gamma-cyclodextrin and pyrene

A ratiometric, versatile, and selective fluorescent pattern to sense and distinguish proteins on the basis of dissociation of aqueous polymer-pyrene/γ-cyclodextrin (γ-CD) inclusion complexes was developed. First, two kinds of aqueous polymer-pyrene were prepared via atom transfer radical polymerization using pyrene functionalized initiator. Then, the pyrene molecules could be accumulated into γ-CD cavity and form polymer-pyrene/γ-CD complexes, resulting in appearance of excimer emissions. The resultant complexes responded to proteins in two ways: nonmetalloproteins binding to polymer component triggered dissociation of the inclusion complexes, accompanied by alteration of pyrene excimer/monomer emission and ratiometric fluorescent intensity changes; the presence of metalloproteins could quench pyrene excimer/monomer emission because of energy transfer. Moreover, the fluorescent responses of the inclusion complexes to different proteins could be modulated by changing polymer type and chain length, resulting in a tunable selectivity and sensitivity. The proposed fluorescent inclusion complexes could provide a promising platform for sensing proteins.

Topics: Concanavalin A; Fluorescence; gamma-Cyclodextrins; Humans; Molecular Structure; Myoglobin; Pepsin A; Polymers; Pyrenes; Serum Albumin; Water

A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis.

Topics: beta-Thalassemia; DNA; DNA, Circular; Escherichia coli; Fluorescent Dyes; gamma-Cyclodextrins; Genotype; Humans; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotides; Point Mutation; Polymorphism, Single Nucleotide; Pyrenes

In this paper, the development is described of an efficient pyrene excimer signaling-based fluorescent sensor for the measurement of mercury ions in aqueous solutions based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and the inclusion interaction of γ-cyclodextrin. Introduction of cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of Hg(2+) and enhance the fluorescence intensity of the pyrene excimer. To demonstrate the feasibility of the design, a bis-pyrene-labeled thymine-rich DNA strand was used as the detection probe. In the presence of Hg(2+), stem-close-shaped DNA strands can be formed with the cooperation of γ-cyclodextrin and ideally predominantly emit the excimer fluorescence. The selectivity of the sensor for Hg(2+) against other biologically and environmentally related metal ions is outstanding due to the high specificity of T-Hg(2+)-T formation. In addition, the pyrene excimer has a long fluorescence lifetime, which can tolerate intense background fluorescence interference from complex biological components, making it potentially applicable in the analysis of complex biological samples.

Topics: Base Sequence; Cations, Divalent; DNA; Drinking Water; Fluorescent Dyes; gamma-Cyclodextrins; Limit of Detection; Mercury; Pyrenes; Rivers; Spectrometry, Fluorescence; Thymine

The chiral microenvironmental properties of bovine serum albumin, cyclodextrins and their mixtures were comparatively investigated based on pyrene as the fluorescence probe. On this basis, a time resolved fluorescence technique to discriminate D- and L-tryptophan (Trp) without separation was developed. This method was based on the quenching rate difference between d- and l-Trp to pyrene in various media including bovine serum albumin, cyclodextrins and their mixtures. Pyrene, tryptophan and bovine serum albumin (or cyclodextrin) could form ternary complexes, which provided a possibility for the discrimination of D- and L-Trp enantiomers. The results indicated that the chiral discrimination of Trp enantiomers could be obtained in the mixture system of gamma-cyclodextrin and BSA system, in which the fluorescence lifetime difference (Deltatau) and the lifetime difference percentage (Deltatau/tau) were 55.8ns and 28.50%, respectively.

Topics: Animals; Cattle; Chemistry Techniques, Analytical; Cyclodextrins; Dose-Response Relationship, Drug; Fluorescence; gamma-Cyclodextrins; Pyrenes; Serum Albumin, Bovine; Stereoisomerism; Tryptophan

Recently, it was demonstrated that gamma-cyclodextrins (gamma-CDs) greatly accelerates transfer of hydrophobic pyrene-labeled and other fluorescent phospholipid derivatives from vesicles to cells in culture (). To understand better the characteristics of this process, we studied the interaction of gamma-CD with pyrene-labeled phosphatidylcholines (PyrPCs) using a variety of physical methods. Either one or both of the acyl chains of PC was labeled with a pyrene moiety (monoPyrPCs and diPyrPCs, respectively), and the length of the labeled chain(s) varied from 4 to 14 carbons. Fluorescent binding assays showed that the association constant decreases strongly with increasing acyl chain length. PyrPC/gamma-CD stoichiometry was 1:2 for the shorter chain species, but changed to 1:3 when the acyl chain length exceeded 8 (diPyrPCs) or 10 (monoPyrPCs) carbons. The activation energy for the formation of diPyr(10)PC/gamma-CD complex was high, i.e., +92 kJ/mol, indicating that the phospholipid molecule has to fully emerge from the bilayer before complex formation can take place. The free energy, enthalpy, and entropy of transfer of monoPyrPC from bilayer to gamma-CD complex were close to zero. The absorption, Fourier transform infrared, and fluorescence spectral measurements and lifetime analysis indicated that the pyrene moiety lies inside the CD cavity and is conformationally restricted, particularly when the labeled chain is short. The acyl chains of a PyrPC molecule seem to share a CD cavity rather than occupy different ones. The present data provide strong evidence that the ability of gamma-CD to enhance intermembrane transfer of pyrene-labeled phospholipids is based on the formation of stoichiometric complexes in the aqueous phase. This information should help in designing CD derivatives that are more efficient lipid carriers then those available at present.

Topics: Acylation; Cyclodextrins; Fluorescent Dyes; gamma-Cyclodextrins; Kinetics; Lipid Bilayers; Phosphatidylcholines; Pyrenes; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Temperature; Time Factors

Topics: Cyclodextrins; Endocrine Glands; gamma-Cyclodextrins; Pyrenes; Sensitivity and Specificity; Spectrometry, Fluorescence; Tosyl Compounds