利用核酸i-motif结构对质子的超敏感特性,选择富含胞嘧啶C的核酸链及其互补链,以及染料SYBR Green Ⅰ对单双链DNA的不同荧光响应特性,研制了一种灵敏的新型pH响应荧光传感器.当pH值从5.0变为7.5时,SYBR Green Ⅰ的荧光强度增大了4倍以上,pH传感范围是5.0～7.5,并显示出很高的pH分辨率(0.1个pH单元).该pH传感器具有简单、快速、成本低等优点,有望用于相关生物过程中微小pH值变化的传感分析.

Metal ions sensing play critical roles in environmental monitoring and in biology. In this assay, we report the development of a facile fluorometric method for the sensing of Ag+ ions via the in situ formation of metal coordination polymer, based on the selective interactions of GSH with Ag+. The formation of coordination polymer with net multiple negative charges in an aqueous buffer solution (Tris-HAc, pH 9.0) resulted in aggregation and fluorescence quenching of a cationic perylene probe. The difference in emission intensity spurred us to develop a new strategy for sensing Ag+ ions. The proposed Ag+ detection method is simple, convenient, selective and sensitive, and can be used for Ag+ detection in lake water samples.

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC50 values are 3.23 μg mL(-1) and 0.303 μg mL(-1) for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.

A novel label-free fluorescence recovery assay for the sensing of a DNA binding protein has been developed. A transcription factor c-Jun protein, and a 21 base pair duplex DNA containing the c-Jun protein binding site (J-DNA) were selected. J-DNA was mixed with a cationic fluorescent perylene probe (compound 1), and induced aggregation of the probe. Quenching of the probe's fluorescence was observed. However, when c-Jun protein was mixed with the J-DNA, c-Jun bound to the duplex DNA, which reduced the degree of the induced perylene probe aggregation, and a turn on fluorescence signal was observed. The recovered fluorescence intensity was directly related to the amount of c-Jun added. The method is highly selective, six non-DNA binding proteins and one randomly selected 21 base pair duplex DNA (con-1) were tested. No noticeable compound 1 fluorescence recovery was observed. Mutations were also introduced to the c-Jun recognition sequence and much reduced fluorescence recovery was observed. Our assay is label-free, convenient, inexpensive, and fast. It can be used in biomedical research such as high throughput screening of drugs targeted at DNA-binding proteins.

A novel method for the sensing of acetylcholinesterase (AChE) activity and inhibitor screening based on the formation of metal coordination polymer has been developed. Acetylthiocholine (ATCh) was selected as the substrate. In the presence of AChE, ATCh was hydrolyzed to thiocholine and acetate. Thiocholine interacted with Ag(I) to form a metal coordination polymer. A positively charged perylene probe (probe 1) was employed. The fluorescence of probe 1 was very efficiently quenched by a polyanion [PVS, poly(vinyl sulfonate)]. In the presence of acetylcholinesterase, the positively charged metal coordination polymer newly formed in situ would interact with PVS, probe 1 monomer molecules were released, and a turn on fluorescence signal was detected. The assay is highly sensitive, a limit of detection of 0.04 mU/mL AChE was obtained. The assay is also highly selective, a number of potential interference proteins (enzymes) were tested, and none of them show noticeable interference. Sensing of AChE inhibitor was also demonstrated. Our assay is fairly simple and inexpensive. We envision that it could be used for the sensitive detection of other hydrolytic enzyme activities with properly selected substrates and for the screening of potential inhibitor drugs.

A choline labeled pyrene probe (Py-Ch) was designed and synthesized. Poly(vinylsulfonate) (PVS) could induce Py-Ch aggregation. The aggregation and deaggregation process could be finely controlled by the acetylcholinesterase (AChE) enzymatic hydrolysis of Py-Ch. The resulting excimer-monomer transition provided a facile way for real-time AChE activity fluorometric assay and inhibitor screening.

A new approach for the sensitive and selective sensing of a protein has been developed. Cytochrome c could quench the fluorescence of a fluorophore labeled aptamer efficiently. The specific binding between the aptamer and the target protein resulted in a turn-on emission signal, which could be used for protein quantification.

A tetracationic perylene probe (probe 1) was designed and synthesized. Probe 1 was used for the real-time fluorescence turn-on assay of alkaline phosphatase (ALP) activity and inhibitor screening. Probe 1 monomer fluorescence could be very efficiently quenched by ATP through the formation of an ATP/probe 1 complex. ALP triggered the degradation of ATP, the breakdown of the ATP/probe 1 complex, and the recovery of the probe 1 monomer fluorescence. In the presence of an ALP inhibitor, a decrease in fluorescence recovery was observed.

A single stranded oligonucleotide could induce aggregation of a perylene probe, the probe's monomer fluorescence was efficiently quenched. However, when the oligonucleotide was 5'-phosphorylated by polynucleotide kinase, it could be very efficiently degraded by lambda exonuclease, probe monomers were released, and a turn on fluorescence signal was detected.

In the present work, we have studied the aggregation of gold nanoparticles (Au-NPs) induced by a cationic perylene probe (compound 1). The compound 1 free monomer contains a large planar aromatic ring structure that could be efficiently adsorbed onto the surface of the Au-NPs. The strong π–π stacking and hydrophobic interactions among compound 1probe molecules adsorbed on adjacent nanoparticles, and the neutralization of the Au-NP surface citrate ion negative charges induced rapid aggregation of the Au-NPs, and evident UV-vis spectra and solution color changes. The use of this observation for a label-free selective sensing of mercury ion has been demonstrated.

A fluorophore labeled oligonucleotide could induce aggregation of a positively charged perylene probe. The perylene aggregate could very efficiently quench the fluorescence of the labeled fluorophore. Based on this observation, a new method for the highly sensitive and selective detection of a protein has been developed.

We have developed a simple, inexpensive, and label-free method for the selective detection of adenosine. Klenow fragment polymerase (KF polymerase) is a commonly-used 5' to 3' DNA polymerase, it also has 3' to 5' exonuclease activity that can digest single-stranded DNA. An adenosine binding DNA aptamer was employed, the aptamer was split into two pieces of single-stranded DNA (aptamer-A1 + aptamer-A2). Without the addition of adenosine, aptamer-A1 and aptamer-A2 existed as single-stranded DNA which could be efficiently degraded by the exonuclease activity of KF polymerase. Much reduced background fluorescence was obtained when SYBR Green dye was added. However, in the presence of adenosine, aptamer-A1 and aptamer-A2 bound to adenosine, and hybridization of the complementary sequences resulted in the formation of a duplex DNA structure, which could initiate DNA polymerization. The addition of SYBR Green dye resulted in a very high fluorescence enhancement, which could be used for the quantification of adenosine.

<正>核酸作为生命的最基本物质之一,是遗传信息储存、复制和传递的主要载体,在生长、遗传、变异等一系列生命现象中起着决定性的作用。因此,核酸的检测及其性质、结构等的研究具有重要意义。现已发现多种疾病与核酸存在密切的关

A new approach has been developed for the highly sensitive and selective sensing of a protein. Lysozyme binding to its aptamer prevents SSB protein binding, and the subsequent binding of the free SSB protein to a molecular beacon results in a turn-on fluorescence signal, which can be used for lysozyme quantification.

The pyrene probe and pyrene-labeled oligonucleotides (ODNs) probe are expected to be candidates as fluorescent probe for DNA assay. In particular, label-free detection is a very hot because of its simpleness, speediness and cheapness. Herein, we have investigated the use of a pyrenylakylammonium salt, a novel fluorescent probe for the detection of one single nucleotide polymorphism (SNP) in double stranded DNA. After S1 nuclease digestion, the pyrene probes bind electrostatically to the perfect complement DNA and emit a strong excimer emission. However, treatment of the non-complementary DNA with S1 nuclease caused nucleotide fragments of less than 5 bases, which could not induce excimer emission. By comparing ratio of excimer to monomer fluorescence between normal and mutant DNA after S1 nuclease digestion, One-base mutation in DNA was detected easily. This new method may be applied to the detection of SNP.

Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.