Homogeneous assay for evaluation of aptamer-protein interaction

We introduce Amplified Luminescent Proximity Homogenous Assay (ALPHA) to assess the K(D) value of aptamer-protein complexes as demonstrated through the study of apple stem pitting virus coat protein-specific aptamers. This method can be used as a simple, cost-effective method for screening aptamer-target protein interactions during aptamer selection.     

Fluorescence polarization for single nucleotide polymorphism genotyping

Single nucleotide polymorphisms (SNPs) are the most abundant variations in the human genome and have become the primary markers for genetic studies for mapping and identifying susceptible genes for complex diseases. Methods that genotype SNPs quickly and economically are of high values for these studies because they require a large amount of genotyping. Fluorescence polarization (FP) is a robust technique that can detect products without separation and purification and it has been applied for SNP genotyping. In this article the applications of FP in SNP genotyping are reviewed and one of the methods, the FP-TDI assay, is discussed in details. It is hoped that readers could get useful information for the applications of FP in SNP genotyping and some insights of the FP-TDI assay.     

Fluorescence chemosensors with pyrene and their interaction with nucleotide phosphate

A group of fluorescence chemosensor with pyrene, compounds (Ⅰ), (Ⅱ) and (Ⅲ), were synthesized The fluorescence spectra and the lifetime of these compounds were carefully measured. The fluorescence quenching spec tra of pyrenyl butyric acid, compounds (Ⅰ), (Ⅱ) and (Ⅲ) by different nucleotide phosphates, AMP ADP, ATP dTTP, were also recorded and studied. The quenching and the stability constants were calculated by Stern-Volmer equa tion and eq. (2), respectively. The mechanism of interaction between fluorescence chemosensor and nucleotide phos phate was didscussed based on the comparison of the results obtained with the CPK model of free molecules of these com pounds in the ground state.     

Nanosensing at the single cell level

This article presents an overview of the development, operation, and applications of optical nanobiosensors for use in in vivo detection of biotargets in individual living cells. The nanobiosensors are equipped with immobilized bioreceptor probes (e.g., antibodies, enzyme substrate) selective to specific molecular targets. Laser excitation is transmitted into the fiber producing an evanescent field at the tip of the fiber in order to excite target molecules bound to the bioreceptors immobilized at the fiber tips. A photometric system detects the optical signal (e.g., fluorescence) originated from the analyte molecules or from the analyte–bioreceptor reaction. Examples of detection of biospecies and molecular signaling pathways of apoptosis in a living cell are discussed to illustrate the potential of the nanobiosensor technology for single cell analysis.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemilu- minescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)32 (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA–ECL assay. The method is useful in SNP analysis due to its sensitivity, safety, and simplicity.     

Electrical conductance of conjugated oligomers at the single molecule level

We determine and compare, at the single molecule level and under identical environmental conditions, the electrical conductance of four conjugated phenylene oligomers comprising terminal sulfur anchor groups with simple structural and conjugation variations. The comparison shows that the conductance of oligo(phenylene vinylene) (OPV) is slightly higher than that of oligo(phenylene ethynylene) (OPE). We find that solubilizing side groups do neither prevent the molecules from being anchored within a break junction nor noticeably influence the conductance value.     

Apache Spark based kernelized fuzzy clustering framework for single nucleotide polymorphism sequence analysis

This paper introduces a kernel based fuzzy clustering approach to deal with the non-linear separable problems by applying kernel Radial Basis Functions (RBF) which maps the input data space non-linearly into a high-dimensional feature space. Discovering clusters in the high-dimensional genomics data is extremely challenging for the bioinformatics researchers for genome analysis. To support the investigations in bioinformatics, explicitly on genomic clustering, we proposed high-dimensional kernelized fuzzy clustering algorithms based on Apache Spark framework for clustering of Single Nucleotide Polymorphism (SNP) sequences. The paper proposes the Kernelized Scalable Random Sampling with Iterative Optimization Fuzzy c-Means (KSRSIO-FCM) which inherently uses another proposed Kernelized Scalable Literal Fuzzy c-Means (KSLFCM) clustering algorithm. Both the approaches completely adapt the Apache Spark cluster framework by localized sub-clustering Resilient Distributed Dataset (RDD) method. Additionally, we are also proposing a preprocessing approach for generating numeric feature vectors for huge SNP sequences and making it a scalable preprocessing approach by executing it on an Apache Spark cluster, which is applied to real-world SNP datasets taken from open-internet repositories of two different plant species, i.e., soybean and rice. The comparison of the proposed scalable kernelized fuzzy clustering results with similar works shows the significant improvement of the proposed algorithm in terms of time and space complexity, Silhouette index, and Davies-Bouldin index. Exhaustive experiments are performed on various SNP datasets to show the effectiveness of proposed KSRSIO-FCM in comparison with proposed KSLFCM and other scalable clustering algorithms, i.e., SRSIO-FCM, and SLFCM.     

Microfluidics-based technologies for the analysis of extracellular vesicles at the single-cell level and single-vesicle level

Extracellular vesicles(EVs) are membrane vesicles secreted by cells, playing critical roles in mediating intercellular communications for various physiological and pathological processes. Most of the EV analysis is currently performed at the bulk level, obscuring the origin of the EVs and diverse characteristics of the individual extracellular vesicle. Technologies to analyze the extracellular vesicles at the single-cell and single-vesicle levels are needed to evaluate EV comprehensively and dec...     

Electrochemical analysis of single nucleotide polymorphisms of p53 gene

Single nucleotide polymorphisms (SNPs) of cancer repression gene p53 were analyzed electrochemically with ferrocenyl naphthalene diimide (1) as a hybridization indicator. The SNPs studied were the transition to A from G in the codon for amino acid at positions 175, 248 or 273 and the transversion to C from G in the codon for the amino acid at position 72. Thus, 20-meric oligonucleotides carrying the SNP site were used both as a sample and a probe with the latter immobilized on an electrode. Even one base difference on the p53 gene resulted in a significant difference in the current response of 1 and the magnitude of the response correlated with the amount of the DNA hybrid on the electrode. Moreover, when PCR products of exon 4, on which the P72/R72 SNP resides, of the p53 gene were analyzed by this method, the heterozygote and homozygotes were discriminated with modest precision.     

Robust electrochemical system for screening single nucleotide polymorphisms

A highly sensitive and selective electrochemical DNA signaling scheme, which identifies the point mutation existing in target DNA sequence, is developed based on the combination of label-free hairpin probe (HP)/DNA endonuclease with zirconia (ZrO(2)) nanoparticle film, representing a promising screening platform for the accurate diagnosis of infections and genetic diseases as well as for environmental and forensic applications.     

Fluorescence anisotropy as a method to examine the thermodynamics of enantioselectivity

A new method is evaluated whereby enantioselective binding is examined by use of steady-state fluorescence anisotropy measurements. A theoretical treatment is presented that relates fluorescence anisotropy measurements to chiral selectivity and allows the estimation of thermodynamic parameters of chiral recognition. It is shown that the natural logarithm of the ratio of anisotropy values of two enantiomers varies as a function of temperature in a manner where the slope and intercept are directly related to the differential enthalpy (delta deltaH degrees) and entropy (Tdelta deltaS degrees) of the enantioselective interaction. The developed method was evaluated by examining the enantioselective interactions of several chiral molecules with beta-cyclodextrin and a molecular micelle.     

Fluorescence excitation and emission spectroscopy on single MEH-PPV chains at low temperature

Fluorescence emission and excitation spectra of single MEH-PPV polymer molecules dispersed in thin PMMA films have been recorded at 1.2 and 20 K. We observe single as well as multichromophore emission in single chain emission spectra, whereby the relative fractions depend on the two different molecular weights (50 and 350 kDa) studied. The molecular weight also affects the distribution of peak emission maxima, which is monomodal (bimodal) for the low (high) molecular weight. The appearance of an additional "red" subpopulation for the high molecular weight sample is attributed to interactions of multiple chromophores from a sufficiently flexible single chain. The comparison of emission spectra appearing in the "blue" as well as "red" subpopulations suggests that these intrachain interactions rather lead to ground-state aggregates than excimers. Independent of the molecular weight, large variations in spectral shape and apparent line width in the emission spectra have been observed. Occasionally, we find very narrow purely electronic zero-phonon lines both in emission and in excitation spectra, with line widths down to the instrumental resolution. In accordance with earlier literature data it is argued that linear electron-phonon coupling should be quite strong for MEH-PPV in PMMA, leading to only a small fraction of chromophores exhibiting zero-phonon lines. In addition, spectral diffusion, which manifests itself by several time-dependent line shifting and broadening phenomena, contributes to the substantial variations of spectral shapes. Excitation experiments with particularly stable chromophores provide an upper limit for the optical line width (approximately 0.1 cm(-1)), which at 1.2 K can actually approach the lifetime-limited homogeneous width. Raising the temperature to 20 K leads to line broadening and typically, to disappearance of zero-phonon lines. The failure to observe zero-phonon lines of chromophores supposedly serving as donors in intramolecular energy transfer is tentatively attributed to fast transfer rates, resulting in strongly broadened lines which escape detection even at 1.2 K.     

DNA molecule manipulation by motor proteins for analysis at the single-molecule level

Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale. Figure DNA molecule manipulation by motor proteins for analysis at the single-molecule level     

Activation analysis for mercury in biological samples at nanogram level

A new method has been devised for determining mercury in samples of biological origin. It is based on complete ignition of the sample in a silica tube, trapping volatile interfering activities such as bromine or chlorine, and selectively adsorbing mercury on a strip of filter paper which has been previously impregnated with elemental selenium. This strip is later counted for quantitative evaluation. The versatility of the method has been demonstrated by the analysis of a wide range of samples such as water, cellulose, flour, fish solubles or animal blood samples with mercury contents between 1 and 200 ng g of sample.