Construction of Electrochemical Aptamer Sensor Based on Pt-Coordinated Titanium-Based Porphyrin MOF for Thrombin Detection

In this work, a Pt-coordinated titanium-based porphyrin metal organic framework (Ti-MOF-Pt) was prepared by embedding single-atom Pt through strong interactions between the four pyrrole nitrogen atoms in the rigid backbone of the porphyrin. The synthesized Ti-MOF-Pt was characterized by TEM, XRD, FTIR and BET. Then, the Ti-MOF-Pt has been used for glassy carbon electrode surface modification and consequently used for construction of a thrombin aptamer sensor. The high surface area provides by MOF and excellent electrochemical property provided by Pt enhance the sensing performance. After optimization of amount of aptamer, hybridization time and specific reaction time, the fabricated aptamer sensor exhibited a linear relationship with the logarithm of the thrombin concentration in the range of 4 pM to 0.2 μM. The detection limit can be calculated as 1.3 pM.

FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

FISH-TAMB, a fixation-free mRNA fluorescent labeling technique, to target transcriptionally active members in microbial community

Keystone species or ecological engineers are vital to the health of an ecosystem, however, often their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, and the fast hybridization time of as short as 15 minutes at target cells’ growth temperature. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A ( mcr A) expressed in 1) Escherichia coli containing a plasmid with a partial mcr A gene of the methanogen Methanosarcina barkeri ( E. coli mcr A + ); 2) M. barkeri ; and 3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA, thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

Label-Free Electrochemical Detection of DNA Hybridization Related to Anthrax Lethal Factor by using Carbon Nanotube Modified Sensors

Background: Anthrax Lethal Factor (ANT) is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals. In this paper, pencil graphite electrodes GE were modified with single-walled and multi-walled carbon nanotubes (CNTs) for the detection of hybridization related to the ANT DNA for the first time in the literature. Methods: The electrochemical monitoring of label-free DNA hybridization related to ANT DNA was explored using both SCNT and MCNT modified PGEs with differential pulse voltammetry (DPV). The performance characteristics of ANT-DNA hybridization on disposable GEs were explored by measuring the guanine signal in terms of optimum analytical conditions; the concentration of SCNT and MCNT, the concentrations of probe and target, and also the hybridization time. Under the optimum conditions, the selectivity of probe modified electrodes was tested and the detection limit was calculated. Results: The selectivity of ANT probes immobilized onto MCNT-GEs was tested in the presence of hybridization of probe with NC no response was observed and with MM, smaller responses were observed in comparison to full-match DNA hybridization case. Even though there are unwanted substituents in the mixture samples containing both the target and NC in the ratio 1:1 and both the target and MM in the ratio 1:1, it has been found that ANT probe immobilized CNT modified graphite sensor can also select its target by resulting with 20.9% decreased response in comparison to the one measured in the case of full-match DNA hybridization case Therefore, it was concluded that the detection of direct DNA hybridization was performed by using MCNT-GEs with an acceptable selectivity. Conclusion: Disposable SCNT/MCNT modified GEs bring some important advantages to our assay including easy use, cost-effectiveness and giving a response in a shorter time compared to unmodified PGE, carbon paste electrode and glassy carbon electrode developed for electrochemical monitoring of DNA hybridization. Consequently, the detection of DNA hybridization related to the ANT DNA by MCNT modified sensors was performed by using lower CNT, probe and target concentrations, in a shorter hybridization time and resulting in a lower detection limit according to the SCNT modified sensors. In conclusion, MCNT modified sensors can yield the possibilities leading to the development of nucleic acid sensors platforms for the improvement of fast and cost-effective detection systems with respect to DNA chip technology.

An Impedimetric Biosensor Based on Ionic Liquid-Modified Graphite Electrodes Developed for microRNA-34a Detection

In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent activation of the PGE surface using covalent agents (CAs), the ionic liquid (IL) was immobilized onto the surface of the chemically activated PGE by passive adsorption. The electrochemical and microscopic characterization of the IL/CA/PGEs was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM). DNA probe concentration, miRNA target concentration, and also the hybridization time and wet adsorption time were optimized by using the EIS technique. Then, the hybridization occurred between specific DNA probes and miRNA-34a was immobilized onto the surface of the IL/CA/PGEs. The impedimetric detection of miRNA-DNA hybrid was performed by EIS. The detection limit (DL) was calculated in a linear concentration range of 2–10 µg/mL miRNA-34a target, and it was found to be 0.772 µg/mL (109 nM) in phosphate buffer solution (PBS) and 0.826 µg/mL (117 nM) in diluted fetal bovine serum (FBS). The selectivity of impedimetric biosensor for miRNA-34a was also tested against to other non-complementary miRNA sequences both in buffer media, or diluted FBS.

Optimization and Application of Fluorescence in Situ Hybridization Assay for Detecting Polyphosphate - Accumulating Microorganisms

Laboratory-scale sequencing batch reactors (SBRs) were operated on activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Polyphosphate-accumulating microorganisms (PAOs) play an important role during the enhanced biological phosphorus removal (EBPR) process. Fluorescence in situ hybridization (FISH) was applied to assess the proportions of microorganisms in the sludge. The aim of this study was to optimize hybridization of PAOMIX and RHC439 probes by orthogonal design. Orthogonal optimization test of the four factors were conducted under the individual three levels. The optimal hybridizition conditions were as follow: hybridization temperature 46°C, hybridization time 2.5h, washing time 15min, formamide concentration 35%(PAOMIX probe); hybridization temperature 50°C, hybridization time 2.5h, washing time 20min, formamide concentration 20% (RHC439 probe).

Microscopic Detection of DNA Hybridization using Miniaturized Diamond DNA-FETs

Miniaturized DNA sensitive field-effect transistors (DNA-FET) have been realized using single crystalline diamond grown by plasma-enhanced chemical vapor deposition (CVD). To bond DNA to diamond, amine linker-molecules are covalently attached by photochemical means to H-terminated diamond surfaces. Using hetero-bifunctional cross-linker and thiol-modified single-strand (ss) cancer marker DNA (CK20), the gate of diamond FETs is modified to sense hybridization of DNA, forming double-strand (ds) DNA molecules on the gate. The density of DNA bonded to diamond has been adjusted to about 1012 cm−2 and the experiments have been performed in phosphate buffer with different ionicity to control the Debye length of the Helmholtz layer. By hybridization, a gate-potential shift of 64 mV is detected in case of the 100 Å Debye lengths, while 46 mV is detected for 10 Å. This is discussed with respect to DNA related variations of charge and pH by hybridization. Time resolved experiments reveal exponential hybridization dynamics with a time constant of 600 s. The sensitivity limit of our experiment is about 1 nM.

ELECTROOSMOTIC MIXING INDUCED BY NON-UNIFORM ZETA POTENTIAL AND APPLICATION FOR DNA MICROARRAY IN MICROFLUIDIC CHANNEL

In this study, we describe a modification of the conventional microarray format. 20-mer oligonucleotide probes and singly labeled 20-mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, has been used to elucidate the performance of this hybridization approach. DNA microarray is integrated with microfluidic channel on a poly(methyl methacrylate) (PMMA) to generate non-uniform zeta potential inside the channel. A microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber The electroosmotic mixing effect induced by the non-uniform zeta potential is utilized to enhance the DNA-DNA hybridization. The flow field in microfluidic channel is measured by particle image velocimetry (PIV). The enhanced signal to noise (S/B) ratio and reduced hybridization time is observed when the electroosmotic mixing is applied in the DNA-DNA hybridization.

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

ABSTRACT Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

Detection of Two Orchid Viruses Using Quartz Crystal Microbalance-Based DNA Biosensors

We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5′-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses.