Tetramethyl-6-carboxyrhodamine Research Articles

Fluorescent dual-mode assay of plant viral disease with polymerase chain reaction amplification

Plant viral diseases pose serious risk to the quality and yield of different crops. Herein, we described a highly sensitive and accurate immunosensor for early diagnosis of plant viral disease by using dual-signal readout with polymerase chain reaction (PCR) amplification. The sensor utilizes 6-carboxyfluorescein group (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) labeled primers as signal probes and their fluorescence signals were effectively quenched by graphene oxide (GO). In the presence of target plant viral related nucleic acid, the cDNA template could initiate the formation of long duplex PCR products with multiple signal probes. Such duplex PCR products cannot be quenched with GO, resulting in an obvious fluorescence signal. The results showed that FAM and TAMRA signals were proportional to the logarithm of the target cDNA concentration in the range of 10 fM–100 pM. The detection limit for the target cDNA was 0.08 fM for FAM and 0.07 fM for TAMRA, respectively. The practical application of our proposed strategy was successfully demonstrated by detection of emerging tobacco mosaic virus (TMV) infections in tobacco samples collected in the field, illuminating the possibility of applying this sensing platform for early diagnosis of plant disease.

Simultaneous detection of mercury (II), lead (II) and silver (I) based on fluorescently labelled aptamer probes and graphene oxide

ABSTRACT We have developed a fluorescence quantitative analysis method for the simultaneous detection of Hg2+, Pb2+ and Ag+ based on fluorescently labelled nucleic acid aptamer probes and graphene oxide (GO). By this method, three nucleic acid aptamer probes (PHg, PPb, PAg) were designed. The carboxyl fluorescein (FAM), tetramethyl-6-carboxyrhodamine (TAMRA) and cyanine-5 (Cy-5) were respectively selected as fluorophore of aptamer probes, and GO was chosen as quencher. In general, these probes were on free single-stranded state and adsorbed on the surface of GO via π-π interactions, which brought fluorophores of probes and GO into close proximity. Due to the fluorescence resonance energy transfer occurred between fluorophores and GO, the fluorescence was quenched and fluorescence signals were all weak. Under the optimal condition, fluorescence intensities of three fluorophores exhibited a good linear dependence on corresponding ions concentration. The detection limit for Hg2+, Pb2+ and Ag+ were 0.2, 0.5 and 2 nmol/L (3σ, n = 11). Average recoveries of this method were 97.56–104.92%, which indicated the method had a high accuracy and low detection limit. In addition, this proposed method has good selectivity, and there was no crosstalk effect among these probes.

Simultaneous detection of lead (II) and mercury (II) ions using nucleic acid aptamer molecular beacons

ABSTRACT We have developed a fluorescence quantitative analysis method for the simultaneous detection of Hg2+ and Pb2+ based on nucleic acid aptamer molecular beacon (MB) probes. In this analytical method, two MB probes for Hg2+ (PHg) and Pb2+ (PPb) were designed. The carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were selected as fluorophores of PHg and PPb, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were selected as organic quenchers, and several continuous nucleotides with guanine (G base) were connected to organic quenchers. The aptamers are put in as a part of stem and loop. In general, the fluorescence of fluorophores was dually quenched by BHQ and G bases, so the fluorescence signals were weak. In the presence of Hg2+ and Pb2+, PHg and PPb bonded with them, and the stem-loop structure of MB was destroyed and the fluorescence recovered. Under the optimal conditions, the fluorescence intensity of FAM had a good linear relationship with the concentration of Hg2+ in the range from 0.7 nmol/L to 84 nmol/L, and that of TAMRA and Pb2+ in the range from 0.2 nmol/L to 24 nmol/L. The detection limit of Hg2+ is 0.36 nmol/L and that of Pb2+ is 0.16 nmol/L (3σ, n = 11). The relative standard deviations (RSD) for determination of Hg2+ and Pb2+ were both lower than 5%, the average recoveries of this method in real samples were 96.55–102.78%, which indicated that the method had a high accuracy.

The G-BHQ synergistic effect: Improved double quenching molecular beacons based on guanine and Black Hole Quencher for sensitive simultaneous detection of two DNAs

We designed two double quenching molecular beacons (MBs) with simple structure based on guanine (G base) and Black Hole Quencher (BHQ), and developed a new analytical method for sensitive simultaneous detection of two DNAs by synchronous fluorescence analysis. In this analytical method, carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were respectively selected as fluorophore of two MBs, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were respectively selected as organic quencher, and three continuous nucleotides with G base were connected to organic quencher (BHQ-1 and BHQ-2). In the presence of target DNAs, the two MBs hybridize with the corresponding target DNAs, the fluorophores are separated from organic quenchers and G bases, leading to recovery of fluorescence of FAM and TAMRA. Under a certain conditions, the fluorescence intensities of FAM and TAMRA all exhibited good linear dependence on their concentration of target DNAs (T1 and T2) in the range from 4 × 10−10 to 4 × 10−8molL−1 (M). The detection limit (3σ, n = 13) of T1 was 3 × 10−10M and that of T2 was 2×10−10M, respectively. Compared with the existing analysis methods for multiplex DNA with MBs, this proposed method based on double quenching MBs is not only low fluorescence background, short analytical time and low detection cost, but also easy synthesis and good stability of MB probes.

Partitioning of pyrimidine single stranded oligonucleotide using polyethylene glycol – sodium sulfate aqueous two-phase systems; experimental and modeling

In this work partitioning of TAMRA (Tetramethyl-6-Carboxyrhodamine) - oligonucleotide in systems containing 18% (w/w) polyethylene glycol (PEG 600, PEG 1000, PEG 1500 and PEG 4000) and 15% (w/w) sodium sulfate (designated as 18/15) was investigated. To find the optimum condition, effective parameters on biomolecule partitioning including polymer molecular weight, pH, temperature and addition of a second salt were studied. The results showed that as pH increases and molecular weight of polymer decreases, the affinity of TAMRA oligonucleotide to accumulate in the top PEG-rich phase increases. In order to find the temperature effects, partitioning of TAMRA oligonucleotide was studied at two different temperatures 20 and 30 °C). At this range, no significant change was observed. Meanwhile, it is found that addition of 0.5% (w/w) of KH2PO4 as the second salt reversed the partitioning behavior of the oligonucleotide. A similar trend with smaller gradients was observed when KH2PO4 was substituted by KCl; whereas, substitution by MgSO4 increased the affinity of the oligonucleotide to partition to top phase. The best results achieved using PEG 600 at pH = 6. In this case, almost 100% of TAMRA oligonucleotide was recovered in the upper phase. Finally, the partitioning of the biomolecule was correlated using a model based on UNIFAC-FV group contribution. The results showed that the model can correlate the partitioning of the oligonucleotide with an acceptable accuracy.

Tetramethyl-6-carboxyrhodamine quenching-based aptasensing platform for aflatoxin B1: Analytical performance comparison of two aptamers

In this study, a simple TAMRA (tetramethyl-6-carboxyrhodamine) quenching-based aptasensing platform was designed for the detection of aflatoxin B1 (AFB1). Here, we compared the analytical performance of two aptamer sequences: seqA and seqB. The AFB1 detection was based on the interactions of FAM (carboxyfluorescein)-labeled aptamer with TAMRA-labeled DNA complementary strand in the presence and absence of target analyte. Under optimized experimental conditions, TAMRA-labeled strand quenched the fluorescence response of FAM-labeled aptamer due to the noncovalent interaction between the two DNA strands. The binding of AFB1 induced the complex formation and weakened the interaction between FAM-labeled aptamer and TAMRA-labeled complementary strand, resulting in the fluorescence recovery. By using this principle concept, an assay was constructed for the detection of AFB1. The method exhibited good sensitivity, good selectivity with a limit of detection of 0.2 ng ml−1, and a wide linear range from 0.25 to 32 ng ml−1. For real sample application, the aptasensors were tested in beer and wine samples, with good recovery rates obtained for AFB1 detection.

Robust fluorescence sensing platform for detection of CD44 cells based on graphene oxide/gold nanoparticles

Gold-coated graphene oxide hybrid material (GO/AuNPs) has exceptional physical and chemical properties like π–π stacking interaction and plays a role in quencher of fluorescence dye. Therefore, GO/AuNPs could enhance the signal-to-background ratio with fluorescence dye that was the point in this fluorescent biosensor. In this study, tetramethyl-6-carboxy-rhodamine (TAMRA)-labeled aptamers that specifically interact with the hyaluronic acid binding domain of CD44 were used as targets to investigate the applicability of the method. GO/AuNPs–TAMRA-aptamer complexes could detect CD44 target cancer cells within a concentration range of 1×101 to 1×107 CFU/mL. A linear relationship was observed between target cell concentration and relative fluorescence intensity. The more mounted up CD44 target cell concentrations, relative fluorescence intensity of GO/AuNPs–TAMRA-aptamer complexes was increased even more, which was superior to that of GO alone. Sensitivity of the detection system displayed a low detection limit of 1×101 CFU/mL. Additionally, this method is specific in that fluorescence is not much enhanced in CD44 negative cancer cell line. Thus, the fluorescence sensing based on GO/AuNPs could be developed to receptive and robust detection tool for various target molecules.

Multiplexed DNA detection with a composite molecular beacon based on guanine-quenching

We developed a multiplexed DNA detection method with a composite molecular beacon (MB) probe based on guanine-quenching by synchronous fluorescence analysis. It is demonstrated by two types of tumor-suppressor genes namely exon segments of p16 (T1) and p53 (T2) genes. The composite MB probe includes two loops and two stems, and two fluorophores of 6-carboxyfluorescein group (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) are connected to the two ends of molecular beacon. Every stem portion of MB include four continuous nucleotides with guanine (G) base as quencher, every loop portion is a probe sequence that is complementary to a corresponding target sequence. In the absence of target DNA, the composite MBs are in the stem-closed form, the fluorescence of FAM and TAMRA are quenched by G bases. At this time, the fluorescence signals of FAM and TAMRA are all very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strands, FAM and TAMRA are separated from G bases, and the fluorescence of FAM and TAMRA recovers simultaneously. Thus, the simultaneous detection of two targets of DNA can be realized by measuring fluorescence signals of FAM and TAMRA, respectively. Under the optimum conditions, the fluorescence intensities of FAM and TAMRA all exhibit good linear dependence on their target DNA concentration in the range from 5 × 10(-11) to 5.5 × 10(-9) M. The detection limit of T1 is 4 × 10(-11) M (3σ), and that of T2 is 3 × 10(-11) M. This composite MB can be applied to detect the real sample, and can be applied to detect two aleatoric sequences of DNA. Compared with previously reported methods of detecting multiplexed target DNA with MBs, the proposed method has some advantages including easy synthesis of composite MB probes, low detection cost and shorter analytical time.

Characterization of TAMRA- and biotin-conjugated peptide arrays for on-chip matrix metalloproteinase activity assay

Peptide arrays have been widely used for high-throughput determination of protease activities in cells and tissues because specific peptides have high binding affinity for the active site of enzymes. Designing peptide substrate probes for enzyme activity assays have been considered to be important; however, the significance of its reporter tag for detecting enzymatic reactions is relatively underestimated. Thus, we investigated the effect of the reporter tag of peptide substrate probes on on-chip protease activity assays. We optimized and characterized proteolytic activity assay of matrix metalloproteinase-3 using direct and indirect substrate probes, tetramethyl-6-carboxyrhodamine (TAMRA)- and biotin-conjugated peptide arrays, respectively. Proteolytic activity assays using both substrate probes demonstrated similar sensitivity, ratio of maximal to minimal FI, IC50 of GM6001, and inter-array reproducibility. However, biotin-conjugated substrate arrays showed a wider dynamic range than TAMRA-conjugated substrate arrays. Thus, this comparative study provides a wealth of information for developing optimal probes necessary for effective analysis of enzyme activity and kinetics.

Detection of EGFR Mutation in Tissue Samples of Non–small-cell Lung Cancer by a Fluorescence Polarization Assay

It is important to identify epidermal growth factor receptor (EGFR) mutations, which have a good value for the individualized management of patients with non-small-cell lung cancer. A novel method for detecting the mutations on exons 19, 21 of EGFR in primary carcinoma samples by a fluorescence polarization (FP) assay was developed in this research. Firstly, 2 pairs of general primers of exons 19, 21 of EGFR were, respectively, used to amplify the target regions in each exon in 2 reactions. Then, 2 probes specific for wild or mutation exons 19, 21 of EGFR were labeled with tetramethyl 6-carboxyrhodamine or 6-carboxyfluorescein hybridized, respectively, with their target amplicons, and the hybridization resulted in an increase in the FP values. Exon 19 deletion and exon 21 missense mutation were determined by the analysis of the FP values. EGFR mutations in 372 non-small-cell lung cancer samples were analyzed in parallel with an FP assay and a sequencing assay. There was no significant difference between the mutation status results obtained with the FP assay and the results obtained with the sequencing assay. The minimum detection level established with this assay was 40 copies/uL. Reliable results could be obtained when more than 30 ng of DNA was tested by a FP assay. An FP assay was able to detect the mutation DNA of EGFR even when its content was as low as 10%. An FP assay allowed the semiautomated detection of EGFR mutations in solution, and it was much simpler and cost effective than the traditional methods.

Self-assembled complex of probe peptide –E. Coli RNA I conjugate and nano graphene oxide for apoptosis diagnosis

Caspase-3 plays an important role in the initiation and propagation of apoptosis which is involved in various kinds of diseases including neurodegenerative diseases and inflammatory diseases. The capase-3 cleavable site of Asp-Glu-Val-Asp (DEVD) connected to a partial sequence of Escherichia coli RNAI was labeled with tetramethyl-6-carboxyrhodamine (TAMRA) as an optical probe. Graphene oxide (GO) was synthesized by a modified Hummer's method and exploited for the preparation of nano-sized GO (NGO) conjugated with polyethylene glycol (PEG). After binding the NGO-PEG by π-π stacking, the quenched fluorescence of TAMRA-DEVD-single stranded DNA (ssDNA) conjugate was recovered via the enzymatic cleavage by caspase-3 in live A549 cells. The comparative study with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay clearly confirmed the specific detection of apoptosis by the non-covalent TAMRA-DEVD-ssDNA/NGO-PEG complex. Furthermore, the self-assembled NGO complex was successfully exploited for in vivo diagnosis of apoptosis-related diseases like hypoxic-ischemic encephalopathy (HIE) and liver cirrhosis.

Monitoring of proteolytic enzyme activity using phase transition-based peptide arrays

We have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine the quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (Km), maximum rate of enzymatic reaction (Vmax), and half maximal inhibitory concentration (IC50) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies.

Locally enhanced concentration and detection of oligonucleotides in a plug-based microfluidic device

We propose a novel technique that allows oligonucleotides with specific end-modification within a plug in a plug-based microfluidic device to undergo a locally enhanced concentration at the rear of the plug as the plug moves downstream. DNA was enriched and detected in situ upon exploiting a combined effect underlain by an entropic force induced through fluid shear (i.e. a hydrodynamic-repellent effect) and the interfacial adsorption (aqueous/oil interface) attributed to affinity. Flow fields within a plug were visualized quantitatively using micro-particle image velocimetry (micro-PIV); the distribution of the fluid shear strain rate explains how the hydrodynamic-repellent effect engenders a dumbbell-like region with an increased concentration of DNA. The concentration of FAM (6-carboxy-fluorescein)-labeled DNA (FC-DNA) and of TAMRA (tetramethyl-6-carboxyrhodamine)-labeled DNA (TC-DNA), respectively, and the hybridization of probe DNA (modified with FAM) with target DNA (modified with TAMRA) were investigated in devices; a confocal fluorescence microscope (CFM) was utilized to monitor the processes and to resolve the corresponding 2D patterns and 3D reconstruction of the DNA distribution in a plug. TC-DNA, but not FC-DNA, concentrating within a plug was affected by the combined effect so as to achieve a concentration factor (C(r)) twice that of FC-DNA because of the lipophilicity of TAMRA. Using fluorescence resonance-energy transfer (FRET), we characterized the hybridization of the DNA in a plug; the detection limit of a system, improved by virtue of the proposed technique (the locally enhanced concentration), for DNA detection was estimated to be 20-50 nM. This technique enables DNA to concentrate locally in a nL-pL free-solution plug, the locally enhanced concentration to profit the hybridization efficiency and the detection of DNA, prospectively serving as a versatile means to accomplish a rapid DNA detection in a small volume for a Lab-on-a-Chip (LOC) system.

Detection of p16INK4a Promoter Methylation Status &1QJ;in Non-small Cell Lung Cancer by a Fluorescence Polarization Assay

The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.

Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor

An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3′-terminal, and a fluophor modified at 5′-terminal. It was found that the guanine bases at the 3′-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5′-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP C in the range from 1.1 to 44.7 μg/mL with a limit of detection of 0.3 μg/mL (3 σ).

Highly sensitive and selective oligonucleotide sensor for sickle cell disease gene using photon upconverting nanoparticles

We report the design of an oligonucleotide sensor for the detection of point mutation associated with sickle cell disease. The sensor was based on luminescence resonance energy transfer between a donor and an acceptor. Photon upconverting nanoparticles (NaYF 4 doped with Yb 3+ and Er 3+) were used as the donor and a conventional fluorophore, N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA), as the acceptor. The sensor could detect the perfectly matched target, in the background of the mismatched target or other oligonucleotides of random sequences. The detection limit of this sensor towards perfectly matched target was calculated to be 120 femtomoles, with no photobleaching. Oligonucleotide sensors of such design demonstrate high sensitivity and specificity.

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5′-CTGAT TAGAG AGAGAA-TAMRA-3′ and 5′-TET-ATGTC TGAGC TGCAGG-3′) and target DNA (3′-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5′) were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 × 10 −6 to 1.0 × 10 −7 M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.