.     

Indirect fluorimetric detection of proteins via postcolumn mixing with fluorescence probe in size-exclusion chromatography

Summary Proteins were visualized by postcolumn mixing with 2-p-toluidinyl-6-naphthalene sulfonate or 1-anilino-8-naphthalene sulfonate in size-exclusion chromatography. The indirect detection is based on fluorescence enhancement of the fluorescence probe owing to hydrophobic interaction with proteins. Bovine serum albumin gave the highest signal intensity among the proteins examined.     

A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid

An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a “caged” inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.     

S,O-doped carbon nitride as a fluorescence probe for the label-free detection of folic acid and targeted cancer cell imaging

A novel nanoprobe based on S,O-doped carbon nitride quantum dots (S,O-CNQDs) was designed and synthesized. The as-prepared S,O-CNQDs exhibits good biocompatibility and strong fluorescence at excitation 360 nm. It is found that folic acid (FA) could efficiently quench the fluorescence of S,O-CNQDs. The obtained S,O-CNQDs is capable of acting as a sensitive and selective probe for FA detection in the range 5.0–83.3 μM with a detection limit of 90 nM. The as-prepared probe has been successfully utilized for the detection of FA in various real samples with satisfactory recoveries (98.8–107 %) and small relative standard deviation (<5%). The reaction mechanism between S,O-CNQDs and FA has been discussed. In addition, FA-S,O-CNQDs formed through a classical cross-linking reaction between FA and S,O-CNQDs easily accesses and penetrates into HepG2 cells with high folate receptors expression. FA-S,O-CNQDs with low cytotoxicity and good biocompatibility shows great potential in FA detection and targeted imaging of cancer cells.     

Synchronous fluorescence determination of protein with functionalized CdS nanoparticles as a fluorescence probe

Nanometer-sized fluorescent particles have been successfully synthesized. A synchronous fluorescence method, with high sensitivity and selectivity, has been developed for rapid determination of protein with functionalized CdS as a fluorescence probe. When Δλ=260 nm, maximum synchronous fluorescence is produced at 274 nm at pH 7.0. Under optimal conditions, the calibration graphs are linear over the range 0.1-3.0 μg ml⁻¹ for bovine serum albumin (BSA), 0.1-11.0 μg ml⁻¹ for γ-globulin (γ-G) and 0.1-1.4 μg ml⁻¹ for human serum albumin (HSA), respectively. Limits of determination were 0.01 μg ml⁻¹ for BSA, 0.019 μg ml⁻¹ for γ-G and 0.021 μg ml⁻¹ for HSA, respectively. The relative standard deviations of seven replicate measurements were 1.8% for 1.0 μg ml⁻¹ BSA, 2.2% for 1.0 μg ml⁻¹ γ-G and 2.3% for 1.0 μg ml⁻¹ HSA.     

Determination of ultra trace amounts of protein by 4-chlorosulfo-(2′-hyaroxylphenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] as the fluorescence spectral probe in AOT microemulsion

Experiments indicated that protein can enhance the fluorescence of the 4-chlorosulfo-(2′-hydroxylophenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] in the presence of bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) microemulsion. Based on this, a sensitive and reproducible fluorometric method for the determination of micro amount protein was developed. The calibration curves of four proteins were given. Under the optimum experimental conditions, the enhanced fluorescence intensity of the system was in proportional to the concentration of protein in the range of 0.1–11 μg mL⁻¹ for bovine serum albumin (BSA), 1.0–10 μg mL⁻¹ for human serum albumin (HSA), 1.0–50 μg mL⁻¹ for ovalbumin (Ova) and 2.5–18 μg mL⁻¹ for γ-globulins (γ-G). Their detection limits were 0.070, 0.071, 0.33 and 0.22 μg mL⁻¹, respectively. The ClSARP-Ti(IV) complex as a spectral probe can be used to the determination of protein in milk powder and oatmeal yielding with satisfactory results. Therefore, the proposed method is one of the most sensitive methods available. In addition, the interaction mechanism of this system is studied by multi-techniques.     

A label-free electrochemical assay for quantification of gene-specific methylation in a nucleic acid sequence

A label-free electrochemical method is developed for simple and convenient quantification of gene-specific DNA hypermethylation in a DNA sequence without PCR amplification, bisulfite conversion or labeling processes.     

Determining PPARγ-ligand binding affinity using fluorescent assay with cis-parinaric acid as a probe

Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (Kd and Ki) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, Kd value of a probe can be obtained by binding assay, and Ki value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R]t), ligand([L]t) and probe([P]t) are required for the data fitting. In this work, Ki values of some typical ligands of PPARγ were successfully determined by use of our equations, among which the Ki value of PPARγ-LY171883 was reported for the first time.     

CdTe量子点同步荧光法测定人血清白蛋白

以巯基乙酸(HS-CH2COOH)为稳定剂,水相中合成了CdTe量子点.在pH 6.40的0.002 mol/L KH2PO4-Na2HPO4缓冲溶液中,固定波长差为220 nm时一定量蛋白质的加入能明显增强量子点的同步荧光强度,并且荧光峰强度增加值与血清白蛋白浓度间存在良好的线性关系,据此建立了一种高灵敏度的测定微量蛋白质的方法.该方法测定人血清白蛋白的线性范围为0.08～2.80 μg/mL,检出限为0.032 μg/mL,10次重复测定1.80 μg/mL的血清白蛋白相对标准偏差为1.1%,已用于实际样品的测定.     

A new fluorescence probe based on diarylethene with a benzothiazine unit for selective detection of Cd2+

A novel photochromic diarylethene derivative containing a benzothiazine unit has been synthesized. Its photochromism and fluorescent selectivity to metal ions were studied in detail in methanol solution. Under the stimulation of Cd²⁺ ions, the derivative showed a significant fluorescence enhancement and obvious red shift, accompanied by the fluorescent color changed from dark purple to bright blue. The 1: 1 stoichiometry between the derivative and Cd²⁺ was verified by titration experiments and high resolution mass spectrometry. In addition, a molecular logic circuit was designed with the emission intensity at 476?nm as output and the stimuli of Cd²⁺/EDTA and UV/vis as inputs.     

Bioanalytical applications of isothermal nucleic acid amplification techniques

The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.