PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids

PCR-free nucleic acid detector based on nanopore sensing

The development of a rapid, sensitive, and cost-effective nucleic acid (NA) detection platform is highly desired for a range of diverse applications. We designed and developed an optical label free, PCR independent and potentially low-cost device for sequence-specific nucleic acid detection. The detection is based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid (PNA) probe) conjugates upon hybridization with target nucleic acid. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pore, which they obstruct, leading to an indefinite, electrically detectable blockade of the pore. In the present of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade was not seen. We show application of this platform to detection of the anthrax lethal factor sequence. Next, we demonstrated the operation of our device with longer DNA targets, and we described the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. Also, target DNA fragments at 10 fM concentration (approximately 6 105 molecules) were detected against a DNA background simulating the non-complementary genomic DNA present in real sample. In addition, no false positive responses were obtained with non-complementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentration based on recognition of species-specific 16S rRNA sequences. To investigate that we detected the specific sequence of 16S rRNA of non pathogenic E.Coli K-12 at 10fM detection limit. Two different non-pathogenic bacteria were used as negative experimental control and the universal PNA probe complementary to all three bacteria was used as positive experimental control. We could successfully detect E.Coli K-12 16S rRNA with no false negative and only one false positive at 3.5 x 104 colony forming units (CFU). This detection limit is below the threshold concentration limit for detecting the pathogenic E.Coli in urine and therefore our device has a potential to be used for detecting the clinically significant pathogens

Other title: Using eDNA to determine Hellbender distribution

"March 10th, 2017."; Includes bibliographical references (page 19).; Interim report.; State Job Number 135321Environmental DNA (eDNA) methods are non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. In this project, we have evaluated the efficacy of eDNA sampling to detect populations of the eastern hellbender indirectly from their aquatic environments. We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods

A label-free and low-power microelectronic impedance spectroscopy for characterization of exosomes.

Electrical Impedance Spectroscopy (EIS) is a non-invasive and label-free technology that can characterize and discriminate cells based on their dielectric properties at a wide range of frequency. This characterization method has not been utilized for small extracellular vesicles (exosomes) with heterogenous and nano-scale size distribution. Here, we developed a novel label-free microelectronic impedance spectroscopy for non-invasive and rapid characterization of exosomes based on their unique dielectric properties. The device is comprised of an insulator-based dielectrophoretic (iDEP) module for exosomes isolation followed by an impedance spectroscopy utilizing the embedded micro-electrodes. This device is capable of distinguishing between exosomes harvested from different cellular origins as the result of their unique membrane and cytosolic compositions at a wide range of frequency. Therefore, it has the potential to be further evolved as a rapid tool for characterization of pathogenic exosomes in clinical settings

Sequence-specific DNA detection at 10 fM by electromechanical signal transduction.

Target DNA fragments at 10 fM concentration (approximately 6 × 10(5) molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences

Sequence-specific Nucleic Acid Detection from Binary Pore Conductance Measurement

We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence

Sequence-specific Nucleic Acid Detection from Binary Pore Conductance Measurement

We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence

Sequence-specific Nucleic Acid Detection from Binary Pore Conductance Measurement

We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence

Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction

Target DNA fragments at 10 fM concentration (approximately 6 × 10⁵ molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences