Fluorophore-labeled Probes Research Articles

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5-Pyrene-Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers.

Oligodeoxyribonucleotides modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non-stringent conditions, relative to the corresponding LNA-free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore-labeled probes for SNP-discrimination studies.

A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells.

We report a supersandwich fluorescence in situ hybridization (SFISH) strategy for highly sensitive and selective in situ visualization of mRNA expression patterns at the single-cell level. This strategy uses two fluorophore-labeled signal probes to generate a supersandwich product, which in turn generates numerous signal probes located at the target mRNA position, resulting in the in situ fluorescence signal amplification.

A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces.

Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections.

Electrostatic nucleic acid nanoassembly enables hybridization chain reaction in living cells for ultrasensitive mRNA imaging.

Efficient approaches for intracellular delivery of nucleic acid reagents to achieve sensitive detection and regulation of gene and protein expressions are essential for chemistry and biology. We develop a novel electrostatic DNA nanoassembly that, for the first time, realizes hybridization chain reaction (HCR), a target-initiated alternating hybridization reaction between two hairpin probes, for signal amplification in living cells. The DNA nanoassembly has a designed structure with a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically assembled outer layer of fluorophore-labeled hairpin DNA probes. It is shown to have high efficiency for cellular delivery of DNA probes via a unique endocytosis-independent mechanism that confers a significant advantage of overcoming endosomal entrapment. Moreover, electrostatic assembly of DNA probes enables target-initialized release of the probes from the nanoassembly via HCR. This intracellular HCR offers efficient signal amplification and enables ultrasensitive fluorescence activation imaging of mRNA expression with a picomolar detection limit. The results imply that the developed nanoassembly may provide an invaluable platform in low-abundance biomarker discovery and regulation for cell biology and theranostics.

Composition and microstructure of colostrum and mature bovine milk fat globule membrane

The microstructures of colostrum and mature bovine milk fat globule membrane (MFGM) were investigated using confocal laser scanning microscopy (CLSM) at different temperatures, and the relationships between microstructure variations and the chemical compositions of the MFGM were also examined. Using a fluorophore-labeled phospholipid probe, we found that non-fluorescent domains on the MFGM were positively correlated with the amount of sphingomyelin at both room (20°C) and physiological (37°C) temperatures. However, at the storage temperature (4°C), there were more non-fluorescent domains on the MFGM. These results indicate that the heterogeneities in the MFGM are most likely to be the result of the lateral segregation of sphingomyelin at the room and physiological temperatures, and at the storage temperature, phospholipids with saturated fatty acids affect the formation of these domains.

A facile fluorescence method for versatile biomolecular detection based on pristine α-Fe2O3 nanoparticle-induced fluorescence quenching

This work investigated the interactions of α-Fe2O3 nanoparticles (NPs) with different structural nucleic acids and their fluorescence quenching ability towards fluorophore-labelled nucleic acid probes. Different from bulk α-Fe2O3 samples, nanoscale α-Fe2O3 particles exhibit the unique properties of strong adsorption and fluorescence quenching to fluorophore-labelled single-stranded DNA (ssDNA) probes. Based on these findings, a facile fluorescence method was developed for versatile quantification of nucleic acids. The size scale of NPs makes a significant impact on this sensing platform. Better selectivity was given by bigger NP (50–100nm)-based nucleic acid-sensing platform compared with smaller NP (30nm)-based one. In the 50–100nm α-Fe2O3 NP-based sensing platform, single nucleotide mismatch or single base-pair mismatch can even be effectively discriminated. The targets of micro-RNA (miRNA), ssDNA and double-stranded DNA (dsDNA) are sensitively detected with detection limits of 0.8nM, 1.1nM and 0.64nM (S/N=3), respectively. Significantly, α-Fe2O3 NPs possess different affinities towards ssDNA probes with different lengths, and can be used as a universal quencher for ssDNA probes labelled with different fluorescent dyes. On the basis of these properties, the pristine α-Fe2O3 NPs hold the potential to be widely utilized in the development of novel biosensors with signal amplification or simultaneous multiple target detection strategies.

Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays

In this work, two different configurations for novel amplified fluorescence polarization (FP) aptasensors based on nicking enzyme signal amplification (NESA) and graphene oxide (GO) enhancement have been developed for ultrasensitive and selective detection of biomolecules in homogeneous solution. One approach involves the aptamer-target binding induced the stable hybridization between an aptamer probe and a fluorophore-labeled DNA probe linked to GO, and forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. The second analytical method involves the target induced the assembly of two aptamer subunits into an aptamer-target complex, and then hybridizes with a fluorophore-labeled DNA probe linked to GO, forming a nicking site-containing duplex DNA region. The formation of the duplex DNA region in both methods triggers the NESA process, resulting in the release of many short DNA fragments carrying the fluorophore from GO, generating a significant decrease of the FP value that provides the readout signal for the amplified sensing process. By using the NESA coupled GO enhancement path, the sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude over traditional aptamer-based homogeneous assays. Moreover, these aptasensors also exhibit high specificity for target molecules, which are capable of detecting target molecule in biological samples. Considering these qualities, the proposed FP aptasensors based NESA and GO enhancement can be expected to provide an ultrasensitive platform for amplified analysis of target molecules.

DNA detection using origami paper analytical devices.

We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.

Mechanisms of DNA Sensing on Graphene Oxide

Adsorption of a fluorophore-labeled DNA probe by graphene oxide (GO) produces a sensor that gives fluorescence enhancement in the presence of its complementary DNA (cDNA). While many important analytical applications have been demonstrated, it remains unclear how DNA hybridization takes place in the presence of GO, hindering further rational improvement of sensor design. For the first time, we report a set of experimental evidence to reveal a new mechanism involving nonspecific probe displacement followed by hybridization in the solution phase. In addition, we show quantitatively that only a small portion of the added cDNA molecules undergo hybridization while most are adsorbed by GO to play the displacement role. Therefore, it is possible to improve signaling by raising the hybridization efficiency. A key innovation herein is using probes and cDNA with a significant difference in their adsorption energy by GO. This study offers important mechanistic insights into the GO/DNA system. At the same time, it provides simple experimental methods to study the biomolecular reaction dynamics and mechanism on a surface, which may be applied for many other biosensor systems.

Early Detection ofTrichinella spiralisin Muscle of Infected Mice by Real-Time Fluorescence Resonance Energy Transfer PCR

Real-time fluorescence resonance energy transfer (FRET) PCR and melting curve analysis using newly developed fluorophore-labeled hybridization probes were applied for the detection of Trichinella spiralis DNA in muscle of mice following oral inoculation with 300 T. spiralis larvae. The developed assay could detect and differentiate T. spiralis, Trichinella papuae, and Trichinella pseudospiralis DNAs by the different melting temperatures (Tm). The assay had a detection limit of 5 × 10(2) positive control plasmid copies, which was equivalent to 1 ng of T. spiralis DNA spiked into 250 mg of muscle sample. No fluorescence signal was detected when the technique was applied to the DNA of 27 parasites other than Trichinella spp. The assay could detect T. spiralis DNA in muscle at 7, 14, and 21 days postinoculation. The range, mean ± standard deviation, and median of the Tm values of all positive muscle tissue samples were 60.4-60.8, 60.6 ± 0.2, and 60.5, respectively. This assay provides an effective tool for the specific, sensitive, and high-throughput detection of T. spiralis DNA in muscle during the early stage of infection. In addition, the technique can be useful for epidemiologic surveillance in naturally infected wildlife.

Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

Near-Infrared Optical Imaging for Monitoring the Regeneration of Osteogenic Tissue-Engineered Constructs

Millions of cases of bone injury or loss due to trauma, osteoporosis, and cancer occur in the United States each year. Because bone is limited in its ability to regenerate, alternative therapy approaches are needed. Bone tissue engineering has the potential to correct musculoskeletal disorders through the development of cell-based substitutes for osteogenic tissue replacement. Multiple medical imaging techniques such as magnetic resonance microscopy (MRM) were investigated recently; these techniques are able to provide useful information on the anatomical and structural changes of developing bone. However, there is a need for noninvasive approaches to evaluate biochemical constituents and consequent compositional development associated with growing osteogenic constructs. In this study, near-infrared (NIR) optical imaging with a bone-specific NIR-targeted probe, IRDye® 800CW BoneTag™ (800CW BT), was applied in this study to longitudinally visualize regions of mineralization of tissue-engineered bone constructs in vivo. A fluorescent cell-based assay was performed to confirm the preferential binding of 800CW BT to the mineralized matrix of differentiated osteogenically driven human mesenchymal stem cells (hMSCs) in vitro. The hMSCs were seeded onto a biocompatible gelatin scaffold, allowed to develop, and implanted into a mouse model. Engineered constructs were examined in vivo using NIR imaging for bone mineralization, paired with MRM for verification of developing tissue. Results showed that NIR imaging with 800CW BT labeling can effectively assess the calcification of the developing osteogenic constructs, which is consistent with the analysis of excised tissue using NIR microscopy and histology. In conclusion, this study evaluated bone-like function of regenerating bone through tracking calcium deposition via NIR optical imaging with a fluorophore-labeled probe in a noninvasive manner.

Base recognition of gap sites in DNA–DNA and DNA–RNA duplexes by short oligonucleotides

To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites.

Molecular Beacons: A Novel Optical Diagnostic Tool

As a result of the efforts of the Human Genome Project and the rise in demand for molecular diagnostic assays, the development and optimization of novel hybridization probes have focused on speed, reliability, and accuracy in the identification of nucleic acids. Molecular beacons (MBs) are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Because of the high specificity and sensitivity characteristics, MBs have been used in variety of fields. In this review, MBs are introduced and discussed as diagnostic tools in four sections: several technologies of MBs will be illustrated primarily; the limitation of MBs next; the third part is new fashions of MBs; and the last one is to present the application of MBs in disease diagnosis.

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

Interferometric silicon biochips for label and label‐free DNA and protein microarrays

Protein and DNA microarrays hold the promise to revolutionize the field of molecular diagnostics. Traditional microarray applications employ labeled detection strategies based on the use of fluorescent and chemiluminescent secondary antibodies. However, the development of high throughput, sensitive, label-free detection techniques is attracting attention as they do not require labeled reactants and provide quantitative information on binding kinetics. In this article, we will provide an overview of the recent author's work in label and label-free sensing platforms employing silicon/silicon oxide (Si/SiO(2)) substrates for interferometric and/or fluorescence detection of microarrays. The review will focus on applications of Si/SiO(2) with controlled oxide layers to (i) enhance the fluorescence intensity by optical interferences, (ii) quantify with sub-nanometer accuracy the axial locations of fluorophore-labeled probes tethered to the surface, and (iii) detect protein-protein interactions label free. Different methods of biofunctionalization of the sensing surface will be discussed. In particular, organosilanization reactions for monodimensional coatings and polymeric coatings will be extensively reviewed. Finally, the importance of calibration of protein microarrays through the dual use of labeled and label-free detection schemes on the same chip will be illustrated.

Graphene oxide-hairpin probe nanocomposite as a homogeneous assay platform for DNA base excision repair screening

Uracil-DNA glycosylase (UDG) as one of the most important base excision repair enzymes plays a crucial role in protecting the genome from endogenous DNA damage and sustaining the genome integrity. Quantitative activity analysis of UDG is a central challenge and of fundamental importance in bioanalysis. Here, we proposed a novel biosensor constituted by adsorbing a fluorophore-labeled hairpin probe onto the surface of graphene oxide (GO) as a homogeneous assay platform for sensitive UDG activity assay. Active UDG could excise the uracil base in the hairpin probe, and further hydrolysis of the leaving abasic site gave rise to high fluorescence. Thus, it provided a convenient approach for UDG activity quantification. Because of the unique ability of GO in universal fluorescence quenching, a low background fluorescence signal can be obtained for the efficient fluorescence resonant energy transfer from the fluorophore-labeled on the hairpin probe to GO sheet. A quite wide dynamic range from 0.0017U/mL to 0.8U/mL was achieved for UDG assay and the detection limit was estimated to be 0.0008U/mL. The results indicated that this strategy offers a simple, cost-effective, highly sensitive and selective homogeneous detection platform for UDG activity assay related biochemical studies.

A Novel Fluorescence Sensor for Cocaine with Signal Amplification through Cycling Exo-Cleaving with a Hairpin Probe

A novel fluorescence assay, based on the signal amplification through cycling reactions of aptameric recognition and nucleic acid exo-cleaving, was established for the sensitive detection of cocaine. In this assay, a new single fluorophore-labeled DNA-hairpin probe was used. This smart probe was established based on the significant nucleobase quenching between the nucleobases and the fluorophore. In the presence of cocaine and its aptamer, the structure of the smart probe changed to recover the fluorescence. In order to enhance the sensitivity of this assay, exonuclease III was introduced to enable the inputted cocaine to react with multiple probes in a recycling manner. Under the optimal conditions, a linear range for cocaine from 4.0 × 10−9 to 8.0 × 10−8 M with a detection limit of 1.76 × 10−9 M (3σ, n = 11) was obtained.

Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

Schistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

Semi-synthesis of bioactive fluorescent analogues of the cytotoxic marine alkaloid discorhabdin C

Semi-synthetic N-13 alkylated analogues of the cytotoxic marine alkaloid discorhabdin C have been found to exhibit cytotoxicity towards tumour cell lines at comparable levels to that of the natural product. Incorporation of an ethylenediamine linker facilitated the synthesis of a variety of fluorophore-labelled probes, of which dansyl analogue 20 exhibited biological activity, providing a tool for mechanism of action and cellular localization studies. An alternative probe design was also exemplified, whereby a bioactive alkyne-terminated analogue (24) was found to undergo Huisgen 1,3-dipolar cycloaddition ‘click’ reactions with fluorescent azides, enabling studies directed towards activity-based protein profiling.