Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindic... more Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results ident...
Malaria is a parasite with a complex lifecycle, and commonly used antimalarial agents from the ar... more Malaria is a parasite with a complex lifecycle, and commonly used antimalarial agents from the artemisinin family have varied effectiveness over different stages of this lifecycle. The pharmacokinetic profile of the artemisinins is also strongly influenced by the parasite burden and lifecycle stage. This work introduces a new pharmacokinetic and pharmacodynamic model incorporating these interdependent drug and lifecycle features, for orally administered artesunate and its principal metabolite dihydroartemisinin. This model, like the underlying system whose features it attempts to capture, is quite complex and cannot be solved analytically like standard linear first-order compartmental models previously used for pharmacokinetic modelling of these drugs. Therefore, understanding, inference and validity are explored through use of the modern statistical technique of a Sequential Monte Carlo sampler. Structural, numerical and practical identifiability are important concepts for all mode...
Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. T... more Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6–12 weeks) and old (14–17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme‐mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical pr...
In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bila... more In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid’s acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inf...
Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerou... more Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the...
Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact... more Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modif...
We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid... more We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 x 100-nm2 precision in standard SiNx membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.
5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modifica... more 5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.
The Journal of pharmacology and experimental therapeutics, Jun 30, 2016
GABA-A receptors meet all the pharmacological requirements necessary to be considered important t... more GABA-A receptors meet all the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6 dimethylcyclohexanol diastereomers were investigated on human γ-aminobutyric acid type A (GABA-A, α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis; trans,trans; and cis,trans isomers were isolated from commercially available 2,6 dimethylcyclohexanol and were tested for positive modulation of sub-maximal GABA responses. For example, the addition of 30 μM cis,cis isomer resulted in ~2-3 fold enhancement of the EC20 GABA current. Co-applications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > cis,trans isomer. In molecular modeling studies, the three cycl...
International journal for ion mobility spectrometry : official publication of the International Society for Ion Mobility Spectrometry, 2012
Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring l... more Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chroma...
We investigated experimentally and theoretically the translocation forces when a charged polymer ... more We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA m...
Proceedings of the National Academy of Sciences, 2013
Significance This research highlights the discovery and genome reconstruction of a member of the ... more Significance This research highlights the discovery and genome reconstruction of a member of the globally distributed yet uncultivated candidate phylum TM6 (designated TM6SC1). In addition to the 16S rRNA gene, no other genomic information is available for this cosmopolitan phylum. This report also introduces a mini-metagenomic approach based on the use of high-throughput single-cell genomics techniques and assembly tools that address a widely recognized issue: how to effectively capture and sequence the currently uncultivated bacterial species that make up the “dark matter of life.” Amplification and sequencing random pools of 100 events enabled an estimated 90% recovery of the TM6SC1 genome.
Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindic... more Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results ident...
Malaria is a parasite with a complex lifecycle, and commonly used antimalarial agents from the ar... more Malaria is a parasite with a complex lifecycle, and commonly used antimalarial agents from the artemisinin family have varied effectiveness over different stages of this lifecycle. The pharmacokinetic profile of the artemisinins is also strongly influenced by the parasite burden and lifecycle stage. This work introduces a new pharmacokinetic and pharmacodynamic model incorporating these interdependent drug and lifecycle features, for orally administered artesunate and its principal metabolite dihydroartemisinin. This model, like the underlying system whose features it attempts to capture, is quite complex and cannot be solved analytically like standard linear first-order compartmental models previously used for pharmacokinetic modelling of these drugs. Therefore, understanding, inference and validity are explored through use of the modern statistical technique of a Sequential Monte Carlo sampler. Structural, numerical and practical identifiability are important concepts for all mode...
Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. T... more Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6–12 weeks) and old (14–17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme‐mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical pr...
In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bila... more In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid’s acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inf...
Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerou... more Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the...
Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact... more Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modif...
We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid... more We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 x 100-nm2 precision in standard SiNx membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.
5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modifica... more 5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.
The Journal of pharmacology and experimental therapeutics, Jun 30, 2016
GABA-A receptors meet all the pharmacological requirements necessary to be considered important t... more GABA-A receptors meet all the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6 dimethylcyclohexanol diastereomers were investigated on human γ-aminobutyric acid type A (GABA-A, α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis; trans,trans; and cis,trans isomers were isolated from commercially available 2,6 dimethylcyclohexanol and were tested for positive modulation of sub-maximal GABA responses. For example, the addition of 30 μM cis,cis isomer resulted in ~2-3 fold enhancement of the EC20 GABA current. Co-applications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > cis,trans isomer. In molecular modeling studies, the three cycl...
International journal for ion mobility spectrometry : official publication of the International Society for Ion Mobility Spectrometry, 2012
Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring l... more Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chroma...
We investigated experimentally and theoretically the translocation forces when a charged polymer ... more We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA m...
Proceedings of the National Academy of Sciences, 2013
Significance This research highlights the discovery and genome reconstruction of a member of the ... more Significance This research highlights the discovery and genome reconstruction of a member of the globally distributed yet uncultivated candidate phylum TM6 (designated TM6SC1). In addition to the 16S rRNA gene, no other genomic information is available for this cosmopolitan phylum. This report also introduces a mini-metagenomic approach based on the use of high-throughput single-cell genomics techniques and assembly tools that address a widely recognized issue: how to effectively capture and sequence the currently uncultivated bacterial species that make up the “dark matter of life.” Amplification and sequencing random pools of 100 events enabled an estimated 90% recovery of the TM6SC1 genome.
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Papers by Adam Hall